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Cytochrome c cationic oligopeptide conjugates: their cellular uptake, intracellular processing and biological activity

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Cytochrome c cationic oligopeptide conjugates: their cellular uptake, intracellular processing and biological activity

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CYTOCHROME C CATIONIC OLIGOPEPTIDE CONJUGATES:
THEIR CELLULAR UPTAKE, INTRACELLULAR PROCESSING
AND BIOLOGICAL ACTIVITY

by

Maureen P. Barnes
____________________________________________________________________

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)

May 2007

Copyright 2007 Maureen P. Barnes

ii
Dedication

This Dissertation is Dedicated to My Mother, Colleen McGonegal.
May She Rest in Peace.

iii
Acknowledgments

I respectfully acknowledge and thank my advisor
Dr. Wei-Chiang Shen
for his guidance and invaluable help, without which this work could not have been
accomplished.

I would like to thank my committee members
Dr. Judy Garner, Dr. Sarah Hamm-Alvarez, Dr. Curtis Okamoto,
& Dr. Clay Wang
for their insightful recommendations and constant willingness to assist in my
research.

I would also like to thank
Daisy Shen and my many labmates
for their friendship and their sharing of scientific deliberation.

My appreciation goes to
My closest friends, especially Anita McElroy and Amy Bauer
who always believed I had what it took to climb this mountain and emerge a better
person.

I am very grateful to
My family, my brother and sisters
who have loved me and supported me from the start.

My sincerest thanks goes to
My dearest father, Steele McGonegal
For the endless encouragement and invaluable scientific knowledge that you have
imparted to me since childhood. I think I played my cards right.

My deepest admiration and gratitude goes to my beloved husband
Edward Barnes III
without your constant support, strength, understanding and immeasurable love, this
dream would never have been realized. Thank you, from the bottom of my heart.

iv
Table of Contents

Dedication…………………………………………………………………………… ii
Acknowledgments…………………………………………………………………... iii
Table of Contents…………………………………………………………………… iv
List of Figures………………………………………………………………………. vii
Abstract……………………………………………………………………………... ix
Preface
1. Synopsis of Discipline………………………………………………………......... xi
2. Scope of Project…………………………………………………………………... xii

Chapter One: Preparation of Conjugates.
1.1. INTRODUCTION…………………………………………………………….. 1
1.1.1. Use of Oligopeptides as Carriers…………………………………………. 1
1.1.2. Cytochrome C as a Potential Cargo………………………………………. 2
1.1.3. NHS-Ester Cross-linkers……………………...…………………………... 5
1.2. EXPERIMENTAL……………………………………………………….……. 8
1.2.1. Materials…….…………………………………………………………….. 8
1.2.2. Preparation of Cytochrome C-Oligopeptide Conjugates……….…………. 8
1.2.2.1. Disulfide Cross-Linker……….………………………………………8
1.2.2.2. Thioether Cross-Linker………….…………………………………... 9
1.2.3. Purification and Characterization of Conjugates……………….…………. 10
1.2.3.1. Optimization of Conjugates……………….………………………… 10
1.2.3.2. Size Exclusion Chromatography………………….…………………. 10
1.2.3.3. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis……... 11
1.2.3.4. Amino Acid Analysis………………………………….…………….. 11
1.2.4. Radioactive Labeling of Cytochrome C and Conjugates………….………. 11
1.3. RESULTS……………………………………………………………….……... 13
1.3.1. Purification and Characterization of Conjugates….………………………. 13
1.3.1.1. Ultraviolet Absorbance of G-25 Filtered Conjugates……….………. 13
1.3.1.2. SDS-PAGE…….……………………………………………………. 13
1.3.1.3. Amino Acid Analysis…..……………………………………………. 16
1.3.2. Measurement of Radioactive Label….……………………………………. 16
1.4. DISCUSSION…….…………………………………………………………… 18
1.4.1. Reducible Conjugates……….…………………………………………….. 19
1.4.2. Non-Reducible Conjugates…………………………….………………….. 19
1.5. CONCLUSION…….………………………………………………………….. 21

v
Chapter Two: Intracellular Processing of Cytochrome C and Cytochrome C
Conjugates.
2.1. INTRODUCTION…………………………………………………………….. 22
2.1.1. Background Information…….…………………………………………….. 22
2.1.2. Degradation of Cytochrome C…….………………………………………. 23
2.2. EXPERIMENTAL…………………….………………………………………. 26
2.2.1. Materials…….…………………………………………………………….. 26
2.2.2. Cell Culture…….………………………………………………………….. 26
2.2.3. Cellular Processing of Conjugates and Cytochrome C…….……………… 27
2.2.3.1. Cellular Uptake….…………………………………………………... 27
2.2.3.2. Subcellular Fractionation….………………………………………… 28
2.2.3.2.1. Analysis of Third Peak in Subcellular Fractionation….……… 31
2.2.3.3. Degradation Pathways………………………………………………. 31
2.3. RESULTS……………….……………………………………………………... 33
2.3.1. Enhanced Cellular Uptake by Conjugates…….…………………………... 33
2.3.2. Involvement of Endocytosis vs. Transduction…………………………….. 37
2.3.3. Origination of Third Peak……………………………………….………… 42
2.3.4. Sensitivity of Internalization to Endosomal Acidification…………….…... 44
2.3.5. Proteasome Inhibitor Assay……………………………………………….. 46
2.4. DISCUSSION….…………………………………………………………….... 48
2.4.1. Enhanced Cellular Uptake by Conjugates……….………………………... 48
2.4.2. Intracellular Processing……………………………………………………. 50
2.4.3. Degradation Pathways…………………………………………………….. 52
2.5. CONCLUSION.……………………………………………………………….. 55

Chapter Three: Biological Activity of Cytochrome C and Cytochrome C
Conjugates.
3.1. INTRODUCTION….………………………………………………………….. 57
3.1.1. Cytochrome C’s Role in Apoptosis…….…………………………………. 57
3.1.2. Glutathione………………………………………….……………………... 62
3.2. EXPERIMENTAL…….………………………………………………………. 65
3.2.1. Materials……….………………………………………………………….. 65
3.2.2. Cell Culture…………………….………………………………………….. 65
3.2.3. Biological Activity of Conjugates and Unmodified Cytochrome C….…… 66
3.2.3.1. Cytotoxicity………………………………….……………………… 66
3.2.3.2. Apoptotic Effect…………….……………………………………….. 66
3.2.4. Effect of Degradation Inhibitors on Apoptosis………….………………… 68
3.2.4.1. Proteasome Inhibition…….…………………………………………. 68
3.2.4.2. Endosomal Acidification Inhibition…………………….…………… 68
3.2.5. Disulfide Bond Reduction……………………….…………………………69
3.2.5.1. Glutathione Assay……………….…………………………………... 69
3.3. RESULTS………….…………………………………………………………... 72
3.3.1. Cytotoxicity by Pierce Protein Assay…….……………………………….. 72

vi
3.3.2. Annexin V / Propidium Iodide Assay………………….………………….. 77
3.3.3. Acridine Orange Assay and Laser Scanning Microscopy Images……..….. 81
3.3.4. Proteasome Inhibitor Assay………………………….……………………. 85
3.3.5. Endosomal Acidification Inhibitor Assay……………….………………… 87
3.3.6. Glutathione Assay……………….………………………………………… 90
3.4. DISCUSSION……….………………………………………………………… 92
3.4.1. Biological Activity……….………………………………………………... 92
3.4.2. Degradation and Reduced Activity………………………….…………….. 96
3.4.3. Inhibition of Disulfide Bond Reduction…………….…………………….. 99
3.5. CONCLUSION………….…………………………………………………….. 100

Chapter Four: Summary and Future Perspectives.
4.1. Summary of Dissertation…….……………………………………………......... 102
Scheme 4.1. Summary of the major findings from this thesis.………................... 105
4.2. Future Perspectives……..……………………………………..………………... 106

References (Numerical)…………………………………………………………….. 108
References (Alphabetical)………………………………………………………….. 116

vii
List of Figures

1.1. Structure of cytochrome c……………………………………………………… 4
1.2. Chemical structure of cross-linkers………...……………………..…………… 6
1.3. Reaction chemistry of cross-linkers………………………………………….… 7
1.4. SDS-PAGE gel……………………………………………...…………………. 15
1.5. Elution profile for radiolabeled conjugates and cytochrome c…………….…... 17
2.1. Structure of MG132………………………………………………………….… 25
2.2. Representation of elution profile for subcellular fractionation……………..….. 30
2.3. Cellular uptake of conjugates and cytochrome c in CHO cell line…………….. 34
2.4. Cellular uptake of conjugates and cytochrome c in U937 cell line………...….. 35
2.5. Cellular uptake of conjugates and cytochrome c in HeLa cell line……………. 36
2.6. Subcellular fractionation of high vs. low conjugates in CHO cell line………... 38
2.7. Subcellular fractionation of C(K)
9
vs. C(R)
9
conjugates in CHO cell line….…. 39
2.8. Example elution profile of oligoarginine conjugate with degradation peak….... 40
2.9. Subcellular fractionation of reducible vs. non-reducible conjugates in HeLa
cell line……………………………………………………………………………….. 41
2.10. Spiked cell elution from S500 gel…………………………………………….... 43
2.11. TCA precipitation of cytochrome c………………………………………….… 45
2.12. Subcellular fractionation with MG132 in HeLa cell line……………………… 47
3.1. Structure of apoptosome……………………………………………………..… 60
3.2. Apoptotic cascade initiated by cytochrome c……………………...…………... 61
3.3. Structure of glutathione……………………………...……………………….... 64

viii
3.4. Cytotoxicity in CHO cell line…………………………………...……………... 74
3.5. Cytotoxicity in U937 cell line…………………………………..…………….... 75
3.6. Cytotoxicity in HeLa cell line……………………………………..………….... 76
3.7. Representation of Annexin V / Propidium Iodide Flow Cytometry results…… 78
3.8. Apoptosis in U937 cell line…………..……………………………………..…. 79
3.9. Apoptosis in HeLa cell line..………………………………………………..…. 80
3.10. Acridine orange staining in U937 cell line…………………………………..… 83
3.11. LSM and acridine orange staining in HeLa cell line...……………………….... 84
3.12. Apoptosis with MG132 in HeLa cell line……………………………….…..…. 86
3.13. Apoptosis with ammonium chloride in HeLa cell line……………………..….. 88
3.14. Apoptosis of cytochrome c with multiple inhibitors in HeLa cell line……..…. 89
3.15. Apoptosis with acetaminophen in HeLa cell line……………………...……..... 91

ix
Abstract

Peptides characterized as protein transduction domain peptides (PTD) or
membrane transduction peptides (MTP) have recently attracted attention as a novel
approach for the efficient intracellular delivery of oligopeptides, oligonucleotides
and other bioactive macromolecules, primarily into the cytoplasm of mammalian
cells. However, the effect of the linkage between cargo and its cationic oligopeptide
carrier on the internalization, intracellular processing and biological activity of the
cargo has not been fully deliberated. In this thesis, the cellular processing of
conjugates of PTDs and the nuclear encoded apoptotic protein cytochrome c
prepared with either a disulfide or a thioether linkage is studied and the conjugates
used as a tool to learn more about the intracellular fate of cationic oligopeptides.
The results from this work, performed in multiple cell lines, including CHO, U937
and HeLa, show that the uptake of the conjugates is increased when compared to that
of cytochrome c alone. In HeLa cells, apoptotic activity of cytochrome c is observed
only in the thioether conjugate, but not in the cytochrome c or the disulfide
conjugate. However, apoptosis is restored in the disulfide conjugate with the
addition of the proteasome inhibitor, MG132. Furthermore, the addition of MG132
resulted in an increase of apoptotic activity in cytochrome c. Conceivably, MG132
can protect cytochrome c, either in the native form or as a product released from the
disulfide conjugate, from degradation by proteasomes. This model is further
supported by the finding that treatment with acetaminophen, a glutathione depleting

x
agent, markedly enhances the apoptotic effect of the disulfide conjugate, but not of
the cytochrome c or the thioether conjugate. This result suggests that not only does
the preservation of the cargo-carrier complex not hinder the cargo’s biological
activity, it is required for the retention of the conjugate’s biological activity.

xi
Preface

1. Synopsis of Discipline

PTD peptides are typically classified as cationic in nature and primarily contain
arginine and lysine residues. They include the basic domain of the nuclear
transcription activator Tat (47-57) (YGRKKRRQRRR) encoded by HIV-1,
Drosophila Antennapedia, Antp (43-58) (RQIKIYFQNRRMKWKK), and small
oligoarginine (R)
n
and oligolysine (K)
n
peptides. The field of peptide based drug
design is dependant upon the use of carriers for drug delivery. Many promising
drugs fail early in clinical trials due to the limitations in their bioavailability. Current
methods using large cationic peptides (up to 100,000 D) of polylysine and
polyarginine have encountered several complications including cytotoxicity and
protein precipitation. These methods also rely on the ability of the carrier peptide to
escape the endosome before lysosomal degradation. PTD sequences are typically
less than 20 amino acids long and do not share the problems of cytotoxicity or
protein precipitation found with the large molecular weight cationic peptides. PTD-
conjugates offer a unique and efficient transduction mechanism for direct
cytoplasmic transport, which can be used to deliver macromolecules and hydrophilic
drugs that are dependant on intracellular delivery.
Cytochrome c (cyt c) is a ~12 kDa protein with a highly conserved amino acid
sequence across the spectrum of species, to include plants, animals, and many
unicellular organisms, making it useful in studies of evolutionary divergence.
1,2,3,4

xii
Horse heart cytochrome c, used in all experiments explained here, and the human
cytochrome c are highly homologous. They differ in only 6 of 104 amino acid
residues.
5

Cytochrome c is a nuclear encoded protein located in the inner membrane space
of all mitochondria. The molecule typically functions in the electron transport
system of oxidative phosphorylation to release energy in the form of ATP. When
released from the mitochondria in response to pro-apoptotic stimuli, cytochrome c
plays a pivotal role in the regulation of apoptosis. The apoptotic function of
cytochrome c makes it an intriguing peptide for cytoplasmic delivery, though on its
own it is ineffective when added in cell cultures, presumably due to the
impermeability across the cell membrane at any significant concentrations.
Conceivably, PTD-mediated membrane transport should be capable of overcoming
the membrane barrier to result in cytochrome c activity.

2. Scope of Project

To understand the optimal complex between a cationic oligopeptide carrier and
its cargo, the fate of this complex must be understood. We have successfully
prepared cytochrome c conjugated to cysteine-terminal oligoarginine, C(R)
9
and
cysteine-terminal oligolysine, C(K)
9
via a disulfide linkage at multiple degrees of
modification as a means to establish the optimal conjugate to use as a tool in
determining the transport and intracellular processing of membrane transduction
peptides. In addition, cytochrome c has been conjugated to cysteine-terminal

xiii
oligoarginine, C(R)
9
via a thioether linkage, for comparison to the disulfide linked
complex. The analysis of a reducible versus a non-reducible conjugate of the
cationic oligopeptide C(R)
9
and cytochrome c further provides us with a means to
investigate the intracellular processing of transduced proteins in the cytoplasmic
compartment. Using this tool, we have investigated the cellular uptake, and
intracellular processing of oligopeptide conjugates versus the unconjugated
cytochrome c. Furthermore, through the analysis of degradation pathways of
cytochrome c, we have learned that the preservation of the conjugate, and not release
of the cargo from the carrier as once thought, is necessary to retain the cargo’s
biological activity in the cytoplasm. The discovery of this information can lead to
the optimization of oligopeptide’s as a carrier of therapeutic agents into the
cytoplasm of cells and their use as a drug delivery system.

1

Chapter One
Preparation of Conjugates

1.1. INTRODUCTION

1.1.1. Use of Oligopeptides as Carriers

Cationic oligopeptides have recently been studied for their ability to traverse
cellular membranes. Though the actual mechanism of entry has still not been
elucidated, it is clear that the high cationic charge allows entry through adsorptive
endocytosis, as well as transduction directly to the cytosol.
6,7
The Tat peptide and
oligoarginine peptides have been shown to deliver a wide range of molecules, from
small molecular weight molecules and proteins to liposomes, both in vitro and in
vivo.
8,9,10,11,12,13
However, the attachment of large macromolecules to the peptides
can not only change the internalization properties of the conjugates, but can also
change the intracellular activity of the macromolecule.
14,15,16
These complications
have not been sufficiently studied. In order to establish the boundaries of
oligoarginine delivery systems for macromolecular cargo, the optimal carrier peptide
model for the delivery of the macromolecule cytochrome c was developed and these
limitations researched.

2
1.1.2. Cytochrome C as a Potential Cargo
Cytochrome c has been studied extensively for its ability to propagate the
apoptotic cascade leading to controlled cell death. The apoptotic function of
cytochrome c makes it an intriguing peptide for cytoplasmic delivery of conjugates.
The biological activity allows for an endpoint measure of the conjugate’s successful
entry into the cytoplasm as well its intracellular processing. Past experiments have
attempted to conjugate cytochrome c to biotin as a means to study this protein’s role
in apoptosis.
17,18
There has been success with this conjugation, but only at a low
molar ratio of 1.4:1, biotin:cytochrome c, and the subsequently biotinylated
cytochrome c is assayed by utilizing harsh methodology such as electroporation, due
to its inability to permeate the cell to any extensive degree on its own. The over-
modification of cytochrome c with biotin has resulted in diminished biological
activity of the molecule. Attempts to induce apoptosis, via a highly modified
biotinylated cytochrome c of 4-5 moles biotin per mole cytochrome c added directly
to cytoplasmic extracts, have failed.
17
These findings suggest that the lack of activity
may be the result of over-modification of the molecule by biotin labeling. Using the
cationic peptides oligoarginine and oligolysine, the limitations of conjugation to
cytochrome c via NHS-cross-linkers that will result in a reducible and a non-
reducible linkage via high and low molar ratios will be analyzed.
The structure of cytochrome c has been extensively studied, lending to a broad
structural database of the peptide.
1,19,20,21,22
It consists of a single polypeptide chain
and a heme group that is almost completely buried inside the protein, covalently

3
anchored by two thioether bonds through two cysteine residues, making it difficult to
displace.
23
Due to their location deep within the molecule, the cysteine residues are
not available for thiol linkage. However, there are approximately 19 naturally
occurring, variable lysine residues available for modification through a NHS-ester
cross-linking reaction. The amine containing

cytochrome c can be conjugated to a
cross-linker via the amide bond. This cross-linker would serve as a means to create
either a disulfide bond between cytochrome c and the oligopeptide or a non-
reducible thioether bond between the two. The amino acid structure of cytochrome c
is shown in Figure 1.1.

4

Figure 1.1. Structure of cytochrome c. The nineteen lysine residues are shown in
pink and the two cysteine residues, bound to the Heme, are shown in yellow. Full
circles around the amino acid imply invariable residues (required for proper folding
and function), partial circles imply conserved residues (partially required) and no
circle implies variable residues. All nineteen lysine residues are variable, and can be
altered with no consequence to cytochrome c’s function.

5
1.1.3. NHS-Ester Cross-linkers
N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP) is a widely used
heterobifunctional cleavable cross-linking reagent. It contains one amine reactive N-
hydroxysuccinimide (NHS) residue and one sulfhydryl reactive pyridyl disulfide
residue. These functional groups allow for a two step reaction between an amine-
containing protein, such as cytochrome c, that is not able to form a disulfide linkage
on its own, and a free sulfhydryl-containing protein, such as C(R)
9
or C(K)
9
to form
a disulfide bond between the cytochrome c and the oligopeptide.
24
The chemical
structure of SPDP is shown in Figure 1.2 and the chemical reaction is shown in
Figure 1.3.
Sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate (Sulfo-SMPB, also referred
to as SMPB) is a heterobifunctional cross-linker that contains a N-
hydroxysuccinimide (NHS) ester that will react with a primary amine at pH 7-9 as
explained above, and a maleimide that will react with sulfhydryl groups at pH 6.5-
7.5 to form stable thioether bonds. These functional groups allow for a two step
reaction between an amine-containing protein and a free sulfhydryl containing
protein, such as C(R)
9
, to form a stable thioether bond through a Michael addition
reaction. The chemical structure of SMPB is shown in Figure 1.2 and the chemical
reaction is shown in Figure 1.3.

6

SPDP
N-Succinimidyl 3-(2-pyridyldithio) propionate

Sulfo-SMPB
Sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate

Figure 1.2. Chemical structure of cross-linkers.

7
A)

B)

Figure 1.3. Chemistry reaction scheme of A) SPDP conjugation taken from Pierce
Biotechnology Instructions for SPDP conjugation and B) SMPB conjugation.

8
1.2. EXPERIMENTAL

1.2.1. Materials

N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP), sulfosuccinimidyl 4-[p-
maleimidophenyl]butyrate (Sulfo-SMPB) and the Micro BCA protein assay reagent
kit were obtained from Pierce Chemical Company (Rockford, IL). Oligopeptides
C(R)
9
and C(K)
9
were synthesized by Genemed (San Francisco, CA). Equine
cytochrome c, glycylglycine, dithiothreitol (DTT) and Sephadex G-25 gel matrix
were all purchased from Sigma (St. Louis, MO). The Na
125
I radioactive label was
obtained from ICN (Irvine, CA) and the molecular weight membrane was purchased
from Spectrum.

1.2.2. Preparation of Cytochrome C-Oligopeptide Conjugates

1.2.2.1. Disulfide Cross-Linker

The disulfide bonded conjugate was prepared by combining the amine-
containing protein equine cytochrome c with SPDP at 1:2 (low conjugate) and 1:5
(high conjugate) molar ratios, respectively, in phosphate buffer, pH 7.0, at 4°C, to
reduce hydrolysis, for 30 minutes. During this incubation, the amine reacts with the
cross-linker via the NHS-ester group. Glycylglycine was added to stop the reaction
and the conjugate was dialyzed in a 3,500 molecular weight cutoff membrane to
remove excess SPDP in 1x phosphate buffered solution (PBS) overnight. A small
aliquot of the 2-pyridyl disulfide-activated conjugate was treated with 25 mM DTT

9
for 10 min

at room temperature to reduce SPDP by pyridine-2-thione cleavage, and
the concentration of moles cross-linker to moles cytochrome c was estimated
spectrophotometrically at 343 nm (Molar extinction coefficient = 8.08 x 103 M
-1

cm
-1
). The 2-pyridyl disulfide-activated conjugate was then combined with 4x molar
excess of the free sulfhydryl-containing C(R)
9
(low and high conjugate) or C(K)
9

(low conjugate only) protein for one hour at room temperature to form the disulfide
bond and purified by size exclusion chromatography using a Sephadex G-25 gel
matrix. A combination of ultraviolet spectroscopy absorbance, to determine
cytochrome c protein concentration, and Pierce protein assay results, to determine
total protein concentration in the conjugate, was used to calculate the final molar
ratio of cytochrome c to oligopeptide. The conjugate was also sent for amino acid
analysis to confirm these results.

1.2.2.2. Thioether Cross-Linker

The thioether linked conjugate was prepared by combining cytochrome c with
Sulfo-SMPB in the same manner as the SPDP conjugate at 1:2 and up to 1:10 molar
ratios, respectively. After overnight dialysis in 1x PBS to remove free SMPB and a
recovery assay to determine the cytochrome c concentration in the conjugate, the
cytochrome c-SMPB conjugate was combined with 4x molar excess C(R)
9
for 30
minutes at room temperature and purified using a Sephadex G-25 gel matrix. No
reduction assay (such as the DTT assay for the SPDP conjugate) was performed on
the SMPB conjugates, given that there is no leaving group in the thioether reaction.

10
Instead, the conjugates were sent to the Life Science Research Center at National
Tsing Hua University in Hsinchu, Taiwan and were evaluated by amino acid analysis
to determine the exact molar ratio of the conjugate.

1.2.3. Purification and Characterization of Conjugates

1.2.3.1. Optimization of Conjugates

The SPDP and SMPB conjugates were prepared at multiple cross-linker molar
excess ratios compared to cytochrome c. Various molar ratios of cytochrome c:
cross-linker were attempted from a 1:1 molar ratio up to a 1:10 molar ratio, at
various temperatures and timed incubations, as well as purified by different means
(dialysis and both G-50 and G-25 columns) in order to obtain the purest low
modified conjugate of 1:2 (SPDP) and 1:0.5 (SMPB) as well as a highly modified
conjugate of 1:5 (SPDP only) that would optimize future experiments such as uptake
and cytotoxicity. The two discrete linker modifications were prepared with
oligoarginine only, not oligolysine, and will serve as a comparison to one another in
these experiments to determine the most favorable conjugate with respect to
efficiency of transduction and ability to promote biological activity.

1.2.3.2. Size Exclusion Chromatography

The final conjugate of cytochrome c to oligopeptide was purified by passing
through a 10 mL Sephacryl G-25 column (fractionation range: 1kDa - 5kDa) with
PBS mobile phase. The conjugate eluted at the column void volume and the free

11
oligopeptide is sufficiently separated, typically eluting after fraction 10. Absorbance
of the fractions, 20 total, was measured at 220, 280 and 450 nm to determine the
location of the peak for cytochrome c as well as that of the free oligopeptide. A
standard curve of cytochrome c was measured at 450 nm and the fractions compared
to it in order to determine the final concentration of cytochrome c in the conjugates.

1.2.3.3. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

The size and linkage of a select few of the conjugates were further characterized
using 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
with a 6.5% stacking gel, with and without DTT. The procedure was described
previously.
25

1.2.3.4. Amino Acid Analysis

The oligoarginine low conjugates of SPDP and SMPB were dried in a speedvac
for stability during shipment and sent to the Life Science Research Center at
National Tsing Hua University in Hsinchu, Taiwan and were analyzed by amino acid
analyzer in order to determine the ratio of cytochrome c to oligoarginine in the
SMPB conjugate as well as to confirm the ratio in the SPDP conjugate.

1.2.4. Radioactive Labeling of Cytochrome C and Conjugates

The tyrosine moiety of the conjugated cytochrome c was labeled with Na
125
I
using the Chloramine-T method.
26
The labeled conjugates were purified by size

12
exclusion chromatography using a Sephadex G-25 gel matrix. Cytochrome c alone
was also iodinated in this manner to serve as a comparison for the conjugates.

13
1.3. RESULTS

1.3.1. Purification and Characterization of Conjugates

1.3.1.1. Ultraviolet Absorbance of G-25 Filtered Conjugates

The SPDP conjugate final ratios were estimated to be 2 or 5 moles C(R)
9
per
each mole of cytochrome c and 2 moles C(K)
9
per mole of cytochrome c. The
SMPB conjugate final ratio was not estimated by these means. The final ratio was
determined solely by the amino acid analysis.

1.3.1.2. SDS-PAGE

This analysis was performed on the low SPDP oligoarginine conjugate, a high
SMPB oligoarginine conjugate (1:10 cytochrome c:oligoarginine) and cytochrome c
alone. The DTT addition in the SDS-PAGE was expected to affect only the SPDP
conjugate, given that the reducing agent breaks disulfide bonds, which are not
available for reduction in the cytochrome c or SMPB conjugate samples. The results
shown in Figure 1.4 show that the cytochrome c band emerged on the gel at
approximately 12 kDa for samples both with and without DTT. The SPDP with
DTT band is visible at approximately 12 kDa and without DTT is visible at
approximately 15 kDa. This result is expected since the DTT will reduce the SPDP
conjugate to cytochrome c and C(R)
9
, which more than likely ran off the gel and
therefore is not visible. The molecular weight of the SPDP conjugate without DTT
reduction should be equivalent to one cytochrome c and two C(R)
9
molecules,

14
approximately 15 kDa. The SMPB conjugate failed to completely enter the running
gel, instead remaining in the stacking gel, perhaps due to an over-modification of this
conjugate by positively charged C(R)
9
residues. Additionally, the inability of the
conjugate to enter the gel could be a result of large aggregates formed by the
conjugate. The maleimide residue of the SMPB cross-linker is commonly used for
the modification of cysteines in proteins. However, it is also known to modify lysine
and tyrosine moieties, of which there are many in cytochrome c.
27
With a large
concentration of SMPB in the solution, large aggregates could have been formed
through the lysine or tyrosine residues of cytochrome c, resulting in a complex too
large to enter the gel. The light band seen at approximately 12 kDa in both SMPB
samples, suggests that not all of the cytochrome c was modified by the cross-linker.

15

1 2 3 4 5 6 7

80
49

35
29

21

7

Figure 1.4. SDS-PAGE gel. Lane 1 molecular weight marker, Lane 2 cytochrome c
with DTT, Lane 3 cytochrome c without DTT, Lane 4 SPDP conjugate with DTT,
Lane 5 SPDP conjugate without DTT, Lane 6 SMPB conjugate with DTT, Lane 7
SMPB conjugate without DTT.

16
1.3.1.3. Amino Acid Analysis

The Life Science Research Center at National Tsing Hua University determined
the amount of each amino acid in pmol concentration after acid hydrolysis of the
protein. Using the amino acid alanine as an internal standard due to its high stability,
the number of arginine residues in each sample was determined, and therefore the
extent of modification. Also determined was the number of lysine and leucine
residues for use as references, due to the fact that lysine is the most abundant amino
acid in cytochrome c and that leucine is a stable amino acid. Once the number of
arginine residues on each sample was determined, we subtracted two (to correct for
the number of arginines on cytochrome c) and divided by nine (to result in the
number of (R)
9
residues per sample). Results showed that the reducible conjugate
has two (R)
9
residues, supporting previous findings by ultraviolet absorbance, and
that the non-reducible conjugate has less than one, 0.5, (R)
9
residue.

1.3.2. Measurement of Radioactive Label

The labeled conjugates and cytochrome c eluted at the column void volume.
Free
125
I is sufficiently separated from the labeled sample, usually eluting after
fraction 10 (data not shown). The typical elution profiles are shown in Figure 1.5.
The concentration of the samples was determined by specific radioactivity.

17
0.0E+00
2.0E+05
4.0E+05
6.0E+05
8.0E+05
1.0E+06
1.2E+06
1.4E+06
1.6E+06
02 4 6 8 1
Fraction (mL)
CPM
0
A
0.0E+00
1.0E+05
2.0E+05
3.0E+05
4.0E+05
5.0E+05
02 46 8 1
Fraction (mL)
CPM
0
D

0.E+00
1.E+05
2.E+05
3.E+05
4.E+05
5.E+05
6.E+05
7.E+05
8.E+05
9.E+05
02 4 6 8 1
Fraction (mL)
CPM
0
B
0.E+00
1.E+05
2.E+05
3.E+05
4.E+05
5.E+05
6.E+05
7.E+05
8.E+05
9.E+05
02 4 6 8 1
Fraction (mL)
CPM
0
E

0.E+00
1.E+05
2.E+05
3.E+05
4.E+05
5.E+05
6.E+05
7.E+05
8.E+05
9.E+05
02 4 6 8 1
Fraction (mL)
CPM
0
C

Figure 1.5. Elution profile for radiolabeled conjugates and cytochrome c. (A)
Cytochrome c, (B) 1:2 SPDP K
9
conjugate, (C) 1:2 SPDP R
9
conjugate, (D) 1:5
SPDP R
9
conjugate and (E) 1:0.5 SMPB R
9
conjugate.

18
1.4. DISCUSSION

Cationic oligopeptides of both lysine and arginine residues have been used to
facilitate transport of bioactive molecules into the cell through endocytosis and
transduction, respectively.
8,28,29
The differences in internalization mechanisms have
been attributed to the guanidine moiety of the arginine. This structure allows
arginine to form bidentate bonds with anionic charges on the cell surface, rather than
simple charge interactions as with the lysine residues. The experiments depend on
the internalization of the conjugate by transduction directly to the cytosol to avoid
lysosomal degradation. With transduction efficiency relying solely on the guanidine
structure of the oligoarginine peptide, and endocytosis dependent on the number of
positive charges, we have optimized the conjugate to decrease the molar ratio of
arginine to cytochrome c in order to maintain transduction capabilities while
retaining cytochrome c structure and activity in the cytoplasm. The oligolysine
conjugate will be used as a comparison to the oligoarginine conjugate to determine
the difference in intracellular processing.
Studies employing RNA-binding peptides such as HIV-1 Rev and human T-cell
lymphotrophic virus (HTLV)-II Rex show that these peptides, containing more than
seven and less than fifteen arginine residues, are translocated through cell
membranes as efficiently as the well characterized Tat peptide and are localized in
the cytosol.
30
Internalization rates decrease with a smaller number of arginine
residues, terminating altogether in peptides only having three or less arginine
residues. These findings suggest a less modified conjugate will not lose transduction

19
efficiency while possibly optimizing the activity due to retention of the cytochrome
c’s amino acid structure.

1.4.1. Reducible Conjugates

Conjugates of either oligoarginine or oligolysine, and cytochrome c were
successfully prepared at a 1:2 molar ratio, respectively through a reducible disulfide
linkage. These conjugates will be used as a tool to observe the difference in
intracellular processing of transduced and endocytosed conjugates with PTD
peptides. A higher modified conjugate of cytochrome c:oligoarginine at a 1:5 molar
ratio was also successfully prepared in order to observe the difference in efficiency
of uptake and the retention of biological activity compared to a smaller conjugate
with less cationic residues. The disulfide bond will allow these conjugates to be
reduced once they are internalized, freeing the homologous cytochrome c peptide.

1.4.2. Non-Reducible Conjugates

For observation in the HeLa cell line, a non-reducible conjugate of cytochrome
c:oligoarginine at a 1:0.5 molar ratio was successfully constructed through a stable
thioether linkage. This conjugate will be compared to the 1:2 cytochrome
c:oligoarginine reducible conjugate as a means of determining the importance of the
conjugate linkage to uptake and biological activity. To account for the 50%
modification of cytochrome c in the non-reducible conjugate, the concentration was
doubled in all experiments to ensure an equal concentration of modified cytochrome
c versus the reducible conjugate. In all subsequent experimental sections, the

20
concentration of conjugate is given as the final concentration of modified
cytochrome c. The non-reducible conjugate’s molar ratio is lower than that of the
reducible conjugate, most likely due to difference in ease of the two reactions. The
2-pyridyldithio group of the SPDP reagent is highly selective for sulfhydryl groups,
and the reaction is rapid and efficient. However, the maleimide group of the SMPB
cross-linker is not as specific for sulfhydryl groups and the addition reaction can be
slow. These differences in the cross-linkers result in a simpler reaction with SPDP
as compared to SMPB.

21
1.5. CONCLUSION

Our results demonstrate that cytochrome c can be successfully conjugated to
oligolysine and oligoarginine through a reducible disulfide bond with the SPDP
cross-linker. Additionally, the conjugation with oligoarginine can also be achieved
through a non-reducible thioether bond with the SMPB cross-linker. The disulfide
complex between cytochrome c and the cationic oligopeptide arginine can be
attained at a low and a high molar modification of both 1:2 and 1:5 cytochrome
c:oligoarginine. Conjugates of cytochrome c and oligolysine were prepared at a 1:2
molar ratio, respectively with a reducible bond. Conjugates of cytochrome c and
oligoarginine were prepared at a 1:2 and 1:5 molar ratio, respectively with a
reducible disulfide bond and a 1:0.5 molar ratio with a non-reducible thioether bond.

22
Chapter Two
Intracellular Processing of Cytochrome C
and Cytochrome C Conjugates

2.1. INTRODUCTION

2.1.1. Background Information

Successful uptake of cytochrome c has only previously been achieved through
either inefficient or harsh methodologies such as microinjection, pinocytic loading
and electroporation.
18,31,32,33,34
The method of microinjection imposes many
limitations, including a restriction on the number of cells that can be analyzed to the
order of hundreds or less. In addition, inducing pinocytosis with hyper-osmotic
buffer alters the cell’s natural state by stimulating uptake of extracellular fluid.
Through the passive uptake of the cytochrome c-oligoarginine conjugates by
transduction, the cell is not disrupted by any unnatural influences and a true
representation of a large number of cells can be obtained.
The potential usefulness of membrane transduction peptides in the cytoplasmic
delivery of cargo molecules with a large range of molecular weights has recently
been assessed. The methods by which the efficiency of these carriers was
determined, such as confocal microscopy and flow cytometry, have not been
completely effective. Due to the high cationic charge on most of these PTDs, they
are internalized concurrently through adsorptive endocytosis and transduction, which
is not clarified in these assays. To further complicate matters, the high surface

23
binding of cationic oligopeptides tends to result in overestimation of internalization
efficiency. Peptide binding to small membrane fragments may result in false
cytoplasmic localization, while binding to intact plasma membrane may give false
vesicular localization. The novel method developed in our laboratory quantitatively
determines the membrane transduction efficiency of cationic oligopeptides, allowing
for selective measurement of transduction without the interference from concurrent
adsorptive endocytosis.
35
The procedure has been found to obtain results that are
consistent with the qualitative findings reported by others.

2.1.2. Degradation of Cytochrome C
In addition to the method of uptake, we analyzed possible intracellular
degradation pathways of cytochrome c and the low modified oligoarginine reducible
and non-reducible conjugates in HeLa cells. The two primary pathways of
intracellular degradation are the proteasome and the lysosomes. Of these, the
proteasome has been implicated as the dominant pathway in the degradation of
cytochrome c.
23,36,37
The proteasome is a cytosolic organelle that functions to control
the degradation of proteins within a cell. They are 2000 kDa cylindrical complexes
that constitute nearly 1% of the total cellular protein. Located abundantly throughout
the cytoplasm, proteasomes regulate many cellular processes, including protein
degradation in an ATP-dependent pathway primarily following ubiquitination of
lysine residues on a substrate protein.
38
It has been shown that cytosolic cytochrome
c is cleaved by this organelle, either due to unfolding of the molecule or

24
ubiquitination.
23,36
The high content of lysine residues in cytochrome c renders the
molecule susceptible to ubiquitination, however the exact lysines involved have yet
to be determined. Studies involving yeast Saccharomyces cerevisiae and horse heart
cytochrome c suggest that specific lysine residues are implicated, namely those in the
N-terminal region of the protein.
36, 39
Additionally, it has been shown that good
substrates of the ubiquitin pathway have free NH
2
termini.
39
Many vertebrate
cytochromes c have an acetylated glycine in the first position, however, the
cytochrome c used in the experiments explained in this thesis has a free NH
2

terminus.
The proteasome has multiple active sites, including chymotrypsin-, trypsin- and
caspase-like specificity. Several synthetic and natural inhibitors are available for
these sites. The most widely used of the proteasome inhibitors are the peptide
aldehydes. This class of inhibitors acts primarily on the chymotryptic activity of the
proteasome, and includes MG132 (N-carbobenzoxyl-Leu-Leu-leucinal) (Figure 2.1).
MG132 is a cell penetrable inhibitor that has been found to specifically block the
proteolytic activity of all subunits of the organelle, and is therefore an effective tool
in determining the proteasome’s role in degradation of cytochrome c.
40

25

Figure 2.1. Structure of proteasome inhibitor, MG132.

26
2.2. EXPERIMENTAL

2.2.1. Materials

All medium was obtained from GIBCO-BRL (Carlsbad, CA) or Cellgro
(Herndon, VA). All culture plates were obtained from Corning (Acton, MA).
Fetal bovine serum (FBS), 10x trypsin-EDTA, non-essential amino acids (NEAA),
sodium pyruvate, L-glutamine and penicillin/streptomycin were purchased from
either Sigma (St. Louis, MO) or GIBCO-BRL. FITC-dextran, sodium chloride
(NaOH), ammonium chloride, chloroquine, trichloroacetic acid and 10x protease
inhibitor cocktail were purchased from Sigma. MG132 proteasome inhibitor was
obtained from Calbiochem (La Jolla, CA), and the micro BCA protein assay reagent
kit was obtained from Pierce Chemical Company (Rockford, IL).

2.2.2. Cell Culture

All experiments were performed using Chinese Hamster Ovary (CHO), Human
leukemic monocyte lymphoma (U937) or Human cervical carcinoma (HeLa) cells.
These cell lines were obtained from American Type Culture Collection (ATCC,
Manassas, VA). CHO cells were grown in Ham’s F12 medium, supplemented with
2 mM L-glutamine, and containing 10% FBS. U937 cells were grown in complete
RPMI 1640 medium, supplemented with 2 mM L-glutamine, and containing 10%
FBS. HeLa cells were grown in Eagle’s minimum essential medium (EMEM)
containing 10% FBS, NEAA and sodium pyruvate. All cells were supplemented
with penicillin and streptomycin at 50 units/ml and 50 µg/mL, respectively. The

27
cells were incubated at 37°C, 5% CO
2
and replenished with fresh medium the day
before confluence, at which time the assays were performed.

2.2.3. Cellular Processing of Conjugates and Cytochrome C

2.2.3.1. Cellular Uptake

Confluent CHO or HeLa monolayers grown in 6-well culture plates were
incubated in serum free medium containing 5 µg/mL iodinated unmodified
cytochrome c or its conjugates for one hour at 37°C. The cell pellets were washed
with cold phosphate buffered solution (PBS), isolated following treatment with
trypsin-EDTA, washed with cold PBS, and dissolved in 1N NaOH. The amount of
internalized conjugate was assayed using a Gamma counter (Packard, Downers
Grove, IL), and the total cell protein content was determined by the Pierce protein
assay. This data was used to calculate the mg cytochrome c / mg cell protein. U937
cells were treated somewhat differently due to the fact that they are a suspension cell
line. For the U937 cell line, the cell/medium suspension was collected in a test tube
and centrifuged at 1,000 rpm for 5 minutes and the supernatant removed. The cell
pellets were then washed with cold PBS, treated with trypsin-EDTA, washed again
with cold PBS, dissolved in NaOH and assayed as the other cell lines.

28
2.2.3.2. Subcellular Fractionation

The measurement of cytosolic localization of the conjugates and cytochrome c
was performed as previously reported.
35
Briefly, confluent CHO or HeLa
monolayers grown in T75 flasks were incubated in serum free medium containing 3
µg/mL of
125
I-labeled conjugate or
125
I-labeled cytochrome c, 0.1 mg/mL FITC-
dextran (70 kDa) (FD) and protease inhibitor cocktail (PI) containing 4 µM AEBSF,
2 µM EDTA, 0.3 µM bestatin, 30 nM E-64, 2 µM leupeptin and 0.6 nM aprotinin.
After treatment for 1 hour at 37°C, the monolayers were washed three times with
cold PBS and detached by treatment with trypsin-EDTA at 37°C for 5 min, and the
isolated cell pellets were washed with 0.5 mg/mL heparin-PBS followed by PBS.
The cell pellets were homogenized in buffer (HB) containing 0.25 M sucrose, 2 mM
EDTA, and 10 mM HEPES, pH 7.4 using a Balch cell press (H & Y Enterprises,
Redwood City, CA).
41
The cell homogenate was centrifuged at 600 g at 4°C for 10
min, and the post-nuclear supernatant was fractionated using Sephacryl S-500
(Amersham, Piscataway, NJ) size exclusion chromatography (1x13 cm column
dimensions) (Fractionation range: 40 kDa – 20,000 kDa) with HB as the eluting
buffer. One milliliter fractions were collected and assayed for
125
I using a Gamma
counter (Packard), for FD using fluorescence spectroscopy (Hitachi, Tokyo, Japan)
(Ex 494nm, EM 519 nm), and for protein content using the Pierce protein assay. The
amount of peptide internalized by endocytosis versus transduction was calculated
using the equations previously described.
35
Figure 2.2 is a representation of the
separation of endocytosis and transduction peaks, as well as the FITC internal

29
standard obtained after fractionation of the sample. This procedure was not
performed on U937 cells since the method has not been validated for suspension cell
lines.

30

0
1
2
3
4
1234567 89 101112131415
Fraction (mL)
ng M TP
0.0
0.1
0.2
0.3
0.4
FD Abs
Endocytosis
Transduction
FITC Leakage

Figure 2.2. Representation of elution profile for subcellular fractionation. Iodinated
peptide elutes in the endocytic (vesicular) and/or transduction (cytosolic) peak (blue
triangles) and FITC-dextran elutes in the endocytic peak, with a second, smaller
leakage peak (red squares).

31

2.2.3.2.1. Analysis of Third Peak in Subcellular Fractionation
In order to determine if the third peak was a constitutive characteristic of
the iodinated cytochrome c and conjugate, or a product of cellular incubation, an
assay was performed on CHO cells modifying the subcellular fractionation assay.
Cells were grown to confluence without incubation with cytochrome c, the conjugate
or FD. They were collected and the post-nuclear supernatant fraction was obtained
as described. The fraction was spiked with FD, split into two equal volumes, each of
which were then spiked with 3 µg/mL of either iodinated cytochrome c or iodinated
1:5 cytochrome c-oligoarginine reducible conjugate. These samples were
fractionated, collected and read as in the subcellular fractionation assay.

2.2.3.3. Degradation Pathways

Multiple assays were performed in HeLa cells using inhibitors of different
degradation pathways in order to pinpoint the method of conjugate (specifically low
modified reducible and non-reducible oligoarginine conjugates) and cytochrome c
degradation. For instance, to determine the extent of conjugate degradation within
lysosomes, ammonium chloride was utilized to inhibit the acidification of these
organelles and cells processed by TCA precipitation. In addition, proteasome
involvement was assayed in the same manner using the inhibitor MG132. Briefly,
confluent HeLa monolayers grown in T25 flasks were incubated in serum free
medium containing the inhibitor (10 mM NH
4
Cl and/or 10 µM MG132 proteasome
inhibitor) for 30 minutes at 37°C. At 30 minutes,
125
I-labeled cytochrome c is added

32
to the medium and incubated for 1 hour. After treatment the monolayers were
washed two times with cold PBS and detached by treatment with trypsin-EDTA at
37°C for 5 minutes, and the isolated cell pellets were again washed two times with
cold PBS, lysed with 0.1% Triton X 100 and precipitated with 5% TCA on ice for 30
minutes. The cell precipitate was centrifuged and the pellet was separated from the
supernatant and both were assayed for
125
I. It is generally believed that proteasome
activity is essential for cell proliferation and therefore, its inhibition may result in
various degrees of anti-proliferative activity in cultured cells.
42
However, exposure
to MG132 does not affect cell viability or growth for 10-20 hours.
43,44,45
For the
determination of transduction in the presence of a proteasome inhibitor, cells will be
incubated for less than 2 hours. Therefore, during such a short time, it is unlikely
that the anti-proliferative activity will have a significant effect on membrane
transduction.
Degradation through the endocytic process was also evaluated utilizing
chloroquine, an inhibitor of endocytosis. HeLa cells were assayed by TCA
precipitation as explained in the presence of MG132 and/or 200 µM chloroquine.
Also, proteasome involvement in the intracellular processing of both conjugates and
cytochrome c alone was further elucidated by performing cytoplasmic localization
assays in HeLa cells as described above in the presence or absence of 10 µM
MG132.

33
2.3. RESULTS

2.3.1. Enhanced Cellular Uptake by Conjugates

On its own, cytochrome c has very little uptake in CHO, U937 or HeLa cells.
The addition of the cationic oligopeptides C(K)
9
and C(R)
9
by the reducible disulfide
linkage considerably increased the amount of cytochrome c located in the cell in the
CHO cell line. The 1:2 molar ratio conjugates of oligolysine and oligoarginine
increase uptake of cytochrome c by approximately 4-fold and 5-fold respectively.
The more modified 1:5 molar ratio conjugate of oligoarginine enhances uptake by
twelve times over cytochrome c in CHO cells. Uptake into the U937 cell line is only
enhanced by the oligolysine conjugate at a rate of almost three times higher than the
unmodified peptide. The lack of enhanced uptake by the oligoarginine conjugate
could possibly be due to rapid turnover of the transduced conjugate not experienced
by the oligolysine conjugate which is presumed to be mainly endocytosed. HeLa
cells were the only cell line assayed with both the reducible and the non-reducible
conjugates of oligoarginine. For both, uptake was increased approximately 8-fold.
These results are shown in Figures 2.3-2.5.

34

(A)

0.E+00
1.E-05
2.E-05
3.E-05
4.E-05
5.E-05
6.E-05
Cyt C (R)9 Conj (R)9
mg molecule/mg cell protein

(B)

0.0E+00
5.0E-06
1.0E-05
1.5E-05
2.0E-05
2.5E-05
Cyt C (R)9 Conj
mg molecule/mg cell protein
0.0E+00
5.0E-06
1.0E-05
1.5E-05
2.0E-05
Cyt C (K)9 Conj
mg molecule/mg cell protein

Figure 2.3. Cellular uptake in CHO cells. (A) Uptake of SPDP 1:5 molar ratio
conjugate compared to cytochrome c alone and C(R)
9
alone, (B) Uptake of SPDP 1:2
molar ratio conjugates versus cytochrome c alone. CHO cell monolayers incubated
with 5 µg/mL
125
I cytochrome c, conjugate or YG(R)
9
, washed, trypsinized,
dissolved in NaOH and assayed for
125
I and protein content. Data are presented as
average ± standard deviation with n = 3.

35

0.E+00
1.E-04
2.E-04
3.E-04
4.E-04
Cyt C (K)9 Conj (R)9 Conj
mg molecule/mg cell protein

Figure 2.4. Cellular uptake of SPDP 1:2 molar ratio conjugates versus cytochrome c
alone in U937 cells. Cells incubated with 5 µg/mL
125
I cytochrome c or conjugate,
collected and centrifuged, washed, trypsinized, dissolved in NaOH and assayed for
125
I and protein content. Data are presented as average ± standard deviation with
n = 3.

36

0.0E+00
5.0E-06
1.0E-05
1.5E-05
2.0E-05
2.5E-05
Cyt C SPDP Conj SMPB Conj
mg molecule/mg cell protein

Figure 2.5. Cellular uptake of low molar ratio conjugates of SPDP and SMPB with
C(R)
9
versus cytochrome c alone in HeLa cells. Cell monolayers incubated with 5
µg/mL
125
I cytochrome c or conjugate, washed, trypsinized, dissolved in NaOH and
assayed for
125
I and protein content. Data are presented as average ± standard
deviation with n = 3.

37

2.3.2. Involvement of Endocytosis vs. Transduction

The manner by which the peptides enter the cell is determined by separating the
endocytosed from the transduced peptide in post-nuclear soluble fractions of the cell
homogenate. CHO cells were assayed with only the reducible conjugates of arginine
and lysine versus cytochrome c. Results showed that while both the 1:5 and 1:2
modified oligoarginine conjugates increased cytochrome c uptake by both endocytosis
and transduction, the uptake of the 1:2 modified oligoarginine conjugate was higher
than that of the 1:5 modified oligoarginine conjugate (Figure 2.6). In addition, the
oligolysine conjugate enhances uptake by endocytosis only, as expected (Figure 2.7).
Cytochrome c had low uptake by endocytosis as well as by transduction. In all
samples, including cytochrome c alone, a third peak, presumed to be the result of
degradation, is observed (Figure 2.8). In HeLa cells, both the reducible and non-
reducible oligoarginine conjugates were assayed and resulted in increased uptake of
cytochrome c by endocytosis and transduction (Figure 2.9A). In cytochrome c alone,
there was low uptake by endocytosis and transduction. The third peak is once again
present and its percentage is slightly higher in the cytochrome c and SPDP conjugate
samples when compared to the SMPB conjugate (Figure 2.9B). This peak was
analyzed by inhibiting different pathways of protein degradation.

38

0
5
10
15
20
25
30
35
40
45
Cyt C 1:5 SPDP (R)9 1:2 SPDP (R)9
ng / mg cell protein

Figure 2.6. Cytoplasmic localization by subcellular fractionation of low and high
SPDP oligoarginine conjugates vs. cytochrome c in CHO cells. Cells were incubated
in serum free medium containing 0.1 mg/mL FITC-Dextran, protease inhibitors and 3
µg/mL
125
I cytochrome c or conjugate and processed as described in the experimental
section. Endocytosis (open bars), Transduction (closed bars), Degradation (hashed
bars). Data are presented as average ± standard deviation with n = 3.

39

0
10
20
30
40
50
60
70
80
Cyt C 1:2 SPDP (K)9 1:2 SPDP (R)9
ng / mg cell protein

Figure 2.7. Cytoplasmic localization by subcellular fractionation of low SPDP
oligolysine and oligoarginine conjugates vs. cytochrome c in CHO cells. Cells were
incubated in serum free medium containing 0.1 mg/mL FITC-Dextran, protease
inhibitors and 3 µg/mL
125
I cytochrome c or conjugate and processed as described in
the experimental section. Endocytosis (open bars), Transduction (closed bars),
Degradation (hashed bars). Data are presented as average ± standard deviation with
n = 3.

40

0
1
2
3
123 4567 89 10 11 12 13 14 15
Fraction (mL)
ng Conjugate
0.0
0.1
0.2
0.3
0.4
0.5
1
2 3

Figure 2.8. Example elution profile of oligoarginine conjugate with degradation
peak. Iodinated peptide elutes in the (1) endocytic, (2) transduction and (3)
degradation peaks (blue triangles) and FITC-dextran elutes in the endocytic peak,
with a second, smaller leakage peak (red squares). Cytochrome c elution is
represented by the black dashed line.

41

0
10
20
30
40
50
60
Cyt C SPDP SMPB
ng / mg cell protein
A

0%
10%
20%
30%
40%
50%
60%
Cyt C SPDP SMPB
% Uptake
B

Figure 2.9. Cytoplasmic localization by subcellular fractionation. HeLa cell
monolayers were incubated in serum free medium containing 0.1 mg/mL FITC-
Dextran, protease inhibitors and 3 µg/mL
125
I cytochrome c or conjugate and
processed as described in the experimental section. Endocytosis (open bars),
Transduction (closed bars), Degradation (hashed bars). (A) ng/mg cell protein and
(B) % Uptake. Data are presented as average ± standard deviation with n = 3.

42
2.3.3. Origination of Third Peak

The third peak is not observed when the post-nuclear supernatant is spiked with
cytochrome c or the conjugate and not incubated with the cells (Figure 2.10). It is
determined that the third peak is in fact due to the degradation of cytochrome c
incubated with the cells and not a constitutive characteristic of the molecule.

43

0
50
100
150
200
250
300
0 1 2 3 4 5 6 7 8 9 101112131415
Fraction (mL)
ng
0
250
500
750
1000
Cyt C
Conjugate
FITC (Cyt C)
FITC (Conj)

Figure 2.10. Spiked cell elution from S500 gel. CHO cells were grown to
confluence, collected and the post-nuclear supernatant fraction was obtained and
spiked with FD and 3 µg/mL of either
125
I labeled cytochrome c or 1:5 oligoarginine
reducible conjugate and processed as described in the experimental section.

44
2.3.4. Sensitivity of Internalization to Endosomal Acidification

The role of lysosomes in the degradation of cytochrome c and the oligoarginine
conjugates was analyzed by both ammonium chloride and chloroquine in HeLa cells.
Ammonium chloride inhibits the acidification of lysosomes, thereby inhibiting the
degradation of endocytosed protein. In addition, chloroquine inhibits endocytosis
eliminating subsequent degradation by otherwise endocytosed protein. TCA
precipitation with both of these compounds co-incubated with cytochrome c shows
no difference between them and the controls (Figure 2.11). This suggests that during
the one-hour incubation, the degradation of cytochrome c is not occurring in
endocytic compartments. Figure 2.11 also shows the results of the proteasome
inhibitor MG132 alone and in conjunction with each of the aforementioned
lysosomotropic agents. This data is discussed in the next section.

45

0%
5%
10%
15%
20%
25%
30%
35%
40%
Control NH4Cl MG132 NH4Cl + MG132
A

0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
Control Chloroquine MG132 Chloroquine +
MG132
B

Figure 2.11. TCA precipitation analysis of cytochrome c degradation with inhibitors.
HeLa cell monolayers were preincubated in serum free medium with the inhibitor for
30 minutes, at which time
125
I-labeled cytochrome c was added for one hour. Cells
were washed, trypsinized, lysed with Triton X 100, precipitated with TCA and the
pellet and supernatant assayed for
125
I. (A) Ammonium chloride and MG132, (B)
Chloroquine and MG132. Results are presented as a % degraded peptide. Data are
presented as average ± standard deviation with n = 3.

46
2.3.5. Proteasome Inhibitor Assay

When cells are incubated in the presence of MG132 and assayed for cytochrome
c or its conjugates’ internalization by subcellular fractionation, degradation is
decreased and internalization by transduction is increased in all three samples
(Figure 2.12). This result suggests that cytochrome c and its conjugates are degraded
in proteasomes. However, in the case of the SMPB conjugate and unconjugated
cytochrome c, the result is not as significant as that of the SPDP conjugate;
suggesting degradation through other pathways may be taking place. Among the
three proteins, the SPDP conjugate showed the most significant change in
degradation.
The proteasome was inhibited by MG132 and the degradation of cytochrome c
was determined by TCA precipitation assay. The assay resulted in decreased
degraded protein, by approximately 15%, compared to the control, supporting
previous data that suggests the proteasome is at least partially responsible for
cytochrome c degradation (Figure 2.11). In addition, when MG132 is co-incubated
with ammonium chloride, the results are similar to MG132 alone, due to the
proteasome inhibitor which is not impeded by the lysosomotropic agent. When co-
incubating MG132 and chloroquine, the decrease in degraded protein is not as great,
suggesting chloroquine may impede the proteasome inhibitor’s action. This poses no
difficulties since no other experiments are performed to test MG132 in the presence
of chloroquine.

47
0%
5%
10%
15%
20%
25%
30%
35%
40%
0 MG132 10 MG132
% Uptake
A

*
*
0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
0 MG132 10 MG132
% Uptake
B

0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
0 MG132 10 MG132
% Uptake
C

Figure 2.12. MG132 effect on degradation and internalization. HeLa cell monolayers
were incubated with or without MG132 in the presence of 3 µg/mL cytochrome c or
conjugate and processed as described in the experimental section. Endocytosis (open
bars), Transduction (closed bars), Degradation (hashed bars). (A) SPDP, (B) SMPB,
(C) Cytochrome c. Data are presented as average ± standard deviation with n = 3,
p<0.5.

48
2.4. DISCUSSION

2.4.1. Enhanced Cellular Uptake by Conjugates

The comparison of the high and low modified oligoarginine conjugate in CHO
cells showed that the conjugate with five C(R)
9
residues enhanced overall uptake to a
greater extent than the conjugate with two C(R)
9
residues. Previous findings show
that increasing the number of positive charges in a membrane transduction peptide
will increase internalization by endocytosis.
35
However, subcellular fractionation
assays show that this is not the case with the highly modified conjugate.
Internalization of homoarginine peptides showed that internalization by any means
was only increased with peptides up to 15 arginine residues. Peptides with 16 or
more arginine residues were observed only on the cellular membrane.
46
The trypsin-
EDTA wash should have stripped the membrane of surface bound oligopeptide,
however some could have been left behind. It is also a possibility that the interaction
of arginine with cargo alters the cell surface binding, allowing for enhanced overall
uptake despite limited membrane transduction.
4

A comparison of the equally modified arginine and lysine conjugates in CHO
and U937 cells led to different results. CHO cells show that the oligoarginine
peptide is slightly more efficient at internalizing the conjugate. This supports some
previous findings that show when comparing equivalent length polymers of
oligolysine and oligoarginine, oligoarginine is more efficient at carrying FITC into
cultured cells.
30,47
However, conflicting data shows that short oligolysine peptides of

49
6 to 12 residues are more efficient than oligoarginine peptides at carrying large
macromolecules into cultured cells.
48
U937 cells back these results in that the
oligoarginine conjugate did not enhance uptake when compared to cytochrome c
alone, but that the oligolysine conjugate did. The method of internalization in this
cell line could not be confirmed because the method developed in our laboratory to
separate endocytosis from transduction has not been validated for suspension cell
lines. The efficiency of peptide internalization as well as the optimal number of
cationic residues that is most favorable for that internalization has been shown to be
cell-type dependent.
10,30,46,48,49,50
The differences lie in the cell surface concentration
of both heparan sulfate proteoglycans and glycosaminoglycans, believed to be
surface binding targets for protein transduction domains, as well as the efficiency of
recycling cytosolic contents to the outside of the cell. This could account for the
disparity in uptake by the two cell lines of the same oligoarginine conjugate.
The linkage between the carrier peptide C(R)
9
and cytochrome c did not make a
difference in the overall uptake of the conjugates in HeLa cells. The SMPB and the
SPDP cross-linked conjugates enhanced cytochrome c uptake to a similar extent.
Interaction of the conjugate and the cellular membrane is not completely understood,
however, no evidence has been found to imply that the bond between carrier and
cargo would affect internalization, as long as the oligopeptide is not hindered in the
conjugation. It has been shown that the structure of the oligoarginine peptide chain
could affect internalization by transduction. Though all transduction peptides tested
here were linear, branched chain peptides of the same length were shown to be as

50
effective at transduction, with the advantage of possible organelle-specific
delivery.
49,51
This finding could be utilized in the future with these conjugates to
create more effective complexes with cytochrome c.
An interesting find in these experiments is that although there is low uptake of
the unmodified cytochrome c, membrane transduction is occurring. However,
despite the localization to the cytosol, cytochrome c does not result in apoptosis of
the cells, possibly due to degradation by the proteasome. This theory is assessed
later in this thesis.

2.4.2. Intracellular Processing

The method developed in our laboratory to separate endocytosed peptides from
transduced peptides was used to further elucidate the method of internalization by
the conjugates. This method employs the trypsin-EDTA wash, which decreases
surface binding of peptides. The method goes a step further, exposing the cells to a
heparin wash. Heparin binds strongly to lysine and arginine residues through
electrostatic interactions and acts to further remove surface bound peptides.
52,53

It is probable that this wash successfully decreased 1:5 oligoarginine conjugate left
on the cellular membrane after incubation. Despite its apparent increase in uptake
over the 1:2 oligoarginine conjugate, assays performed in CHO cells show that the
increased number of arginine residues on the highly modified conjugate did not
enhance endocytosis or transduction over the 1:2 oligoarginine conjugate. This
result shows that the 1:2 (R)
9
conjugate is optimal for internalization compared to the

51
1:5 (R)
9
conjugate. Because only a certain number of arginine residues are necessary
to enhance uptake, optimally seven to fifteen, the higher modified conjugate appears
to have too many residues, resulting in a bulky conjugate that is not as efficient as
the lower modified conjugate in uptake by either endocytosis or transduction.
Results of subcellular fractionation with the oligolysine conjugate showed an
increase in endocytosis over the unmodified cytochrome c, with no visible
transduction. This result was expected, since oligolysine has been shown to be
internalized solely by endocytosis.
35
This conjugate will be analyzed for its
biological activity and compared with the oligoarginine conjugate, whose
internalization is the result of both endocytosis and transduction. Additionally, the
third peak, presumed to be degradation from transduced peptide, is present in the
oligolysine conjugate. This is most likely the result of complete degradation of any
free cytochrome c present in the sample as well as cytochrome c that may have
escaped the endosomes after internalization.
Comparison of the reducible and non-reducible conjugates in HeLa cells
revealed that the type of linkage used in the conjugation is not important to the
efficiency of internalization. It was theorized that extracellular reduction of the
disulfide linked conjugate but not the thioether linked conjugate, mediated by protein
disulfide isomerase (PDI), may have an effect on their transport.
54, 55
However, aside
from a slight increase in endocytosis by the SMPB conjugate, there is no significant
difference in the two conjugates’ internalization, particularly by transduction, that
would lead us to believe that PDI has a significant effect on the SPDP conjugate.

52
The subcellular fractionation assays all resulted in an unexpected occurrence.
The fractionation typically produces two peaks representing endocytosis and
transduction. However, the unmodified cytochrome c as well as all of the
conjugates, regardless of linkage or cationic carrier, resulted in a third peak at the
end of the elution profile. The third peak was never seen in fractionations with the
oligopeptide alone. When cells were incubated free of cytochrome c or conjugate
and the isolated post-nuclear supernatant pellet spiked with one or the other prior to
fractionation, the third peak was not visible. This result is a strong indicator that the
third peak is degradation of cytochrome c, and that it is a result of cellular
processing. Additionally, it is of interest to note that the percentage of the third peak
in cytochrome c and the reducible conjugate fractionations is larger when compared
to that of the non-reducible conjugate, presumably because the free cytochrome c as
well as cytochrome c released from the reducible conjugate is recognized and
degraded by the proteasome (Figure 2.9B).

2.4.3. Degradation Pathways

The third peak in the subcellular fractionation assays was presumed to be a
degradation product, in part because it elutes late on the gel. These late eluting
smaller peptides are fragments of the cytochrome c protein, degraded during cellular
incubation. Lysosomal degradation through the endocytic pathway and degradation
within the cytosol by the proteasome are both potential culprits in the break down of
cytochrome c. The proteasome is typically involved in the controlled destruction of

53
intracellular proteins. Its interaction with cytochrome c is well documented.
23,36

However, since cytochrome c is internalized by both endocytosis and transduction,
its fate by both pathways was studied. Inhibitors of these pathways were assayed by
TCA precipitation and their effect on this peak was analyzed.
TCA precipitation of cell-associated cytochrome c with lysosomotropic agents
ammonium chloride or chloroquine treatment was performed. Compounds
internalized via receptor-mediated or fluid phase endocytosis are subjected to
lysosomal acidification.
56,57
Lysosomotropic agents become protonated within the
acidic subcellular compartments, causing them to accumulate and subsequently
elevating the organelle pH of lysosomes and endosomes.
58
This action prevents
degradation of the internalized ligand and in the case of chloroquine, inhibits further
intracellular processing.
59
However, neither of these inhibitors had an effect on the
percentage of degraded cytochrome c. When compared to controls, there was no
change in the percentage of degraded protein in these samples. This result suggests
that cytochrome c is not subjected to degradation by the endocytic pathway within
the first hour of incubation. However, when TCA precipitation of cytochrome c was
performed in the presence of the proteasome inhibitor MG132, there was a
significant decrease in degraded protein of approximately 15%. This result further
supports the belief that the proteasome is at least partially responsible for
cytochrome c degradation and for the third peak. This was confirmed by subcellular
fractionation assays performed with both cytochrome c and the conjugates in the
presence of MG132. The inhibitor resulted in a decrease in the third peak coupled

54
with an increase in the transduction peak. The result was significant in the reducible
conjugate. The reduction in degradation and subsequent increase in transduced
peptide is in all probability due to a decrease in degraded cytochrome c, which exists
in both the cytochrome c and reducible conjugate samples. This trend is also seen,
though not to any significant degree, in the non-reducible conjugate. As stated in
Chapter 1, it appears that during the thioether-linked conjugate preparation, not all of
the cytochrome c was attached to C(R)
9
, which implies the possibility of free
cytochrome c within this conjugate that could show degradation.

55
2.5. CONCLUSION

The results in this chapter demonstrate that the uptake of conjugated cytochrome
c is significantly enhanced in all three tested cell lines, CHO, HeLa and U937 when
compared to the unmodified peptide. However, uptake in the U937 cells was only
increased by the oligolysine conjugate, perhaps due to recycling or reverse
transduction of the oligoarginine conjugate prior to analysis. In CHO cells, where
both the low and high modified oligoarginine conjugate were assayed, results
showed that while the uptake of both was significantly improved, the higher
modified conjugate increased uptake by more than twice that of the lower modified
conjugate. This means that an increasing number of oligopeptides attached to
cytochrome c not only does not impede uptake, but improves uptake, though the
result could be due to surface binding. Additionally, the different linkages do not
affect uptake of the conjugates in HeLa cells. Both the disulfide linked and the
thioether linked complexes were enhanced to an equal degree above cytochrome c
alone.
In determining the method of uptake, results showed that the oligolysine
conjugate is only internalized by endocytosis, while the oligoarginine conjugates are
taken up by both transduction and endocytosis. The high and low modified
oligoarginine reducible conjugate was tested in CHO cells and the lower modified
conjugate proved to be more efficient at internalization, especially by transduction.
This result suggests that while many C(R)
9
residues enhances uptake, a lower
modified conjugate is optimal for internalization by transduction. Additionally, both

56
the disulfide and the thioether linked conjugates enhance uptake by both endocytosis
and transduction into HeLa cells, when compared to unmodified cytochrome c.
An unexpected third peak is seen in the subcellular fractionation assays.
Presumed to be degradation, most likely by the proteasome resulting from the
transduced peptide, degradation inhibitors were assayed for their effect on the peak.
Lysosomotropic agents ammonium chloride and chloroquine did not have an effect
on cytochrome c, suggesting that degradation of the peptide is not occurring in the
lysosomes during the one hour incubation. However, inhibition of the proteasome
results in decreased degradation of cytochrome c, the reducible conjugate and to a
lesser extent the non-reducible conjugate. The decreased degradation also resulted in
an increase in transduced peptide, supporting our theory that the transduced peptide
and not the endocytosed peptide is being degraded by the proteasome.

57
Chapter Three
Biological Activity of Cytochrome C
and Cytochrome C Conjugates

3.1. INTRODUCTION

3.1.1. Cytochrome C’s Role in Apoptosis

Apoptosis is a controlled form of cell death that can be characterized by
morphological changes in the cell. Condensation of nucleoplasm and cytoplasm,
cytoplasmic membrane blebbing and DNA fragmentation are some of the primary
markers indicative of apoptotic cell death.
60,61
Cytochrome c’s function in the
apoptotic cascade was first identified in 1996 by Xiaodong Wang and colleagues, in
a search to discover the mechanism of caspase activation.
62

Cytochrome c is a nuclear encoded protein located in the inner membrane space
of all mitochondria.
63
By accepting electrons from cytochrome b and transferring
them to cytochrome oxidase, cytochrome c typically functions in the electron
transport system of oxidative phosphorylation to release energy in the form of ATP.
In this process the iron of the cytochrome c heme group (which is identical to that of
hemoglobin and myoglobin) shifts from the stable, neutral ferrous state to the ferric
state. When released from the mitochondria in response to pro-apoptotic stimuli,
cytochrome c plays a pivotal role in the regulation of apoptosis. Mitochondria have
been implicated in apoptosis after it was discovered that the Bcl-2 family of proteins
was located in the mitochondrial outer membrane.
64,65
This family of proteins

58
contains members that are either pro- (Bad, Bid, BAK, Bax) or anti- (Bcl-2, Bcl-xl)
apoptotic. The balance between these factors influences whether cytochrome c is
released and the subsequent induction of apoptosis. In response to specific apoptotic
stimuli via the Bcl-2 family of proteins, cytochrome c is released through pores
formed in the mitochondrial membrane, initiating the cascade leading to
programmed cell death.
66
This process involves two classes of the family of cysteine
proteases known as initiator and effector caspases. Once released into the cytosol,
cytochrome c binds to the cytoplasmic scaffolding protein apoptosis protease
activating factor-1 (Apaf-1) complexed with dATP to form the apoptosome
complex.
67
A 27-Å three-dimensional structure of the apoptosome was recently
elucidated through cryo-electron microscopy technology.
68
This finding has shed
light on how the apoptosome assembles and how it interacts with caspase-9 (Figure
3.1). The binding of cytochrome c to Apaf-1 results in the recruitment of the
initiator Procaspase-9 to the apoptosome where the cysteine protease is activated and
released. This initiates the apoptotic cascade through the cleavage and activation of
downstream effector caspases, such as Procaspase-3 and -7.
69,70
In turn these
caspases instigate orderly disassembling of the cell through proteolytic cleavage of
other cellular substrates. Figure 3.2 shows the apoptotic cascade initiated by
cytochrome c.
The role of proteasomes in the down regulation of the apoptotic activity of
cytochrome c is enigmatic. Inhibition of the proteasome has been implicated in the
accumulation of cytochrome c in the cytosol, but the causality behind this is

59
controversial. A wide range of both pro-apoptotic and anti-apoptotic substrates exist
for the proteasome and blockage of the organelle’s activity has resulted in both an
increase in cell survival and an increase in apoptosis.
71,72
Some studies show that the
increase in cytosolic cytochrome c is due to the molecule’s degradation by the
ubiquitin-proteasome degradation pathway, and therefore a blockage of the organelle
leads to a build up of cytochrome c.
36,37
However, the exact interaction between the
proteasome and cytochrome c’s induction of the apoptotic cascade has yet to be
determined.

60

Figure 3.1. Representation of the apoptosome, adapted from review composed by
Andreas Gewies in 2003. Apaf-1 consists of three functional domains, the caspase-
recruitment domain (CARD), a central nucleotide binding domain (not shown) and
several WD40 repeats. Upon association with one cytochrome c the rigid Apaf-1
structure becomes flexible to allow for dATP binding. The apoptosome is composed
of seven molecules of Apaf-1 that form a symmetrical wheel-like structure. The
different molecules interact via their N-terminal CARD domains at the center, with
their C-terminal WD40 repeats extended. Procaspase-9 is recruited to the center of
the apoptosome, where interaction with the CARD domain triggers a conformational
change in the enzyme, subsequently activating it as well as downstream effector
caspases, such as caspase-3 and ultimately leading to apoptosis.

61

Bcl-2
Bax
Bid
Cyt c
dATP
Apaf-1
Cyt c
Caspase-9
Caspase-3
APOPTOSIS

Figure 3.2. Apoptotic Cascade Initiated by Cytochrome c.

62

3.1.2. Glutathione

Glutathione (GSH) is a tripeptide sulfhydryl (-SH) antioxidant, composed of
glutamic acid, cysteine and glycine. Due to its water solubility, it is found primarily
in the cytosol of animals, plants and microorganisms.
73,74
Its high electron-donating
capacity, due to its cysteine residue, combined with its high intracellular
concentration makes GSH a powerful reducing agent, used principally to reduce
oxidized thiols of other proteins in the cytosol of cells. Glutathione exists
intracellularly in two forms, its reduced form (GSH) and its oxidized form of two
glutathione molecules disulfide bonded together (GSSH) which results from GSH
oxidation of other intracellular peptides (Figure 3.3).
75
Glutathione is expected to
play a role in the reductive cleavage of the reducible conjugate’s disulfide bonds
resulting in the release of homologous cytochrome c and ultimately a decrease in the
conjugate’s biological activity. The apoptotic effect of the reducible conjugate at
differing GSH concentrations is assayed to determine if retention of the conjugate in
a reduced GSH environment will improve its biological activity. Acetaminophen is a
powerful reducer of glutathione. It produces a toxic metabolite (N-acetyl-p-benzo-
quinone imine, NAPQI) that is detoxified by glutathione. This process utilizes GSH,
resulting in its depletion within the cell and subsequent inaccessibility to reduce the
disulfide bonds in the reducible conjugate. Functional concentrations of
acetaminophen in HeLa cells have previously been identified and will serve as a
marker for appropriate experimental conditions for glutathione reduction without

63
induction of cell death.
76,77,78
Since depletion of GSH can itself lead to apoptosis,
care will be taken to avoid this occurance.
79

64

(A)

(B)

Figure 3.3. Structure of Glutathione. (A) Oxidized Glutathione (GSSH) and (B)
Reduced Glutathione (GSH).

65
3.2. EXPERIMENTAL

3.2.1. Materials

All medium was obtained from GIBCO-BRL (Carlsbad, CA) or Cellgro
(Herndon, VA). All culture plates were obtained from Corning (Acton, MA).
Fetal bovine serum, 10x trypsin-EDTA, NEAA, Sodium pyruvate, L-glutamine and
penicillin/streptomycin for cell culture were all purchased from either Sigma (St.
Louis, MO) or GIBCO-BRL. The annexin V / propidium iodide kit, acridine orange
stain, ammonium chloride, chloroquine, trichloroacetic acid, 10x protease inhibitor
cocktail, cystine, Ellman’s reagent, acetaminophen and cycloheximide were all
purchased from Sigma. MG132 proteasome inhibitor was obtained from
Calbiochem (La Jolla, CA). The Micro BCA protein assay reagent kit was obtained
from Pierce Chemical Company (Rockford, IL).

3.2.2. Cell Culture

All experiments were performed using Chinese Hamster Ovary (CHO), Human
leukemic monocyte lymphoma (U937) or Human cervical carcinoma (HeLa) cells.
These cell lines were obtained from American Type Culture Collection (ATCC,
Manassas, VA). CHO cells were grown in Ham’s F12 medium, supplemented with
2 mM L-glutamine, and containing 10% FBS. U937 cells were grown in complete
RPMI 1640 medium, supplemented with 2 mM L-glutamine, and containing 10%
FBS. HeLa cells were grown in Eagle’s minimum essential medium (EMEM)
containing 10% FBS, NEAA and sodium pyruvate. All cells were supplemented

66
with penicillin and streptomycin at 50 units/ml and 50 µg/mL, respectively. The
cells were incubated at 37°C, 5% CO
2
and replenished with fresh medium the day
before confluence, at which time the assays were performed.

3.2.3. Biological Activity of Conjugates and Unmodified Cytochrome C

3.2.3.1. Cytotoxicity

To determine the cytotoxicity of the conjugates, cells were grown overnight to
~50% confluence in 24-well culture plates and incubated in serum free medium
containing the indicated µg/mL concentration of conjugate (usually based on
cytochrome c concentration), cytochrome c (equivalent to the conjugate’s
cytochrome c concentration) and/or C(R)
9
(the oligoarginine concentration
equivalent to that in the conjugate concentrations) for one hour at 37°C (Note: for all
experiments, n = 3 wells). At one hour, fetal bovine serum was added to 5% and the
cells were incubated until the PBS control reached confluence. The cells were
washed with PBS, dissolved in 1N NaOH and the total cell protein content was
determined by the Pierce protein assay. The assay was performed on CHO, U937
and HeLa cells.

3.2.3.2. Apoptotic Effect

Apoptosis was assayed by both annexin V / propidium iodide per
manufacturer’s protocol and acridine orange (AO) as previously described, with
slight variation.
31
Briefly, regarding the annexin V / propidium iodide assay, cells

67
were plated in a 24-well culture plate and allowed to attach overnight. Then, cells
were incubated in serum free medium with or without inhibitor, if part of the
experiment, for 30 minutes at 37°C. At 30 minutes, cytochrome c conjugates or the
equivalent amount of cytochrome c and C(R)
9
was added to the medium of the cells.
After incubation for one hour at 37°C, fetal bovine serum was added to 5% and the
cells were incubated for 3 more hours. The cells were washed with PBS and
resuspended in 1x binding buffer at a concentration of approximately 1x10
6

cells/mL. 5 µL of annexin V-FITC and 10 µL of propidium iodide was added to 500
µL of each cell suspension, and the suspension was incubated at room temperature
for 10 minutes. The fluorescence of the samples was determined with a flow
cytometer (USC/Norris Comprehensive Cancer Center Flow Cytometry and Immune
Monitoring Core). The assay was performed on U937 and HeLa cells only.
Briefly, regarding the acridine orange assay, cells were plated in a 12-well
culture plate and allowed to attach overnight. Then cells were incubated in serum
free medium with conjugate, cytochrome c and/or C(R)
9
as in the annexin V /
propidium iodide assay. The positive control cycloheximide (10 µg/mL) was also
used in the HeLa cell acridine orange experiment. After incubation for one hour,
fetal bovine serum was added to 5% and the cells were incubated for 3 more hours.
The cells were washed with PBS and isolated following treatment with trypsin-
EDTA. After another PBS wash the cells were stained with 4 µM acridine orange
solution and placed on ice for 5 minutes before a final PBS wash. The cells were
assayed by fluorescence. Pictures of the HeLa cells were captured prior to staining

68
with a laser scanning light microscope (LSM). Apoptosis assays were performed on
U937 and HeLa cells only. The microscope used for Acridine Orange analysis was
provided by Microscopy Sub-Core at the USC Center for Liver Diseases (NIH
1P30DK48522).

3.2.4. Effect of Degradation Inhibitors on Apoptosis

3.2.4.1. Proteasome Inhibition

Proteasome involvement in the intracellular degradation of both the reducible
and non-reducible oligoarginine conjugates and cytochrome c alone was determined
in HeLa cells by performing TCA precipitation assays as previously described and
annexin V / propidium iodide assays per the manufacturer’s protocol as described
above. For the annexin V / propidium iodide assay, cells were preincubated with
either 0 or 10 µM MG132 proteasome inhibitor in serum free medium for 30 minutes
at 37°C prior to the addition of PBS (control), 80 µg/mL conjugates (based on
cytochrome c) or 80 µg/mL cytochrome c for one hour, followed by the addition of
5% FBS and incubation for 3 hours. The assay was carried out as described above.

3.2.4.2. Endosomal Acidification Inhibition

The apoptotic effect of the reducible and non-reducible oligoarginine conjugates
in the presence of the lysosomotropic agent ammonium chloride was assayed by
annexin V / propidium iodide and flow cytometry in HeLa cells. Cells were
preincubated with 10 mM ammonium chloride for 30 minutes, spiked with 80 µg/mL

69
conjugate (based on cytochrome c concentration) and incubated for one hour before
FBS addition to 5% and incubation for 3 more hours. The annexin V assay was
performed as described above.
The effect of different inhibitors on cytochrome c’s biological activity was
determined by incubating cytochrome c with ammonium chloride, chloroquine,
MG132 or protease inhibitor cocktail and assaying for apoptosis by annexin V /
propidium iodide by flow cytometry. All samples contained 80 µg/mL cytochrome c
and one of the following: 10 µM MG132, 10 mM ammonium chloride, 0.2 mM
chloroquine or 0.2 µL 10x protease inhibitor cocktail containing 4 µM AEBSF, 2
µM EDTA, 0.3 µM bestatin, 30 nM E-64, 2 µM leupeptin and 0.6 nM aprotinin.
The cells were preincubated in serum free medium with the inhibitor at 37°C. After
a 30 minute incubation, cytochrome c was added to the medium and the cells were
incubated for 1 hour. FBS was added to 5% and the cells were incubated for 3 more
hours before processing. The experiment was performed on HeLa cells as described
above.

3.2.5. Disulfide Bond Reduction

3.2.5.1. Glutathione Assay

Before testing the conjugates in the presence of acetaminophen, and varying
concentrations of cystine, the toxicity and effect on intracellular glutathione levels of
both compounds were assayed. To determine the optimum concentration of each on
HeLa cells for glutathione reduction without imposing cell death, we first exposed

70
the cells to increasing concentrations of each and visually observed their morphology
under the microscope for signs of toxicity. Briefly, the cells were grown overnight
in 6 well culture plates to ~50% confluence in MEM medium containing 30 mg/L
cystine (normal cystine concentration) and 10% FBS. The medium was changed to
MEM with increasing concentrations of cystine (either kept at 30 mg/L or increased
to 50 mg/L cystine) or acetaminophen (10 mM, 20 mM or 30 mM) and 10% FBS
and the cells were observed every hour for 6 hours and then again at 24 hours.
Once the optimum acetaminophen and cystine levels were determined, the cells
were grown in medium to enhance (30 mg/L cystine) or decrease (10mM
acetaminophen, 30 mg/L cystine) intracellular glutathione levels. In separate
experiments, the cells were assayed first for their intracellular glutathione
concentration by Ellman’s reagent as previously described and then for apoptosis by
the conjugates and cytochrome c in each medium.
80,81,82
Briefly, regarding the
Ellman’s reagent assay, the cells were grown in T75 flasks in a normal cystine
concentration. The day before the experiment, the cells were fed with either 30
mg/L cystine medium (cells to be tested with acetaminophen and 30 mg/L cystine) or
50 mg/L cystine medium (cells to be tested with 50 mg/L cystine). On the day of the
experiment the medium was changed and the acetaminophen samples were incubated
with medium containing the appropriate acetaminophen concentration for 1 hr at
37°C. The cell pellets were isolated following treatment with trypsin-EDTA at 37°C
for 5 minutes and washed three times with cold PBS. The cell pellets were lysed in 5
mM phosphate buffer and the cell lysates were precipitated with ice-cold 5%

71
trichloroacetic acid solution. The supernatant was separated from the pellet by
centrifugation and extracted with 5x2 mL ether wash before adding Ellman’s
reagent. The resulting solution was mixed and measured for glutathione
concentration at 412 nm.
Apoptosis was assayed by annexin V / propidium iodide per manufacturer’s
protocol as previously described, with slight variation. Briefly, cells were plated in a
12-well culture plate in 30 mg/L cystine MEM medium and allowed to attach
overnight. Then cells were incubated in 10% FBS medium with or without 10 mM
acetaminophen for 1 hour at 37°C. After incubation, the medium was replaced with
serum free medium (still with or without 10 mM acetaminophen) and cytochrome c
conjugate or the equivalent amount of cytochrome c. After incubation for one hour
at 37°C, fetal bovine serum was added to 5% and the cells were incubated for 3 more
hours. The cells were isolated and treated as previously described to stain with
annexin V and propidium iodide and the fluorescence was determined by flow
cytometry (USC/Norris Comprehensive Cancer Center Flow Cytometry and Immune
Monitoring Core).

72
3.3. RESULTS

3.3.1. Cytotoxicity by Pierce Protein Assay

The cytotoxic activity of the 1:2 reducible oligoarginine conjugate was
determined and compared to that of cytochrome c and/or oligoarginine by incubating
the cells in varying concentrations of each (based on cytochrome c) in CHO cells.
The results, expressed as a percentage of control (untreated cells) and presented in
Figure 3.4, show a lack of cytotoxicity in response to the conjugate or any of the
controls. Over-modification of cytochrome c may be correlating to a decrease in
activity once internalized by the cell. However, CHO is not known to be a preferable
model for cytochrome c apoptosis. Therefore, the experiment was performed in two
cell lines proven to be optimal models for cytochrome c apoptosis, U937 and HeLa,
both human cancer cell lines. U937 cells were tested first with increasing
concentrations (based on the oligopeptide concentration) of reducible conjugates of
1:2 oligoarginine and 1:2 oligolysine (Figure 3.5A). The results, expressed as a
percentage of control (untreated cells) showed that the oligolysine conjugate had no
significant cytotoxic effect. However, the oligoarginine conjugate resulted in
cytotoxicity in a dose-dependant manner. The 1:5 oligoarginine reducible conjugate
was compared to the 1:2 oligoarginine reducible conjugate alongside controls in the
U937 cell line (Figure 3.5B). Both conjugates result in at least two times greater
cytotoxicity when compared to the controls.

73
Cytotoxicity in HeLa cells was analyzed with only the low oligoarginine
conjugates, both the reducible and non-reducible, versus cytochrome c alone and
oligoarginine alone. While both conjugates show at least 20% cytotoxicity, the non-
reducible conjugate resulted in slightly higher cell death when compared to the
reducible conjugate (Figure 3.6). Both conjugates were assayed further to determine
if the increased cell death was the result of apoptosis.

74

0
20
40
60
80
100
120
140
0.15 0.5 1.5 5 15
Cytochrome c Concentration (ug/mL)
% Control
Cyt C
C(R)9
Cyt C + C(R)9
(R)9 Conj

Figure 3.4. Cytotoxicity in CHO cells. 1:2 SPDP oligoarginine conjugate. CHO cell
monolayers were incubated in serum free medium containing increasing
concentrations of cytochrome c, conjugate, C(R)
9
or cytochrome c and C(R)
9
for one
hour, at which time FBS was added to 5% and the cells were grown to control
confluence and processed as described in the experimental section. C(R)
9

concentration is equivalent to that in the conjugate. Data are presented as average ±
standard deviation with n = 3.

75
0
20
40
60
80
100
120
06 12 18
Oligopeptide Concentration (ug/mL)
% Control
1:2 (R)9 Conjugate
1:2 (K)9 Conjugate
A

0
20
40
60
80
100
120
Control
Cyt C
(R)9
Cyt C + (R)9
1:2 (R)9 Conj
1:5 (R)9 Conj
% Control
B

Figure 3.5. Cytotoxicity in U937 cells with SPDP conjugates. (A) Oligolysine
conjugate versus oligoarginine conjugate to determine optimum concentration for
future assays. U937 cells were incubated in serum free medium containing
increasing concentrations of each conjugate for one hour, at which time FBS was
added to 5% and the cells were grown to confluence and processed as described in
the experimental section. (B) Cells were treated as in (A), cytochrome c
concentration of 30 µg/mL in all of the samples and oligoarginine concentration in
the controls was equal to that of the 1:5 conjugate in order to maximize the
oligoarginine effect. Data are presented as average ± standard deviation with n = 3.

76

0
20
40
60
80
100
120
0 10204080
Cytochrome c Concentration (ug/mL)
% Control
SPDP Conj
SMPB Conj
Cyt C
(R)9

Figure 3.6. Cytotoxicity in HeLa cells. Low oligoarginine SPDP and SMPB
conjugates. HeLa cell monolayers were incubated in serum free medium containing
increasing concentrations of cytochrome c, conjugate, or (R)
9
for one hour, at which
time FBS was added to 5% and the cells were grown to control confluence and
processed as described in the experimental section. C(R)
9
concentration is
equivalent to that in the conjugates. Data are presented as average ± standard
deviation with n = 3.

77
3.3.2. Annexin V / Propidium Iodide Assay

To determine the manner of cytotoxicity, apoptosis versus necrosis, an apoptosis
assay was performed and the results analyzed by flow cytometry. A representation
of the flow cytometry results prior to analysis are seen in Figure 3.7. The reducible
1:2 oligoarginine conjugate was tested in U937 cells and compared to various
controls (Figure 3.8). Some cell death was observed in the oligoarginine control as
well as the cytochrome c plus oligoarginine control, approximately 31% and 46%
respectively. However, the conjugate resulted in approximately 80% apoptosis,
more than twice that of the controls.
The non-reducible and reducible oligoarginine conjugates were assayed in HeLa
cells. There was no significant apoptosis seen in either the controls or the reducible
conjugate. However, the non-reducible conjugate resulted in 46% apoptosis, a
significantly higher apoptotic effect compared to the reducible conjugate and the
controls (Figure 3.9).

78

Figure 3.7. Representation of annexin V / propidium iodide flow cytometry results.
FL1-H (x axis) reads annexin V absorbance, FL2-H (y axis) reads propidium iodide
absorbance. The lower left quadrant denotes viable cells, the lower right quadrant
denotes early apoptotic cells and the upper right quadrant denotes late apoptotic
cells. These results are from U937 cells treated for 4 hours with either PBS control
(top) or 1:2 oligoarginine conjugate (bottom).

79

0
20
40
60
80
100
120
140
160
180
Control Cyt C (R)9 1:2 (R)9 Conj Cyt C+ (R)9
% Control

Figure 3.8. Apoptosis by flow cytometry in U937 cells. U937 cells were incubated
in serum free medium with 73 µg/mL conjugate (based on cytochrome c
concentration) or equivalent µg/mL cytochrome c and/or 18 µg/mL C(R)
9
(the
oligoarginine concentration equivalent to that in the conjugate) for one hour. At one
hour, FBS was added to 5% and the cells were incubated for 3 more hours and
processed as described in the experimental section. Data are presented as average ±
standard deviation with n = 3.

80

0
20
40
60
80
100
120
140
160
Control
Cyt C
(R)9
SMPB Conj
Cyt + (R)9
SPDP Conj
% Control

Figure 3.9. Apoptosis by flow cytometry in HeLa cells. HeLa cell monolayers were
incubated in serum free medium with 80 µg/mL conjugate (based on cytochrome c
concentration) or equivalent µg/mL cytochrome c and/or 20 µg/mL C(R)
9
(the
oligoarginine concentration equivalent to that in the conjugate) for one hour. At one
hour, FBS was added to 5% and the cells were incubated for 3 more hours and
process as described in the experimental section. Data are presented as average ±
standard deviation with n = 3.

81
3.3.3. Acridine Orange Assay and Laser Scanning Microscopy Images

Acridine Orange (AO) experiments were performed in U937 and HeLa cells as a
supporting assay to the annexin V / propidium iodide assays. After incubation with
the peptides and staining by AO, the cells were observed by fluorescence
microscopy, excited at 525 nm and 630 nm to view both the green (AO-DNA
complex) and the red/orange (AO-RNA complex) fluorescence. In the U937 cells,
red/orange fluorescence, an indicator of apoptosing cells, was seen to a greater extent
in the 1:2 oligoarginine conjugate when compared to the oligolysine conjugate and
the controls (Figure 3.10). These results support the previous cytotoxicity and
annexin V / propidium iodide findings in this cell line. The oligoarginine conjugate,
and not the oligolysine conjugate retains it ability to propagate the apoptotic cascade
in U937 cells.
AO assays in HeLa cells also support the annexin V / propidium iodide results.
The addition of a positive control, cycloheximide, which promotes apoptosis through
the inhibition of protein synthesis in eukaryotic organisms, allowed for comparison
of the apoptosed cells exposed to the SMPB conjugate and known apoptosis with
cycloheximide. AO staining along with laser scanning light microscopy pictures
without fluorescence showed that the non-reducible conjugate results in much higher
apoptosis when compared to the reducible conjugate and the controls, which all
showed little to no apoptosis (Figure 3.11). AO staining in cycloheximide was not as
extensive as in the non-reducible conjugate. This is because cycloheximide induced

82
apoptosis to such a great extent that the majority of the cells were too compromised
to show staining, as seen in the LSM pictures.

83

A B C
D E F

Figure 3.10. Acridine Orange Apoptosis Staining in U937 cells. U937 cells were
incubated in serum free medium with the conjugate or equivalent concentration of
cytochrome c and/or C(R)
9
(the oligoarginine concentration equivalent to that in the
conjugate) for one hour. At one hour, FBS was added to 5% and the cells were
incubated for 3 more hours and process as described in the experimental section. (A)
PBS control, (B) Oligoarginine Control, (C) Cytochrome c plus Oligoarginine
control, (D) Cytochrome c control, (E) Oligolysine Conjugate and (F) Oligoarginine
Conjugate.

A B C G D E F
I H J K L M N

Figure 3.11. Laser Scanning Microscopy (LSM) and Acridine Orange Apoptosis Staining in HeLa cells. LSM pictures before
staining (A-G) and acridine orange staining (H-N). PBS control (A and H), Oligoarginine control (B and I), Cytochrome c plus
Oligoarginine control (C and J), Cytochrome c control (D and K), SPDP conjugate (E and L), SMPB conjugate (F and M) and
Cycloheximide (positive control) (G and N).

84
85

3.3.4. Proteasome Inhibitor Assay

Through continuous inhibition of the proteasome by the peptide aldehyde
inhibitor MG132, cytochrome c released from the disulfide linked conjugate, as well
as cytochrome c on its own, is protected from degradation by this organelle and is
therefore available within the cell to promote apoptosis. In the presence of MG132,
the percentage of cells that apoptosed due to cytochrome c increased by 34%, while
those that apoptosed due to the reducible conjugate increased by 49% (Figure 3.12).
However, no significant effect was seen in the non-reducible thioether linked
conjugate, it retained its biological activity in the presence of active or inhibited
proteasomes with no significant difference. Since cytochrome c is not released from
this cargo-carrier complex it is therefore not available for destruction by the
proteasome.

86

0
25
50
75
100
125
150
175
0 ug/mL MG132 10 ug/mL MG132
% Control

*
*

Figure 3.12. Annexin V assay with cytochrome c, conjugates and MG132. HeLa cell
monolayers were preincubated with either 0 or 10 µM MG132 for 30 minutes, spiked
with 80 µg/mL cytochrome c or conjugate (based on cytochrome c concentration)
and incubated for one hour before FBS addition to 5% and incubation for 3 more
hours. Cells were analyzed by annexin V assay as described in the experimental
section. PBS (closed bars), cytochrome c (open bars), SPDP conjugate (lined bars)
and SMPB (dotted bars). Data are presented as average ± standard deviation with n
= 3, p <0.05.

87

3.3.5. Endosomal Acidification Inhibitor Assay

Ammonium chloride’s effect on the conjugates’ biological activity was analyzed
by the annexin V / propidium iodide assay. Results showed that incubation with
ammonium chloride does not result in a change in apoptosis by the non-reducible
conjugate, but does significantly increase apoptosis in the reducible conjugate
(Figure 3.13). This finding suggests that lysosomal degradation is occurring with the
reducible conjugate only, leading to diminished concentration of this complex and
ultimately a decreased apoptotic effect.
The annexin V / propidium iodide assay with cytochrome c only and the
inhibitors ammonium chloride, chloroquine, protease inhibitor cocktail or MG132
supported the previous TCA results (Figure 3.14). The inhibition of the proteasome
was the only condition that leads to an increase in apoptosis by cytochrome c, similar
to the previous result with MG132. In all other conditions, the cells actually thrived
when compared to the PBS control.

88

0
50
100
150
200
SPDP Control SPDP NH4Cl SMPB Control SMPB NH4Cl
% Control

*

Figure 3.13. Annexin V assay with conjugates and ammonium chloride. HeLa cell
monolayers were preincubated with 0 or 10 mM ammonium chloride for 30 minutes,
spiked with 80 µg/mL conjugate (based on cytochrome c concentration) and
incubated for one hour before FBS addition to 5% and incubation for 3 more hours.
Cells were analyzed by annexin V assay as described in the experimental section.
Data are presented as average ± standard deviation with n = 3, p <0.05.

89

0
25
50
75
100
125
150
Control MG132 NH4Cl ChloroquinePI Cocktail
% Control

Figure 3.14. Annexin V apoptosis assay with cytochrome c. HeLa cell monolayers
were preincubated in serum free medium with 10 µM MG132, 10 mM ammonium
chloride, 200 µM chloroquine or 0.2 µL 10x protease inhibitor cocktail containing 4
µM AEBSF, 2 µM EDTA, 0.3 µM bestatin, 30 nM E-64, 2 µM leupeptin and 0.6 nM
aprotinin for 30 minutes. 80 µg/mL cytochrome c was added and the cells were
incubated for 1 hour before the addition of 5% FBS and further incubation for 3
hours. Cells were analyzed by annexin V assay as described in the experimental
section. Data are presented as average ± standard deviation with n = 3.

90

3.3.6. Glutathione Assay

The optimum concentration of cystine and acetaminophen for a resulting
difference in glutathione levels without causing cellular toxicity was determined to
be 30 mg/L and 10 mM, respectively. After 24 hours in each condition, cells were
still healthy and after one hour incubation in each condition, glutathione
concentrations were decreased by 15% with acetaminophen. The reduction of
glutathione resulted in the enhancement of apoptosis with the reducible conjugate by
almost 200% with little to no effect on cytochrome c or the non-reducible conjugate
(Figure 3.15).

91

0
100
200
300
400
Control Cyt C SPDP SMPB
% Control

Figure 3.15. Annexin V assay with acetaminophen in HeLa cells. HeLa cell
monolayers were incubated in 10% FBS medium containing 30 mg/L cystine and 0
or 10 mM acetaminophen for 1 hour at 37°C. At 1 hour the medium was replaced
with 0% FBS medium (still with or without 10 mM acetaminophen) and 80 µg/mL
conjugate (based on cytochrome c concentration) or equivalent µg/mL cytochrome c
and incubate for one hour. At one hour, FBS was added to 5% and the cells were
incubated for 3 more hours. Cells were analyzed by annexin V assay as described in
the experimental section. Control (open bars) versus acetaminophen (closed bars).
Data are presented as average ± standard deviation with n = 3.

92

3.4. DISCUSSION

3.4.1. Biological Activity

Prior to determining the mechanism of cell death, a cytotoxicity assay was
performed in order to determine the lowest concentration of cytochrome c that would
induce biological activity. CHO cells were excluded since results showed that this
cell line was not an effective model for assaying biological activity of cytochrome c
(Figure 3.4). Therefore, HeLa and U937 cells, which have been used extensively to
assay cytochrome c induced apoptosis, were used for biological activity assays in
this thesis.
62
Additionally, these cell lines have been proven to be good models for
apoptosis with microinjected or pinocytically loaded cytochrome c.
31,32,33
U937 cells
treated with low and high oligoarginine conjugate and low oligolysine conjugate
showed no cytotoxicity with the oligolysine conjugate (Figure 3.5A). While both
oligoarginine conjugates enhanced cytotoxicity by at least 70% (Figure 3.5B).
Previous uptake results showed that the oligolysine conjugate is more efficient at
uptake in this cell line, though presumably all through endocytosis. Though we were
unable to confirm the method of uptake in the suspension cell line, we know that
lysine is predominantly endocytosed and that arginine is predominantly transduced
directly to the cytosol.
35
Therefore, despite its apparent inability to increase uptake
in U937 cells, it is likely that the oligoarginine conjugate is internalized and
localized in the cytosol. The resulting cytotoxicity through both oligoarginine
conjugates is a strong indicator that transduction of the complex is required in order
93

to retain biological activity. This mechanism of internalization not only avoids the
acidic compartments of the endocytic pathway that will likely degrade the conjugate,
more importantly it increases the accessibility of the conjugate to cytosolic
components. Furthermore, the cytotoxicity observed in both conjugates suggests that
the biological activity of cytochrome c is not hindered by the addition of up to 5
C(R)
9
residues.
A comparison of the reducible and non-reducible conjugates’ ability to induce
cytotoxicity revealed that while both conjugates were effective, the thioether linked
conjugate was more cytotoxic. We hypothesized that this difference was due to the
retention of the intact conjugate, which protects cytochrome c from degradation by
the proteasome, allowing it to initiate the apoptotic cascade. To confirm that the
method of cell death is in fact apoptosis, annexin V / propidium iodide assays were
performed.
Apoptosis assays were carried out using the annexin V / propidium iodide assay
which will distinguish between early and late apoptosis. Annexin V is a 35-36 kDa,
calcium dependant phospholipid binding protein with a high affinity for the
membrane phospholipid phosphatidylserine (PS). PS is normally located on the
inner surface of the lipid bilayer in living cells. When cells undergo apoptosis, PS is
translocated to the outside of the cell membrane and becomes available to bind
annexin V.
83
When cells have entered the late stages of apoptosis and the cell
membrane integrity is compromised, propidium iodide is able to enter the cell and
bind cellular DNA.
94

Cytochrome c induced apoptosis usually begins within 30 minutes of exposure
and reaches its apex at approximately 3 hours, so a short incubation time for these
assays was sufficient. However, apoptosis assays were performed at time points
from 1 hour up to 72 hours to determine the optimum time point. After 72 hours,
cells that had survived the initial toxicity had started to proliferate and the percentage
of apoptosis decreased. Maximum apoptosis was seen at 4 hours (data not shown),
therefore subsequent apoptosis assays were carried out at 4 hours of incubation.
The 1:5 oligoarginine conjugate showed a minimal increase in cytotoxicity in
U937 cells, when compared to the 1:2 oligoarginine conjugate. Consequently, only
the lower modified conjugate was utilized in further experimentation due to this
conjugate’s effectiveness in inducing programmed cell death, resulting in
approximately 80% apoptosis, with the added benefit of minimal modification
(Figure 3.8). Additionally, past experiments have shown that seven to fifteen
oligoarginine residues, similar to that in the lower modified conjugate, are optimal
for transduction.
30
This data supports the idea that the use of the less modified
cytochrome c is beneficial for future experiments.
The low modified reducible conjugate was not as effective in the HeLa cell line
when compared to U937 cells, resulting in no significant apoptosis. While the
difference in the biological activity of the SPDP conjugate in the two cell lines is not
completely understood, it is likely the result of unique reductive qualities in each cell
type, one being a suspension cell line and the other an adherent. It is also probable
that the proteasome interaction with cytochrome c varies among the cell lines. These
95

differences will affect the processing and ultimately the biological activity of the
conjugate in each cell type, though these differences have yet to be clearly defined.
To confirm the annexin V findings, we performed a second type of assay to
determine apoptosis. Acridine Orange is a DNA fluorochrome that is able to cross
the intact plasma membrane, binding DNA and RNA (or ssDNA), allowing the
morphology of nuclear chromatin to be visualized by fluorescence microscopy. It is
used in apoptosis studies as DNA fragmentation during this process is a hallmark of
its occurrence.
84,85
AO complexes with the two nucleic acids differently, affecting
the wavelength of the emitted light from each complex, allowing for the two to be
differentiated from one another. The AO-DNA complex emits green light (525 nm),
and the AO-RNA complex emits red/orange light (>630 nm). Additionally, acidic
granules in apoptosing cells will protonate AO, inhibiting it from leaving the cell and
increasing its intracellular concentration, further exaggerating the red/orange
fluorescence in these cells. In both cell lines, AO staining confirmed the annexin V
results. The AO-RNA binding, seen as red in the fluorescence pictures, is present to
a very small extent in all samples in U937 cells, including the control (Figure 3.10).
However, it is seen to an extreme extent when cells are incubated with the
oligoarginine conjugate (Figure 3.10). In the HeLa cells, the red staining of
apoptotic cells in the AO pictures correlates to apoptotic cell morphology seen in the
LSM pictures (Figure 3.11). Only the positive control and the SMPB conjugate
samples result in significant AO-RNA staining, confirming that the SPDP conjugate
is not apoptotic in HeLa cells. As previously stated, the difference in biological
96

activity between the reducible and non-reducible conjugate is thought to be due to
their intracellular processing by the proteasome. The reduction of the SPDP
conjugate frees cytochrome c for degradation, diminishing its biological activity.
However, the retention of the SMPB conjugate may protect cytochrome c from this
degradation, retaining its biological activity. This assumption is supported by the
apoptosis assays that inhibit the proteasome activity (Figure 3.12).

3.4.2. Degradation and Reduced Activity

HeLa cells were used for the comparison of the intracellular degradation of
reducible and non-reducible conjugates. The decision to use HeLa cells for these
studies was due to the findings that CHO cells could not be used for apoptosis assays
and U937 cells could not be used for the subcellular fractionation assays in order to
determine the mechanism of internalization. HeLa cells afforded us the opportunity
to perform all of the pertinent assays in one cell line.
We studied the effect on apoptosis by proteasome inhibition with the reducible
and non-reducible conjugates of cytochrome c as well as unconjugated cytochrome
c. The result showed that only the non-reducible conjugate was unaffected by the
proteasome inhibitor, MG132. This data supports our theory that free cytochrome c,
like that in the unconjugated cytochrome c and the SPDP conjugate, is degraded by
the proteasome. Furthermore, when the proteasome is inhibited, the free cytochrome
c is available to propagate the apoptotic cascade. This finding is a strong indicator
that a non-reducible link between protein transduction domains and their cargo is
97

necessary to maintain the cargo’s biological activity if the cargo is a good substrate
of the proteasome. It is unclear why the retention of the conjugate protects it from
the proteasome, allowing it to induce apoptosis, but many possibilities exist. Though
we are uncertain of the exact lysine residues involved in the cross-linking of
cytochrome c to SMPB, it is probable that those targeted for ubiquitination and
subsequent degradation of cytochrome c are blocked by this conjugation, inhibiting
the ubiquitin tag from marking cytochrome c for destruction. Additionally, the
retention of the conjugate may make it too large to enter the proteasome, allowing it
to avoid degradation by the organelle.
The inhibition of lysosomal degradation with ammonium chloride resulted in an
increase in the SPDP conjugate’s apoptotic activity but did not affect the SMPB
conjugate. We previously observed that lysosomotropic agents did not affect the
biological activity of the unconjugated cytochrome c. However, the SPDP results
suggest that the disulfide linkage alters the fate of cytochrome c internalized through
this pathway. Previous findings with disulfide linked conjugates and lysosomotropic
agents have led to similar results. It has been shown that the biological activity of
disulfide linked immunotoxins is enhanced in a dose dependant manner in the
presence of chloroquine due to increased internalization of the conjugates.
86
The
data indicates that by raising the lysosomal pH, the release of the disulfide linked
conjugate from acidic compartments is enhanced, while the unconjugated molecule
is unaffected and remains within the lysosomes, where it is unable to initiate its
biological activity. In addition, endosome disruption has been shown to increased
98

the cytosolic localization of a Tat-fusion protein with no change in the localization of
the unconjugated protein.
87
This is further evidence that endocytosed disulfide
linked conjugates in the presence of lysosomotropic amines can escape the
organelles and retain their biological activity. Therefore, it is likely that the increase
in apoptosis with the SPDP conjugate is due to that, in the presence of
lysosomotropic amines, the oligoarginine carrier can release the cytochrome c
moiety in the endosomes which can escape into the cytosol; a process which is
analogous to the effect of lysosomotropic amines on immunotoxin. In the SMPB
conjugate, cytochrome c cannot be released from the oligoarginine carrier and
therefore no increase of apoptosis will be observed in the presence of lysosomotropic
amines. Conversely, lysosomotropic amines did not show any effect on the
apoptotic activity of the free cytochrome c, which possibly is due to either the lack of
membrane-binding ligands such as oligoarginine, or the low uptake of free
cytochrome c by HeLa cells.
Additionally, apoptosis assays confirmed previous TCA results that showed only
the proteasome was responsible for cytochrome c degradation. Inhibition with either
ammonium chloride, chloroquine or protease inhibitor did not alter cytochrome c’s
biological activity when compared to the control. However, MG132 showed an
increase of cytochrome c apoptosis to a similar extent as in the previous proteasome
inhibition assays. This finding confirms that degradation of cytochrome c is not
occurring in the lysosomes and that the proteasome is the main pathway for the
intracellular degradation of cytochrome c.
99

3.4.3. Inhibition of Disulfide Bond Reduction

The measurement of intracellular glutathione concentrations can be easily
performed by using Ellman’s reagent.
80,81
The sulfhydryl group of GSH reacts with
Ellman’s reagent (DTNB, 5,5’-dithio-bis-2-nitrobenzoic acid) to produce 5-thio-2-
nitrobenzoic acid (TNB) that has absorbance at 412 nm. The reaction results in a
concurrently produced disulfide between the two reactants that is also reduced,
allowing for the recycling of GSH and further production of TNB. The rate of TNB
production is directly proportional to the concentration of GSH in the sample.
Therefore the measurement of the TNB absorbance provides an accurate assessment
of GSH concentration.
The reduction of the disulfide bond in the SPDP conjugate by glutathione was
decreased to determine if the retention of the conjugate would result in enhanced
apoptosis, similar to that of the SMPB conjugate. Glutathione levels were
diminished in HeLa cells with the addition of acetaminophen and the cells were
incubated with the SPDP conjugate, the SMPB conjugate, or cytochrome c, and
assayed for apoptotic activity. The results showed a significant increase in apoptosis
by the reducible conjugate, with little to no effect in either of the other samples.
Decreasing the reduction of the SPDP conjugate allowed cytochrome c to remain
bound to the oligoarginine carrier and resulted in the conjugate’s renewed ability to
initiate the apoptotic cascade, as observed in the SMPB conjugate. This
demonstrates the need for the preservation of the conjugate once internalized in order
to maintain cytochrome c activity.
100

3.5. CONCLUSION

HeLa and U937 cells were both used for biological activity assays with the
conjugates, though different results were obtained with the reducible oligoarginine
conjugate in the two cell lines. In U937 cells, results showed that the oligolysine
conjugate was not cytotoxic, probably due to its likely uptake by endocytosis,
leading to degradation in the lysosomes and no release of cytochrome c into the
cytosol. However, the reducible oligoarginine conjugate results in an increase in
apoptosis by approximately 80%. We conclude that, in this cell line, transduction of
the conjugate is necessary for the biological activity to be exerted in the cytoplasmic
compartment. However, the effect of the conjugate linkage was not determined.
In HeLa cells, the reducible conjugate was not as successful as in U937 cells, as
it showed no ability to induce apoptosis. However, the non-reducible conjugate
enhanced apoptosis by 46% compared to the controls. This result demonstrates that
in HeLa cells, the linkage of cytochrome c to oligoarginine is an important factor for
the conjugate’s intracellular activity. Further testing determined that the decrease in
apoptosis with both unmodified cytochrome c and the reducible conjugate is due to
diminished intracellular cytochrome c concentrations through proteasome
degradation. When the complex is inhibited from reduction and subsequently the
release of cytochrome c is prevented, the proteasome does not act to degrade
cytochrome c and the disulfide conjugate becomes available to promote apoptosis.
Additionally, when intracellular concentrations of glutathione were decreased and
the disulfide bond in the conjugate is not reduced, apoptosis was increased by
101

approximately 200%. These results indicated that the preservation of the intact
cytochrome c-oligoarginine conjugate is necessary for the retention of the biological
activity of cytochrome c in HeLa cells. Furthermore, when taken together, the
MG132 and acetaminophen data indicates that the proteasomes are directly involved,
not indirectly, in the degradation and subsequently the apoptotic activity of
cytochrome c.
Recent experiments involving chemotherapeutic agents such as Cisplatin,
Etoposide and Fluorouracil in HeLa and U937 cell lines have resulted in a maximum
of 30% cell death.
88
The conjugates prepared in the course of this research have
proven to be more successful, with cell death as high as 80% in the same cancer cell
lines. The complexes between protein transduction domains and cytochrome c
explained in this thesis are promising tools for future drug delivery.

102

Chapter Four
Summary and Future Perspectives

4.1. Summary of Dissertation

The purpose of this project is to create a conjugate between cationic
oligopeptides and cytochrome c to use as a tool to investigate the transport and
intracellular processing of protein transduction domains. We compared
oligoarginine to oligolysine in two different cell lines, as well as the method of
linkage, to determine the optimum conjugate in HeLa cells. While the performance
of the conjugates varied among the cell lines, basic conclusions can be drawn about
their functionality.
The major findings in this thesis are summarized in Scheme 4.1 and in the
following points:
1. Cytochrome c is minimally internalized by membrane transduction, but does not,
on its own, lead to apoptosis. This is likely due to its degradation in the cytosolic
proteasome.
2. Cytochrome c can be successfully conjugated to both C(R)
9
and C(K)
9
through a
disulfide linkage. Furthermore, the resulting conjugate enhances uptake when
compared to the unmodified molecule.
3. Internalization of the oligoarginine conjugate is mediated by both endocytosis
and membrane transduction, while that of the oligolysine conjugate is
predominantly by endocytosis.
103

4. Cytochrome c conjugates of oligoarginine, but not of oligolysine, are able to
elicit a greater apoptotic response due to delivery to the cytosol via membrane
transduction.
5. Cytochrome c can be successfully conjugated to C(R)
9
through both a reducible
disulfide linkage and a non-reducible thioether linkage. Furthermore, the
resulting conjugates enhance uptake when compared to the unmodified molecule.
6. The preservation of the non-reducible complex protects cytochrome c from
degradation and allows it to retain its biological activity, while the dissociation of
the reducible complex leads to the degradation of the released cytochrome c,
inhibiting it from promoting apoptosis in HeLa cells.
7. The proteasome is responsible for degradation of cytochrome c alone, resulting
in lower apoptotic effect. Degradation pathways other than the proteasome are
not responsible for significant degradation of cytochrome c.
8. The proteasome and lysosome are responsible for degradation of cytochrome c in
the reducible SPDP conjugate, resulting in lower apoptotic effect.
9. There is no obvious degradation pathway for the non-reducible SMPB conjugate.
10. Acetaminophen treatment that acts to lower intracellular glutathione
concentrations markedly enhanced the apoptotic effect of the reducible conjugate
with little to no change in the cytochrome c or non-reducible conjugate’s
biological activity.
11. Preservation of the intact conjugate is necessary to maintain the biological
activity of cytochrome c in HeLa cell monolayers.
104

The findings presented in this thesis suggest that oligoarginine is a more reliable
carrier for the successful delivery of biologically active cargo due to its
internalization by membrane transduction to the cytosol of cells. Furthermore, the
intracellular processing of protein transduction domain conjugates is critical in
determining the fate and biological activity of the cargo molecules. The cargo’s
cytosolic stability is crucial in maintaining its biological activity, therefore the
preservation of the conjugate is essential to the success of this potential drug delivery
system for cytochrome c (Scheme 4.1).

105

Scheme 4.1. Summary of the major findings from this thesis.

106

4.2. Future Perspectives

The major findings summarized in this thesis provide the foundation for a novel
drug delivery system. The cytochrome c-cationic membrane transduction peptide
conjugate has proven to be a successful tool for the delivery and subsequent
activation of the cytochrome c cargo. However, more can be done to understand
their processing and ultimately their usefulness in vitro and eventually in vivo. For
example, there are inconsistencies in the method of delivery and intracellular
processing of these conjugates in different cell lines. A better understanding of these
differences would serve to optimize the conjugate and to identify the specificity,
which will result in a more efficient drug delivery system to target cells. It would be
useful to further research the differences in the proteasome - cytochrome c
interaction in U937 cells as compared to HeLa cells to determine if this interface is
the reason why the SPDP conjugate is apoptotic in one cell line and not the other
(under normal conditions). Furthermore, the cell surface concentrations of heparan
sulfate proteoglycans and glycosaminoglycans have been suggested to affect the
internalization of amino acid chains of oligoarginine. A variation in concentration of
these binding sites on U937 versus CHO cell surfaces may be affecting the
internalization of the oligoarginine conjugate, leading to the apparent difference in
its uptake in these cell lines.
Additionally, intracellular expression of regulatory factors could also affect the
processing of the conjugates after cytoplasmic delivery. For example, cytochrome c
microinjected into MCF7 cells deficient in caspase-3 is unable to propagate the
107

apoptotic cascade however, when MCF7 cells are supplemented with caspase-3,
cytochrome c microinjection results in apoptosis. It would be of interest to
determine the caspase-3 concentration in each of the three cell lines tested for this
thesis, to determine if this enzyme is responsible in any way for the effectiveness of
the conjugates. Alternatively, intracellular levels of caspase-3 can be modulated by
either gene transfection or siRNA treatment in order to maximize the apoptotic effect
of cytochrome c conjugates. A firm understanding of these differences among cells
and their impact on the processing of the conjugate may lead to an improved drug
delivery system that could be developed into an effective approach for the treatment
of cancer and other diseases.

108

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Bushnell, G.W., Louie, G.V. and Brayer, G.D. High-resolution three-dimensional
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Caron, N.J., Quenneville, S.P. and Tremblay, J.P. Endosome disruption enhances the
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Chang, S., Phelps, P., Berezesky, I., Ebersberger, M. Jr. and Trump, B. Studies on
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Dallaporta, B., Pablo, M., Maisse, C., Dauqas, E., Loeffler, M., Zamzami, N. and
Kroemer, G. Proteasome activation as a critical event of thymocyte apoptosis. Cell
Death Differ. 7:368-373 (2000).

Danial, N.N. and Korsmeyer, S.J. Cell death: Critical control points. Cell. 116: 205-
219 (2004).

Darzynkiewicz, Z. Differential staining of DNA and RNA in intact cells and isolated
cell nuclei with acridine orange. Methods Cell Biol. 33: 285-298 (1990).

Darzynkiewicz, Z,. Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M.A., Lassota, P.
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DeLeve, L. and Kaplowitz, N. Glutathione metabolism and its role in hepatotoxicity.
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Duke, R.C., Ojcius, D.M. and Young, J.D.E. Cell suicide in health and disease. Sci.
Am. 275: 79-87 (1996).

Drexler, H. Activation of the cell death program by inhibition of proteasome
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Ellman, G.L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82: 70-77 (1959).
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Emert-Sedlak, L., Shangary, S., Rabinovitz, A., Miranda, M., Delach, S.M. and
Johnson, D.E. Involvement of cathepsin D in chemotherapy-induced cytochrome c
release, caspase activation, and cell death. Mol. Cancer. Ther. 4: 733-742 (2005).

Falnes, P., Wesche, J. and Olsnes, S. Ability of the Tat basic domain and VP22 to
mediate cell binding, but not membrane translocation of the diphtheria toxin a-
fragment. Biochemistry 40: 4349-4358 (2001).

Futaki, S. Arginine rich peptides: Potential for intracellular delivery of
macromolecules and the mystery of the translocation mechanisms. Int. J. Pharm.
245: 1-7 (2002).

Futaki, S., Nakase, I., Suzuki, T., Zhang, Y. and Sugiura, Y. Translocation of
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Abstract (if available)

Abstract Peptides characterized as protein transduction domain peptides (PTD) or membrane transduction peptides (MTP) have recently attracted attention as a novel approach for the efficient intracellular delivery of oligopeptides, oligonucleotides and other bioactive macromolecules, primarily into the cytoplasm of mammalian cells. However, the effect of the linkage between cargo and its cationic oligopeptide carrier on the internalization, intracellular processing and biological activity of the cargo has not been fully deliberated. In this thesis, the cellular processing of conjugates of PTDs and the nuclear encoded apoptotic protein cytochrome c prepared with either a disulfide or a thioether linkage is studied and the conjugates used as a tool to learn more about the intracellular fate of cationic oligopeptides. The results from this work, performed in multiple cell lines, including CHO, U937 and HeLa, show that the uptake of the conjugates is increased when compared to that of cytochrome c alone. In HeLa cells, apoptotic activity of cytochrome c is observed only in the thioether conjugate, but not in the cytochrome c or the disulfide conjugate. However, apoptosis is restored in the disulfide conjugate with the addition of the proteasome inhibitor, MG132. Furthermore, the addition of MG132 resulted in an increase of apoptotic activity in cytochrome c. Conceivably, MG132 can protect cytochrome c, either in the native form or as a product released from the disulfide conjugate, from degradation by proteasomes. This model is further supported by the finding that treatment with acetaminophen, a glutathione depleting agent, markedly enhances the apoptotic effect of the disulfide conjugate, but not of the cytochrome c or the thioether conjugate. This result suggests that not only does the preservation of the cargo-carrier complex not hinder the cargo's biological activity, it is required for the retention of the conjugate's biological activity.

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Asset Metadata

Creator Barnes, Maureen P. (author)

Core Title Cytochrome c cationic oligopeptide conjugates: their cellular uptake, intracellular processing and biological activity

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 02/14/2009

Defense Date 12/14/2006

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Barnes,OAI-PMH Harvest

Language English

Advisor Shen, Wei-Chiang (committee chair), Garner, Judy A. (committee member), Hamm-Alvarez, Sarah F. (committee member), Okamoto, Curtis Tashio (committee member), Wang, Clay C. C. (committee member)

Creator Email mbarnes@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m255

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Dmrecord 161201

Document Type Dissertation

Rights Barnes, Maureen P.

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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