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Silencing of expression of the DRIP-80 gene correlates with aberrations in calcium ion distribution in nickel compound- and 3-methylcholanthrene-transformed C3H/10T1/2 mouse embryo fibroblast cel...

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Silencing of expression of the DRIP-80 gene correlates with aberrations in calcium ion distribution in nickel compound- and 3-methylcholanthrene-transformed C3H/10T1/2 mouse embryo fibroblast cel...

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SILENCING OF EXPRESSION OF THE DRIP-80 GENE CORRELATES WITH
ABERRATIONS IN CALCIUM ION DISTRIBUTION IN NICKEL COMPOUND-
AND 3-METHYLCHOLANTHRENE-TRANSFORMED C3H/10T1/2 MOUSE
EMBRYO FIBROBLAST CELL LINES

by

Duy Mai

________________________________________________________________________

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

December 2009

Copyright 2009 Duy Mai
ii

Acknowledgements

I would like to thank my colleagues Hongkyu Lee, M. S., Alana Harrison, M. S.,
Lindy Scott, M. S., Preethi Samala, M. S., and Jimmy Zheng, undergraduate student at
USC, for their help to me and collaboration with me on this project. I would also like to
thank my mentor, Dr. Joseph R. Landolph, Jr., for his guidance on this project.

iii

Table of Contents

Acknowledgements ii

List of Tables v

List of Figures vi

Abstract ix

Chapter One: Introduction 1
Carcinogenesis by Nickel Compounds 1
Identification of Down-Regulation of the DRIP-80 Gene and
Its Role in Vitamin D Regulation of Calcium Ion Homeostasis 7
The Role Ca
2+
ion in Signal Transduction in Mammalian Cells 13

Chapter Two: Materials and Methods 15
Cell Culture 15
Confocal Microscopy 17

Chapter Three: Results 20
Ca
2+
Ion Distributions in Non-Transformed 10T1/2 Cells 20
Ca
2+
Ion Distributions in Nickel-Transformed and in MCA-
Transformed 10T1/2 Cell Lines 21
Variability of Total Cellular Fluorescence Among Cells 24
Nuclear Fluorescence Levels of Non-Transformed, Nickel
Compound-Transformed, and MCA-Transformed Cells 31
Cytoplasmic Fluorescence Levels of Non-Transformed,
Nickel Compound-Transformed, and MCA-Transformed Cells 36
Distribution of Ca
2+
Ion Staining as a Function of Cell Density 42

Chapter Four: Discussions 46
Intracellular Calcium Ion Distribution in Non-Transformed
10T1/2 Cells 46
Calcium Function in Non-Transformed Cells 48
Calcium Distributions in MCA- and Nickel Compound-
Transformed Cells 50
Calcium Distribution in MCA-Transformed Cells 52
Nickel Induced DNA Methylation 53
Calcium Transport Mechanisms 54
iv

Effects of DRIP-80 Repression on Transcription 57
Tissue Specific Effects DRIP-80 Repression 59

Chapter Five: Conclusions 61

Chapter Six: Future Directions 63
References 67

v

List of Tables

Table 1: Ten cDNA Fragments Were Differentially Expressed Between
Non-Transformed, Nickel Compound-Transformed, and MCA-
Transformed Cells 7

Table 2: Transformed Cell Line Designations 16

Table 3: Comparison of Intracellular Calcium Localization Among Various
Cell Lines 23

Table 4: Distribution of Ca
2+
Ion Staining as a Function of density of
Cells Seeded 44

Table 5: Distribution of Ca
+2
Ion Staining as a Function of Density of
MCA Cl 15 cells Seeded 45

vi

List of Figures

Figure 1 : Mechanism of Nickel Transformation 4

Figure 2: Differential Display Gels 6

Figure 3: Mediator Complex 9

Figure 4: Vitamin D Pathway 10

Figure 5: Mediator is a Multi-Subunit Complex 12

Figure 6: Transformed Cell Lines Were Generated From Foci 16

Figure 7: Volocity Software 19

Figure 8: Confocal Microscopy Images of Non-Transformed 10T1/2
Cells Showing Intracellular Ca
2+
Ions Stained With Fluo-3AM Dye 22

Figure 9: Confocal Microscopy Images of MCA- and Nickel Compound-
Transformed Cells Showing Calcium Associated Fluorescence 23

Figure 10: Fluorescence Variability 25

Figure 11: Total Cellular Fluorescence of Non-Transformed 10T1/2 Cells 26

Figure 12: Total Cellular Fluorescence of 10T1/2 Cells With
Predominantly Nuclear Staining 26

Figure 13: Total Cellular Fluorescence of 10T1/2 Cells With
Predominantly Cytoplasmic Staining 27

Figure 14: Total Cellular Fluorescence of MCA Cl 15 Cells 27

Figure 15: Total Cellular Fluorescence of MCA Cl 16 Cells 28

Figure16: Total Cellular Fluorescence of NiS3A1 Cells 28

Figure 17: Total Cellular Fluorescence of NiS3B1 Cells 29

Figure 18: Total Cellular Fluorescence of NiS7A1 Cells 29

Figure 19: Total Cellular Fluorescence of NiO2C3 Cells 30
vii

Figure 20: Average Total Cellular Fluorescence of All Cell Lines 30

Figure 21: Nuclear Fluorescence of 10T1/2 Cells as Percentage of Total
Fluorescence 32

Figure 22: Nuclear Fluorescence of MCA Cl 15 Cells as Percentage of
Total Fluorescence 33

Figure 23: Nuclear Fluorescence of MCA Cl 16 Cells as Percentage of
Total Fluorescence 33

Figure 24: Nuclear Fluorescence of NiO2C3 Cells as Percentage of Total
Fluorescence 34

Figure 25: Nuclear Fluorescence of NiS3A1 Cells as Percentage of Total
Fluorescence 34

Figure 26: Nuclear Fluorescence of NiS3B1 Cells as Percentage of Total
Fluorescence 35

Figure 27: Nuclear Fluorescence of NiS7A1 Cells as Percentage of Total
Fluorescence 35

Figure 28: Nuclear Fluorescence in All Cell Lines as Percentage of Total
Fluorescence 36

Figure 29: Cytoplasmic Fluorescence of 10T1/2 Cells as Percentage of
Total Fluorescence 38

Figure 30: Cytoplasmic Fluorescence of MCA Cl 15 Cells as Percentage of
Total Fluorescence 39

Figure 31: Cytoplasmic Fluorescence of MCA Cl 16 Cells as Percentage of
Total Fluorescence 39

Figure 32: Cytoplasmic Fluorescence of NiS3A1 Cells as Percentage of
Total Fluorescence 40

Figure 33: Cytoplasmic Fluorescence of NiS3B1 Cells as Percentage of
Total Fluorescence 40

Figure 34: Cytoplasmic Fluorescence of NiS7A1 Cells as Percentage of
Total Fluorescence 41

viii

Figure 35: Cytoplasmic Fluorescence of NiO2C3 Cells as Percentage of
Total Fluorescence 41

Figure 36: Cytoplasmic Fluorescence of All Cell Lines as a Percentage of
Total Fluorescence 42

ix

Abstract

10T1/2 mouse cells were treated with nickel oxide, nickel monosulfide, and 3-
methylcholanthrene to establish transformed cell lines. Non-transformed 10T1/2 cells
expressed the vitamin D receptor interacting protein 80 (DRIP-80), while nickel ion-
transformed cell lines did not (2). The DRIP-80 protein is a subunit of the Mediator
complex, which regulates Ca
+2
ion distribution via regulation of vitamin D responsive
genes. Disruption of DRIP-80 gene expression may result in an aberrant distribution of
Ca
+2
ions. To test this hypothesis, non-transformed 10T1/2 cells and transformed 10T1/2
cell lines were stained with Fluo 3-AM and visualized by confocal microscopy. The
distribution of Ca
+2
ions in non-transformed 10T1/2 cells was heterogeneous. Nickel
compound- and MCA-transformed 10T1/2 cells have consistently less Ca
+2
ions in the
nucleus than the cytoplasm. These results suggest that disruption of normal transport of
Ca
+2
ions between nucleus and cytoplasm may contribute to the altered phenotypes in
transformed 10T1/2 cell lines.

1

Chapter One: Introduction

Carcinogenesis by Nickel Compounds

Nickel compounds have wide ranging industrial applications. They are utilized in
the manufacture of stainless steel, jewelry, utensils, electroplating, nickel-cadmium
batteries, coinage, catalysts used in the hydrogenation of fats and oils, ceramic glazes,
and paints (reviewed in 2,3). Manufacture of these nickel-containing products generates
billions of dollars worth of useful commercial products and economic activity per year.
However, nickel refinery workers come into contact with dusts and aerosols of
mixtures of water soluble and water insoluble nickel compounds through inhalation
exposure. In nickel refinery workers who were exposed to mixtures of insoluble and
soluble nickel compounds who also smoked cigarettes, and who were also exposed to
mists of sulfuric acid, epidemiological studies show that there were increased incidences
of nasal, lung, and esophageal carcinomas (3, 4, 5). Exposure to nickel arsenide (Ni
5
As
2
)
has also been associated with respiratory cancers in nickel refinery workers (3). The
International Agency for Research on Cancer (IARC) classified many nickel compounds,
particularly the insoluble nickel compounds, as Group 1 confirmed carcinogens to
humans (6).
In vivo studies have shown that specific insoluble nickel compounds are
carcinogenic in rodents. Injection of nickel subsulfide (Ni
2
S
3
) into rats induces a 100%
incidence of fibrosarcomas at the injection site (7). Inhalation of nickel subsulfide and
2

black and green nickel oxides are also carcinogenic in rodents when administered by the
inhalation route of exposure, causing nasal and lung tumors (1, 8).
In vitro studies have provided model cell culture systems in which to study the
molecular mechanisms of nickel compound-induced neoplastic cell transformation.
Nickel subsulfide, crystalline nickel monosulfide, and green and black nickel oxides
induce anchorage independence, morphological transformation, and neoplastic
transformation in C3H/10T1/2 Cl 8 (10T1/2) mouse embryo fibroblast cells (1, 8).
Morphological transformation also occurs in Syrian hamster embryo (SHE) cells exposed
to nickel subsulfide (9). Nickel compounds also induce anchorage independence in
human fibroblast cells (5). Due to the induction of nasal and respiratory tumors in nickel
refinery workers, it is important to define the molecular mechanisms of nickel
compound-induced neoplastic cell transformation.
Mechanisms of action for morphological and neoplastic transformation of cells by
carcinogenic nickel compounds have been proposed by Landolph et al. (3, 8) and Lu et
al. (10). Insoluble nickel particles induce invagination of the cell membrane, leading to
active phagocytosis of nickel-containing particles. Once phagocytosed into cells, nickel-
containing particles are then solubilized within phagocytic vacuoles as they enter the
lysosomal network, which has an acid pH of 4.5, to generate intracellular divalent nickel
ions. Ni
2+
ions can bind to cytosolic proteins that translocate into the nucleus or bind to
DNA-associated proteins already in the nucleus, such as histones (Figure 1). Then, Ni
2+

ions react with intracellular hydrogen peroxide (itself formed from superoxide, a product
of cellular metabolism), to generate hydroxyl radicals through Fenton-like reactions (8).
Ni
2+
ion stimulation of arachidonic acid metabolism may also play a contributory role in
3

the nickel-induced generation of reactive oxygen species, including superoxide, hydrogen
peroxide, and hydroxyl radicals (reviewed in 5). Reactive hydroxyl radicals generated by
Ni
2+
ions via their reaction with hydrogen peroxide, in proximity to DNA, can induce
chromosomal aberrations, such as dicentrics, translocations, satellite associations, rings,
and chromosome breaks. Ni
2+
ions can also bind to histones and cause the histones to
condense around actively transcribed genes (8, 11, 12). This may lead to methylation of
the promoters of these genes in murine cells, and cause repression of transcription of
these genes (2, 3, 8). Through this latter methylation mechanism, Ni
2+
ions can
extensively alter gene expression in transformed cells. It has been determined that an
estimated total of 130 genes are differentially expressed between nickel compound-
transformed and MCA-transformed cell lines compared to non-transformed 10T1/2 cell
lines, in previous work in our laboratory (2, 8). Our laboratory has hypothesized that
aberrant gene regulation plays a major contributory role in induction and maintenance of
transformed phenotypes in 10T1/2 cells exposed to nickel compounds. (2, 8).
4

Figure 1. Mechanism of Nickel Transformation. Water insoluble nickel-containing
particles are phagocytosed into cells, and then solubilized in phagolysosomes. Ni
+2
ions
bind to proteins in the cytoplasm which translocate to the nucleus or bind to proteins
already present in the nucleus. Ni
+2
ions may generate reactive oxygen species (ROS) in
proximity to DNA, leading to oxidative damage to DNA, and ultimately causing altered
mRNA transcription. (Reprinted from Landolph, 2002, with the permission of Dr. Joseph
R. Landolph, Jr.)

Previous studies in our laboratory showed that treatment of 10T1/2 cells with
nickel subsulfide (Ni
3
S
2
), crystalline nickel monosulfide (NiS), black nickel oxide
(bNiO), and green nickel oxide (gNiO) induced foci of transformed cells. We ring-
cloned the foci, expanded them into transformed cell lines, and measured the biological
characteristics of the transformed cell lines (1). Next, our laboratory studied differential
gene expression between non-transformed 10T1/2 cells and the nickel compound- and
MCA-transformed 10T1/2 cell lines (2, 8). mRNA was extracted from nickel compound-
transformed, MCA-transformed, and non-transformed cell lines. cDNAs were generated
from these mRNAs by random, arbitrarily primed-polymerase chain reaction (RAP-PCR)
and reverse transcription-polymerase chain reaction (RT-PCR), using anchored and
5

arbitrary primers (8, 13 - 16). The resultant cDNA fragments were analyzed on five
differential display gels (8). Differential display analysis of 8% of the total mRNA found
that ten cDNA fragments were differentially expressed between nickel compound- or
MCA-transformed and non-transformed 10T1/2 cells (Table 1) (refs. 2, 8). Homology
comparisons to gene sequences in the NCBI BLAST database of GENBANK identified
the differentially expressed cDNAs as fragments of the calnexin gene, the DRIP-80 gene,
the Ect-2 gene, the Wdr1 gene, the IGFR1 gene, the SNAP C3 gene, the FAD synthetase
gene, and three unidentified genes (Table 1) (17). Genes expressed at higher steady-state
levels in transformed cells are the calnexin gene, the Ect-2 proto-oncogene, and the Wdr1
gene (2). In contrast, the DRIP-80 (MED-17) gene, the IGFR1 gene, the SNAP C3 gene,
and the FAD synthetase gene were under-expressed in nickel compound- and MCA-
transformed 10T1/2 cell lines compared to non-transformed 10T1/2 cells (Table 1) (2, 8).
The expression patterns of all these differentially expressed genes were verified by
reverse Northern blotting analyses. As expected, the results yielded the same pattern of
differential gene expression observed in the differential display gels, confirming the
validity of the results obtained by the differential display gels (2, 8).

6

Figure 2. Differential Display Gels. Fragment R1-2 is expressed in non-transformed
10T1/2 cells, MCA Cl 16 cells, and MCA Cl 15 but not expressed in NiS3A1 cells.
Fragment R1-2 is also under-expressed in other NiS- and NiO-transformed cell lines.
Homology comparisons with sequences in the NCBI BLAST database of GENBANK
identified R1-2 as a fragment of the DRIP-80 gene (p=5.3X10
-34
). (Reprinted from
Verma et al., 2004, with the permission of Dr. Joseph R. Landolph, Jr.)

7

Table 1. Ten cDNA Fragments Were Differentially Expressed Between Non-
Transformed, Nickel Compound-Transformed, and MCA-Transformed Cells. The gene
identity was ascertained by homology comparisons with sequences in the NCBI BLAST
database of GENBANK.

Fragment
Name
Expression of
Gene
Fragments in
10T1/2 cell
lines
Expression of
Gene Fragments
in Nickel
Compound-
Transformed Cell
Lines
Expression of
Gene
Fragments in
MCA-
Transformed
Cell Lines Gene Identity
R1-1
R1-2
Present
Present
Absent
Absent
Present
Present
No similarity
DRIP80 (p=5.3X10
-34
)
R2-1 Absent Absent Present No similarity
R2-2 Present Absent Absent No similarity
R2-3 Present Absent Absent No similarity
R2-4 Present Absent Absent SNAP C3 (p=7.0X10
-3
)
R2-5 Absent Present Present Ect2 (p=5.0X10
-35
)
R3-1 Present Overexpressed Overexpressed Wdr1 (p=0.0)
R3-2 Absent Present Present Calnexin (p=6.2X10
-49
)
R3-3 Present Uderexpressed Present IGFR-1 (p=3.0X10
-56
)

Table 1. This table was reprinted from Verma et al., 2004, with the permission of Dr.
Joseph R. Landolph, Jr.

Identification of Down-Regulation of the DRIP-80 Gene and Its Role in
Vitamin D Regulation of Calcium Ion Homeostasis

The cDNA fragment identified as a fragment of the DRIP (Vitamin D Receptor
Interacting Protein) gene #80 was selected for further investigation in this thesis. Verma
et al. in our laboratory found that the DRIP-80 gene is not expressed in NiO- and NiS-
transformed 10T1/2 cells, but it is expressed in non-transformed and in MCA-
transformed cell lines (Figure 2) (2, 8). Therefore, the mechanism through which DRIP-
80 gene repression occurs may be specific to nickel compounds and not to other chemical
8

carcinogens. However, we have to study transformed cell lines induced by other
carcinogens for DRIP-80 expression to address this point. The DRIP-80 gene maps to
mouse chromosome 9 and human chromosome 11 (18). The DRIP-80 (MED-17) protein
is one of 22 to 28 subunits comprising the Mediator complex (19). Due to the large
number of subunits forming Mediator, this complex presents a large and complicated
surface to which a diverse range of proteins can bind. For instance, Mediator complex
binds to a variety of nuclear receptors, such as the vitamin D receptor (VDR), the
androgen receptor (AR), the thyroid receptor (TR), the peroxisomal proliferating
activated receptor γ (PPARγ), the retinoic acid receptor (RAR), and the steroid receptor
(20-24) (20-24). Mediator also interacts with chromatin remodeling cofactors, Swi and
Snf, to induce promoter accessibility (20, 25). Finally, the Mediator complex also binds
to transcriptional cofactors (both activators and repressors), such as SREBP-1a, TFIIH,
and the p65 subunit of NF-κB, to convey regulatory information from DNA elements to
the transcription apparatus (19, 20). Thus, Mediator acts as an adaptor to integrate a
diverse array of pathways and proteins into a system of regulation which controls gene
transcription.
The first step of Mediator-dependent transcription is the targeting of the Mediator
complex to specific genes by nuclear receptors. Upon binding to their ligands, nuclear
receptors are recruited to DNA response elements proximal to gene promoters (19).
Mediator is then localized to the promoters through binding interactions with the nuclear
receptors. The Mediator-ligand-receptor complex recruits RNA polymerase II along with
transcription cofactors to activate transcription of the target gene (25, 26). Thus, the
9

Mediator complex serves as a molecular bridge which transduces regulatory information
from DNA elements to the transcription apparatus (Figure 3 and Figure 4).

Figure 3. Mediator Complex. Mediator complex recruitment of regulatory factors and
the pre-initiation complex to DNA elements. (Modified from Takagi et al. 2006, with
permission of Roger D. Kornberg)

10

Figure 4. Vitamin D Pathway. Vitamin D binds to the vitamin D receptor (VDR) which
then dimerizes with the retinoid X receptor (RXR). The heterodimer binds to the
vitamin D response element (VDRE) upstream from the promoter of the target gene. The
Mediator complex is recruited, which in turn recruits the general transcription apparatus
(GTA) to initiate transcription. (Reprinted from Malloy et al., 1999, with the permission
of David Feldman)

The role of the Mediator complex in transcription is such an integral part of
normal cell function that Mediator inactivation due to DRIP-80 silencing has significant
physiological effects. DRIP-80 protein is located in the “head” region of the Mediator
complex, where it maintains the structural integrity of the head module by binding six
other Mediator subunits (Figure 5) (19, 23). Therefore, the head region of the Mediator
complex is not expected to form in nickel compound-transformed cells, where the DRIP-
11

80 gene is not expressed. It would be expected that transcription of RNA pol II-
dependent genes would therefore be greatly impaired, because the head region provides
the major contact sites for RNA pol II, TFIIF, and many transcriptional activators (19,
27). Ranish et al. showed that mutations to subunits of the head module led to reduced
RNA pol II binding (28). Takagi et al. disrupted the head module of yeast Mediator
complex using a temperature-sensitive mutant of the DRIP-80 protein (27). He found
that this caused the release of Mediator body and tail modules from gene promoters and
upstream activating sequences, thereby terminating all transcription (27). This
demonstrates that DRIP-80 gene expression and proper formation of the head region is
critical for Mediator-dependent transcription. Thus, silencing of expression of the DRIP-
80 gene in nickel compound-transformed cells would be expected to result in a
“headless” Mediator complex with reduced function and profound consequences to gene
transcription.
12

Figure 5. Mediator is a Multi-Subunit Complex. DRIP-80 (Med 17) plays a structural
role in the head region. DRIP-80 maintains the structural integrity of the Mediator
complex through binding interactions with six other Mediator subunits. Thus, the
absence of DRIP-80 protein will have profound consequences to Mediator function.
(This figure is reproduced from Blazek et al., 2005, with the permission of M.
Meisterernst)

Of interest to our laboratory is the interaction of the vitamin D-vitamin D receptor
(VDR) complex with the Mediator complex. The 1,25-dihydroxyvitamin D
3
(vitamin D)
hormone binds to the VDR, a nuclear receptor, to regulate vitamin D-responsive genes
through the Mediator complex (Figure 4) (29). Experiments show that selective
depletion of Mediator subunits decreases the transcriptional response to vitamin D (20).
The principle genomic function of vitamin D is the regulation of genes involved in
calcium metabolism and calcium homeostasis (21). We hypothesize that nickel ion-
induced silencing of the DRIP-80 gene produces a Mediator complex with reduced
13

function, which would be expected to result in aberrant vitamin D regulation of genes
involved in Ca
2+
homeostasis. Deregulation of expression of genes involved in Ca
+2
ion
homeostasis may lead to alterations of the intracellular Ca
+2
ion distributions in
transformed cells. Changes to the intracellular Ca
+2
distribution could disrupt the
functions of many intracellular compartments, and this would likely lead to
morphological and neoplastic cell transformation.

The Role Ca
+2
Ions in Signal Transduction in Mammalian Cells

Calcium is a biochemical messenger which coordinates the activities of various
intracellular compartments (31). Ca
+2
ions are particularly important to the function of
the nucleus. Active nuclear calcium signaling has been observed in a number of cells,
including pancreatic exocrine cells, neurons, starfish oocytes, and hepatocytes (31).
Although the full extent of the physiological role of nuclear calcium is unknown, Ca
+2

ions control transcription of certain genes and protein translocations across the nuclear
envelope (32). Calcium-dependent gene regulation is involved in cell proliferation,
death, and survival (31-35). Studies have linked nuclear Ca
+2
ions to cellular processes
important to cancer development (10, 32, 36, 37). Ca
+2
ion signaling induces the nuclear
localization of FOXO, a transcription factor and tumor suppressor (36). Nuclear calcium
signaling also promotes the cell cycle progression of mouse embryonic stem cells through
the G
1
/S phase (38). Nickel ion-induced destabilization of calcium homeostasis has been
associated with the induction of DNA single-stranded breaks and sister chromatin
exchange in human blood lymphocytes exposed to nickel compounds (39). Thus, we
14

hypothesized 1) that nickel ion treatment of 10T1/2 cells, which resulted in loss of
expression of the DRIP-80 gene, could lead to permanent changes in the intracellular
distribution of Ca
2+
ions, and 2) that the resultant change in the intracellular distribution
of Ca
+2
ions could contribute to the induction and maintenance of the transformed
phenotypes (focus formation, anchorage independence, and tumorigenicity) in nickel
compound-transformed C3H/10T1/2 mouse embryo cells. To test hypothesis #1, we
investigated the distribution of intracellular Ca
+2
ions in nickel compound- and MCA-
transformed 10T1/2 cell lines verses those in non-transformed 10T1/2 cells. To do this,
we used confocal fluorescence microscopy to visualize Ca
+2
ion distributions between the
nucleus and the cytosol of nickel ion-transformed and MCA-transformed 10T1/2 cell
lines verses Ca
+2
ion distributions in non-transformed 10T1/2 mouse embryo fibroblast
cell lines.

15

Chapter Two: Materials and Methods

Cell culture

The non-transformed C3H/10T1/2 Cl8 (10T1/2) mouse embryo fibroblast cell line
was derived from the embryos of pregnant C3H mice by Reznikoff et al. (40). The
10T1/2 cells are hypertetraploid, contact-inhibited, and non-tumorigenic mouse embryo
fibroblast cells that have low rates of spontaneous focus formation and low rates of
anchorage independent colony formation (8). The low frequency of spontaneous
morphological transformation coupled with the high rate of morphological transformation
when 10T1/2 cells are treated with chemical carcinogens make 10T1/2 mouse fibroblast
cells an ideal in vitro model system in which to study chemically induced morphological,
anchorage-independent, and neoplastic cell transformation (5).
Transformed cell lines were generated from foci of transformed cells, which were
induced by treating 10T1/2 cells with crystalline nickel monosulfide (NiS), 3-
methylcholanthrene (MCA), or green (HT) nickel oxide (NiO) (Figure 6) (8). The
chemical carcinogen, MCA, was used to induce foci and hence to generate transformed
cell lines for comparative purposes. MCA-transformed cells lines were used determine
whether the differential gene expression observed in transformed cells was specific to
carcinogenic nickel compounds or common to other chemical carcinogens, such as MCA.
MCA is a polycyclic aromatic hydrocarbon, a strong animal carcinogen, and a strong
inducer of morphological transformation in 10T1/2 mouse embryo cells (2). The MCA-
transformed cell lines were designated MCA Cl 15 and MCA Cl 16 (Table 2) (1). The
16

transformed cell lines derived from nickel monosulfide-induced foci were designated
NiS3A1, NiS7A1, and NiS3B1 (Table 2). Transformed cell lines created from green
(HT) nickel oxide induced foci were called NiO2C3 and NIO G1-2 (Table 2) (1, 3).

Figure 6. Transformed Cell Lines Were Generated From Foci. Foci are created by
exposure of 10T1/2 cells to crystalline nickel monosulfide (NiS), 3-methylcholanthrene
(MCA), or green (HT) nickel oxide (NiO). This is a picture of a type III focus induced
by treating 10T1/2 cells with 1 μg/ml of MCA. (This figure is reprinted from Muira et
al., 1989, with the permission of Dr. Joseph R. Landolph, Jr.)

Table 2. Transformed Cell Line Designations

Transformed Cell Line
Designation Transforming Agent
NiS3A1 Nickel Monosulfide
NiS7A1 Nickel Monosulfide
NiS3B1 Nickel Monosulfide
MCA Cl 15 3-Methylcholanthrene
MCA Cl 16 3-Methylcholanthrene
NiO2C3 Nickel Oxide
NIO G1-2 Nickel Oxide

Table 2. Transformed cell lines were generated from foci created by exposure of 10T1/2
cells to crystalline nickel monosulfide (NiS), 3-methylcholanthrene (MCA), or green
(HT) nickel oxide (NiO).

17

Non-transformed 10T1/2 cells and transformed cell lines were cultured using
methods described by Reznikoff et al. (40). Cells were cultured in Basal Medium Eagles
(BME) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific Products,
Irvine, California) (1, 40-42). The BME medium was prepared by the Bioreagents Core
Facility of the USC/Norris Comprehensive Cancer Center by purchasing BME powder
from the GIBCO-BRL Company, dissolving this in water, adjusting the pH to 7.2, and
filter-sterilizing the BME. The 10T1/2 cell line was used from passages five to fifteen
and then discarded, after which a new vial of frozen cells was thawed for use, to reduce
the occurrence of spontaneous morphological transformation. Cells were maintained in
the subconfluent, logarithmic phase of growth (approximately 80% confluence). Cells
were cultured in 75 cm
2
tissue culture flasks (Corning Glass Works, Corning, New York).
Cells were grown in dishes and flasks in Forma CO
2
incubators to maintain a humidified
37
o
C atmosphere with 5% CO
2
to 95% air (volume/volume) in which the cells were
cultured for four days prior to visualization by microscopy.

Confocal Microscopy

For confocal microscopy studies, cells were seeded into a LabTek II Chambered
Coverglass with 4 wells of 1 ml capacity each (purchased from Thermo Fisher Scientific,
Rochester, New York). Confocal microscopy provides a noninvasive method for
obtaining high lateral resolution images of intact living cells. The distribution of
intracellular Ca
+2
ions was visualized within living cells using the Fluo 3-AM calcium
binding fluorescent dye according to protocols specified by the Dojindo Company
18

(Dojindo Molecular Technologies, Gaithersburg, MD). To prevent Fluo 3-AM
aggregation, 16.5 mg of pluronic F127 detergent was added to Fluo 3-AM in dry
dimethyl sulfoxide (DMSO) (1 mg Fluo 3-AM in 442 μl DMSO) (Dojindo Molecular
Technologies, Gaithersburg, MD). The mixture was then diluted to create a 4 mM Fluo
3-AM imaging buffer by adding 5 μl of the Fluo 3-AM/DMSO/pluronic F127 mixture to
2500 μl of Hanks’ balanced salt solution (HBSS) (obtained from the Bioreagents Core
Facility at the USC/Norris Comprehensive Cancer Center). Dye was loaded into the cells
by adding 80 μl of warm Fluo 3-AM imaging buffer to each well and incubating the cells
with the dye at 37
o
C for 20 minutes. Next, 400 μl of warm HBSS containing 1% fetal
bovine serum was added, and the cells were further incubated for 40 minutes at 37
o
C.
Cells were finally washed with warm HEPES buffer saline (10 mM HEPES, 1 mM
Na
2
HPO
4
, 137 mM NaCl, 5 mM KCl, 1 mM CaCl
2
, 0.5 mM MgCl
2
, 5 mM glucose, 0.1%
BSA, pH 7.4) (obtained from the Bioreagents Core Facility at the USC/Norris
Comprehensive Cancer Center) to remove extracellular Fluo 3-AM dye. Cells were then
incubated in 1 ml of warm HEPES buffer saline, and then incubated at 37
o
C for an
addition 10 minutes to complete the de-esterification of Fluo 3-AM.
Images were captured using a Zeiss LSM510 Confocal Laser Scan Microscope
(Carl Zeiss MicroImaging, Oberkochen, Germany) with a 25x oil-immersion objective
plan (numerical aperture = 0.8). The Fluo-3AM fluorescent dye was excited with an
argon ion laser set to a wavelength of 488 nm and fluorescent emission was detected at a
wavelength of 520 nm. Images were obtained using the LSM imaging software (Carl
Zeiss MicroImaging, Oberkochen, Germany) and analyzed with the Volocity software
(Improvision, Coventry, United Kingdom). The nuclear and cytoplasmic areas of the
19

cells were highlighted with Volocity (Figure 7). The mean and total fluorescent
intensities were measured within the nuclear and cytoplasmic areas in arbitrary units.
Measurements were then normalized to the background fluorescence.

Figure 7. Volocity Software. The nuclear and cytoplasmic regions of the cell were
highlighted with the Volocity software program. The blue area represents the cytoplasm
of a cell while the green region represents the nucleus. The total fluorescence of these
regions are then measured in arbitrary units and subtracted by the background
fluorescence. The background fluorescence is determined by the fluorescence reading
within the area of the orange box.

20

Chapter Three: Results

Ca
2+
Ion Distributions in Non-Transformed 10T1/2 Cells

Fluorescence images were taken of non-transformed 10T1/2 cells and nickel
compound- and MCA-transformed 10T1/2 cell lines. Some images were excluded from
analysis due to poor picture quality, resulting in difficulty of interpretation. Of the
images analyzed, a large fraction of the non-transformed 10T1/2 cell population had Ca
+2

ion-associated fluorescence predominantly in the cytoplasm (Figure 8a). In these cells,
the majority of the free intracellular Ca
+2
ions are localized around the perinuclear area
with a sharp drop-off of Ca
+2
ion levels approaching the plasma membrane (Figure 8a).
Other non-transformed 10T1/2 cells have predominantly nuclear staining, indicating there
are higher levels of Ca
+2
ions in the nucleus than in the cytoplasm (Figure 8b). The
cytoplasmic Ca
+2
ions are also localized around the peri-nuclear region, although this
region has a lower Ca
+2
ion concentration than the nucleus (Figure 8b). After analyzing
the Ca
+2
ion distributions of 132 non-transformed 10T1/2 cells, we found that 40 or
30.3% had predominantly high nuclear Ca
+2
ion concentrations (State 1), and 92 or
69.7% had Ca
+2
ions predominantly in the cytoplasm (State 2) (Table 3). We suspected
that these two states of Ca
+2
ion distribution- predominantly nuclear (State 1), and
predominantly cytoplasmic (State 2) - might be correlated to different states of the cell
cycle in asynchronous 10T1/2 cells.

21

Ca
+2
Ion Distributions in Nickel-Transformed and in MCA-Transformed 10T1/2
Cell Lines

In contrast to non-transformed 10T1/2 cells, none of the transformed cell lines-
NiO2C3 cells, NiS3A1 cells, NiS3B1 cells, NiS7A1 cells, MCA Cl 15 cells, or MCA Cl
16 cells- had nuclear calcium concentrations exceeding cytoplasmic calcium levels
(Figure 9 A-F, Table 3). All transformed cells lines had higher concentrations of
cytosolic calcium than nuclear calcium; there was very little Ca
+2
ion in the nucleus
(Table 3). In the NiO2C3 cell line (transformed by green NiO), there is a strong
cytoplasmic staining for Ca
+2
ion, but little nuclear Ca
+2
ion staining in the nucleus
(Figure 9A). In the MCA Cl 15 and MCA Cl 16 cell lines (transformed by MCA), there
is cytoplasmic staining for Ca
+2
ions, but less staining for Ca
+2
ions is seen in the nucleus
(Figures 9B and 9C). Similarly, for the NiS3A1 cell line (transformed by crystalline
nickel monosulfide), there is again staining for Ca
+2
ion in the cytoplasm, but little or no
staining for Ca
+2
ion in the nucleus (Figure 9D). In addition, for NiS7A1 cells
(transformed by crystalline nickel monosulfide), there is strong staining for Ca
2+
ions in
the cytoplasm, but the nucleus was only weakly stained for Ca
+2
ions (Figure 9E). Lastly,
in NiS3B1 cells (transformed again by crystalline nickel monosulfide), there is strong
staining for Ca
+2
ions in the cytoplasm, but little or no staining for Ca
+2
ions in the
nucleus (Figure 9F).
The percentages of cells with predominant staining of Ca
+2
ions in the nucleus in
the various cell lines were: 10T1/2, 28.7%; MCA Cl 15, 0%; MCA Cl 16, 0%; NiO2C3,
0%; NiS3B1, 0%; NiS3A1, 0%; NiS7A1, 0% (Table 3). In conclusion, the non-
transformed 10T1/2 cell population has a heterogeneous distribution of Ca
+2
ions.
22

Approximately 30% of 10T1/2 cells have predominantly nuclear Ca
+2
ion staining
compared to 70% of 10T1/2 cells with predominantly cytoplasmic Ca
+2
ion staining
(Figure 8A, Figure 8B, Table 3). In contrast, the MCA- and nickel compound-trans-
formed cells always have higher cytoplasmic Ca
+2
ion concentrations than nuclear Ca
+2

ion concentrations. In the transformed cell lines, 0% of the cells had high Ca
+2
ion
concentration in the nucleus; most of the Ca
+2
ions were in the cytoplasm in 100% of
cells of the transformed cell lines (Figure 9A-9F, Table 3)

Figure 8. Confocal Microscopy Images of Non-Transformed 10T1/2 Cells Showing
Intracellular Ca
2+
Ions Stained With Fluo-3AM Dye. (a) Some 10T1/2 cells have Ca
2+
ion
staining predominantly in the cytoplasm. (b) Other 10T1/2 cells have higher
concentrations of Ca
2+
ion in the nucleus compared to the cytoplasm.

23

Figure 9. Confocal Microscopy Images of MCA- and Nickel Compound-Transformed
Cells Showing Calcium Associated Fluorescence. There are higher calcium
concentrations in the cytoplasm than in the nucleus of (a) NiO2C3, (b) MCA Cl 15, (c)
MCA Cl 16, (d) NiS3A1, (e) NiS7A1, and (f) NiS3B1 cell lines.

Table 3. Comparison of intracellular calcium localization among various cell lines

Cell Types
Number of Cells With
Predominantly Nuclear Staining
Number of Cells With
Predominantly Cytoplasmic Staining
10T1/2 40 (30.3%) 92 (69.7%)
MCA Cl 16 0 8
MCA Cl 15 0 24
NiO2C3 0 4
NiS3A1 0 97
NiS3B1 0 85
NiS7A1 0 62

Table 3. A comparison of calcium localization among cell lines reveals heterogeneity in
calcium distribution in non-transformed 10T1/2 cells. MCA- and nickel compound-
transformed cell lines have intracellular calcium predominantly localized in the
cytoplasm of all cells. No transformed cells with predominantly nuclear calcium staining
have been observed.
24

Variability of Total Cellular Fluorescence Among Cells

The total cellular fluorescence is the sum of nuclear and cytoplasmic
fluorescence. The total fluorescence is highly variable in transformed and non-
transformed cell lines. Even cells within the same viewing field, which were exposed to
identical experimental conditions, show a wide range of fluorescence intensities (Figure
10). We suspect that variation in Ca
+2
ion staining among cells of the same field is likely
the result of variability in the Fluo 3-AM absorption characteristics of individual cells
rather than differences in the total intracellular Ca
+2
ion levels. When fluorescence is
measured in arbitrary units (AU) with the Volocity image analysis software, the total
fluorescence of non-transformed 10T1/2 cells varies greatly (Figure 11). Non-
transformed 10T1/2 cells with predominantly nuclear staining have a range in
fluorescence intensities from a low value of 72,566 AU to a high value of 3,920,840 AU,
a 50-fold difference (Figure 12). Non-transformed 10T1/2 cells with predominantly
cytoplasmic Ca
+2
ion staining also vary substantially in total fluorescence (Figure 13).
The transformed cells also exhibit similar variability in total cellular fluorescence
(Figure 14 to Figure 19). When comparing total fluorescence levels between cell lines,
10T1/2 cells with greater cytoplasmic Ca
+2
ion staining have similar total fluorescence
intensities to that of nickel compound- and MCA-transformed cells (Figure 13 compared
to Figures 14-19 and summarized in Figure 20). Non-transformed 10T1/2 cells with
predominantly nuclear Ca
+2
ion staining (Figure 12) have greater total fluorescence
intensities than either 10T1/2 cells with predominantly cytoplasmic fluorescence (Figure
13) or transformed cells (Figure 14-19, summarized in Figure 20). This indicates that
25

higher levels of total intracellular Ca
+2
ion exist in non-transformed cells in State 1,
where cells have predominantly high levels of nuclear Ca
+2
ions (Figure 8B vs. Figure 8A
and Figures 9A-9F, summarized in Figure 20).

Figure 10. Fluorescence Variability. Although the cells are in the same viewing field
and have been exposed to identical experimental conditions, there is still a wide range of
total fluorescence intensities within populations of NiS3B1 cells. Similar variability in
the total cellular fluorescence intensity exists for the other cell lines as well.

26

Figure 11. The combined nuclear fluorescence and cytoplasmic fluorescence (total
cellular fluorescence) was measured in 132 non-transformed 10T1/2 cells. The total
fluorescence is measured in arbitrary units (AU) and is highly variable in this cell line.

Figure 12. The combined nuclear fluorescence and cytoplasmic fluorescence (total
cellular fluorescence) was measured in 40 non-transformed 10T1/2 cells with mostly
nuclear Ca
2+
ion staining. The total cellular fluorescence is measured in arbitrary units
(AU) and is highly variable in these cells.

0
4
8
12
16
0 100 200 300 400 500
Number of Cells (n = 132)
Total Fluorescence (AU X 10
4
)
Total Cellular Fluorescence of
Non-Transformed 10T1/2 Cells
0
2
4
6
8
0 100 200 300 400 500
Number of Cells (n = 40)
Total Fluorescence (AU X 10
4
)
Total Cellular Fluorescence of 10T1/2 Cells
With Predominantly Nuclear Staining
27

Figure 13. The combined nuclear fluorescence and cytoplasmic fluorescence (total
cellular fluorescence) was measured in 92 non-transformed 10T1/2 cells with mostly
cytoplasmic fluorescence staining. The total cellular fluorescence is measured in
arbitrary units (AU) and is highly variable in these cells.

Figure 14. The combined nuclear fluorescence and cytoplasmic fluorescence (total
cellular fluorescence) was measured in 24 MCA Cl 15 cells. The total cellular
fluorescence is measured in arbitrary units (AU) and is highly variable in this cell line.

0
4
8
12
16
0 100 200 300 400 500
Number of Cells (n = 92)
Total Fluorescence (AU X 10
4
)
Total Cellular Fluorescence of 10T1/2 Cells
With Predominantly Cytoplasmic Staining
0
1
2
3
4
5
6
0 100 200 300 400 500
Number of Cells (n = 24)
Total Fluorescence (AU X 10
4
)
Total Cellular Fluorescence of
MCA Cl 15 Cells
28

Figure 15. The combined nuclear fluorescence and cytoplasmic fluorescence (total
cellular fluorescence) was measured in 8 MCA Cl 16 cells. The total cellular
fluorescence is measured in arbitrary units (AU) and is highly variable in this cell line.

Figure 16. The combined nuclear fluorescence and cytoplasmic fluorescence (total
cellular fluorescence) was measured in 97 NiS3A1 cells. The total cellular fluorescence is
measured in arbitrary units (AU) and is highly variable in this cell line.

0
1
2
3
4
0 100 200 300 400 500
Number of Cells (n = 8)
Total Fluorescence (AU X 10
4
)
Total Cellular Fluorescence of
MCA Cl 16 Cells
0
4
8
12
16
0 100 200 300 400 500
Number of Cells (n = 97)
Total Fluorescence (AU X 10
4
)
Total Cellular Fluorescence of
NiS3A1 Cells
29

Figure 17. The combined nuclear fluorescence and cytoplasmic fluorescence (total
cellular fluorescence) was measured in 85 NiS3B1 cells. The total cellular fluorescence
is measured in arbitrary units (AU) and is highly variable in this cell line.

Figure 18. The combined nuclear fluorescence and cytoplasmic fluorescence (total
cellular fluorescence) was measured in 62 NiS7A1 cells. The total cellular fluorescence
is measured in arbitrary units (AU) and is highly variable in this cell line.

0
4
8
12
16
0 100 200 300 400 500
Number of Cells (n = 85)
Total Fluorescence (AU X 10
4
)
Total Cellular Fluorescence of
NiS3B1 Cells
0
3
6
9
0 100 200 300 400 500
Number of Cells (n = 62)
Total Fluorescence (AU X 10
4
)
Total Cellular Fluorescence of
NiS7A1 Cells
30

Figure 19. The combined nuclear fluorescence and cytoplasmic fluorescence (total
cellular fluorescence) was measured in 4 NiO2C3 cells. The total cellular fluorescence is
measured in arbitrary units (AU) in the Volocity program and is highly variable in this
cell line.

Figure 20. A comparison of the average total cellular fluorescence in all cell lines reveals
that 10T1/2 cells with higher calcium levels in the cytoplasm (c) have fluorescence
intensities similar to those of nickel compound- and MCA-transformed cells. Non-
transformed 10T1/2 cells with higher calcium levels in the nucleus (n) have higher total
fluorescence intensities than transformed cells or 10T1/2 cells with predominantly
cytoplasmic Ca
+2
ion staining.
0
1
2
3
0 200 400 600 800 1000
Number of Cells (n = 4)
Total Fluorescence (AU X 10
4
)
Total Cellular Fluorescence of
NiO2C3 Cells
0
50
100
150
200
250
300
350
10T1/2
(all)
10T1/2
(n)
10T1/2
(c)
MCA15 MCA16 NiS3A1 NiS3B1 NiS7A1 NiO2C3
Total Fluorescence (AUX10
4
)
Cell Lines
Average Total Cellular Fluorescence of All Cell
Lines
31

Nuclear Fluorescence Levels of Non-Transformed, Nickel Compound-
Transformed, and MCA-Transformed Cells

In contrast to the total fluorescence intensity, the ratio of nuclear fluorescence to
the total cellular fluorescence is more constant within the 10T1/2 cell population. The
non-transformed 10T1/2 cells with predominantly cytoplasmic Ca
+2
ion staining have
nuclear fluorescence values ranging from 0% to 25% of the total fluorescence (Figure
21). The nuclei of these cells have an average of 14% of the total cellular fluorescence
(Figure 21). The non-transformed 10T1/2 cells with predominantly nuclear Ca
+2
ion
staining have nuclear fluorescence ranging from 25% to 70% of the total cellular
fluorescence (Figure 21). The nuclei account for an average of 38% of the total cellular
fluorescence in the 10T1/2 cells with predominantly nuclear Ca
+2
ion staining (Figure
21).
The nuclear fluorescence of MCA Cl 15 cells and MCA Cl 16 cells represents an
average of 11% and 7% of their total cellular fluorescence, respectively (Figures 22 and
23). In the NiO2C3 transformed cell line, nuclear fluorescence accounted for 9% of the
total cellular fluorescence (Figure 24). In the transformed cell lines NiS3A1 cells,
NiS3B1 cells, and NiS7A1 cells, the nuclear fluorescence represents 13%, 16%, and 12%
of the total cellular fluorescence, respectively (Figures 25-27). A comparison among all
cell lines show that non-transformed 10T1/2 cells with predominantly nuclear Ca
+2
ion
staining have nuclear fluorescence 2.1-fold higher than 10T1/2 cells with predominantly
cytoplasmic Ca
2+
fluorescent staining (Figure 28). Non-transformed 10T1/2 cells with
predominantly nuclear Ca
+2
ion staining also have nuclear fluorescence two-fold higher
32

than the MCA- and nickel ion-transformed cell lines (Figure 28). Non-transformed
10T1/2 cells with higher levels of cytoplasmic Ca
+2
ions have similar nuclear
fluorescence intensities as the two MCA-transformed and four nickel ion-transformed
cell lines (Figure 28). Thus, the nuclei of non-transformed cells can vary from a state of
low Ca
+2
ion concentration (State 2) to a state where the nuclei contain up to70% of the
total cellular Ca
+2
ions (State 1). In contrast, the nuclear fluorescence of transformed
cells never exceeds 30% of the total fluorescence and resemble non-transformed 10T1/2
cells in State 2.

Figure 21. In the non-transformed 10T1/2 cells, most cells have nuclei accounting for
25% or less of the total cellular fluorescence. However, there is a sizable subpopulation
of 10T1/2 cells with greater than 25% nuclear fluorescence. The intensity of nuclear
fluorescence in 10T1/2 cell with predominantly nuclear Ca
+2
ion staining can vary
greatly, from 25% to 70% of the total fluorescence.

0
10
20
30
40
0 25% 50% 75% 100%
Number of Cells (n = 142)
Nuclear Fluorescence
Nuclear Fluorescence of 10T1/2 Cells as
Percentage of Total Fluorescence
33

Figure 22. In the MCA Cl 15 cell line, all cells have nuclei accounting for 30% or less of
the total fluorescence. The nuclei of MCA Cl 15 cells have an average of 11% of the
total cellular fluorescence.

Figure 23. In the MCA Cl 16 cell line, all cells have nuclei accounting for 10% or less of
the total fluorescence. The nuclei of MCA Cl 16 cells have an average of 7% of the
total cellular fluorescence.

0
3
6
9
12
0% 25% 50% 75% 100%
Number of Cells (n = 24)
Nuclear Fluorescence
Nuclear Fluorescence of MCA Cl 15 Cells as
Percentage of Total Fluorescence
0
2
4
6
8
0% 25% 50% 75% 100%
Number of Cells (n = 8)
Nuclear Fluorescence
Nuclear Fluorescence of MCA Cl 16 Cells as
Percentage of Total Fluorescence
34

Figure 24. In the NiO2C3 cell line, all cells have nuclei accounting for 20% or less of the
total fluorescence. The nuclei of NiO2C3 cells have an average of 9% of the total
cellular fluorescence.

Figure 25. In the NiS3A1 cell line, all cells have nuclei accounting for 30% or less of the
total fluorescence. The nuclei of NiS3A1 cells have an average of 13% of the total
cellular fluorescence.

0
1
2
3
0% 25% 50% 75% 100%
Number of Cells (n = 4)
Nuclear Fluorescence
Nuclear Fluorescence of NiO2C3 Cells as
Percentage of Total Fluorescence
0
10
20
30
40
0% 25% 50% 75% 100%
Number of Cells (n = 97)
Nuclear Fluorescence
Nuclear Fluorescence of NiS3A1 Cells as
Percentage of Total Fluorescence
35

Figure 26. In the NiS3B1 cell line, all cells have nuclei accounting for 30% or less of the
total fluorescence. The nuclei of NiS3B1 cells have an average of 16% of the total
cellular fluorescence.

Figure 27. In the NiS7A1 cell line, all cells have nuclei accounting for 30% or less of the
total fluorescence. The nuclei of NiS7A1 cells have an average of 12% of the total
cellular fluorescence.

0
10
20
30
40
0% 25% 50% 75% 100%
Number of Cells (n = 85)
Nuclear Fluorescence
Nuclear Fluorescence of NiS3B1 Cells as
Percentage of Total Fluorescence
0
4
8
12
16
20
24
0% 25% 50% 75% 100%
Number of Cells (n = 62)
Nuclear Fluorescence
Nuclear Fluorescence of NiS7A1 Cells as
Percentage of Total Fluorescence
36

Figure 28. Non-transformed 10T1/2 cells with a predominantly cytoplasmic Ca
+2
ion
distribution (c)have nuclear fluorescence similar to the nuclear fluorescence of MCA-and
nickel compound-transformed cells. Non-transformed 10T1/2 cells with predominantly
nuclear Ca
+2
ion staining (n) have approximately two-fold higher nuclear fluorescence
intensity than either transformed cells or non-transformed cells in a state of higher
cytoplasmic Ca
+2
ion concentration.

Cytoplasmic Fluorescence Levels of Non-Transformed, Nickel Compound-
Transformed, and MCA-Transformed Cells

The cytoplasmic fluorescence was also compared to the total cellular fluorescence
in all cell lines. The cytoplasmic fluorescence accounts for an average of 86% of the
total fluorescence in 10T1/2 cells with mostly cytoplasmic Ca
2+
ion staining (Figure 36).
However, the cytoplasmic fluorescence accounted for only 62% of the total cellular
fluorescence in non-transformed 10T1/2 cells with predominantly nuclear fluorescence
(Figure 36). The majority of 10T1/2 cells have cytoplasmic fluorescence that account for
0%
25%
50%
75%
100%
10T1/2
(all)
10T1/2
(n)
10T1/2
(c)
MCA15 MCA16 NiS3A1 NiS3B1 NiS7A1 NiO2C3
Nuclear Fluorescence
Cell Lines
Nuclear Fluorescence in All Cell Lines as
Percentage of Total Fluorescence
37

75% to 100% of the total cellular fluorescence, and this subpopulation represents the
10T1/2 cells with predominantly cytoplasmic Ca
+2
ion fluorescent staining (Figure 29).
However, a sizable subpopulation of 10T1/2 cells had less than 75% cytoplasmic
fluorescence out of the total fluorescence, and these are the 10T1/2 cells with
predominantly nuclear Ca
+2
ion fluorescent staining (Figure 29).
In contrast to non-transformed 10T1/2 cells, all MCA Cl 15 cells and MCA Cl 16
cells have cytoplasmic to total fluorescence ratios greater than 75%, with averages of
89% and 93%, respectively (Figure 30, 31, and 36). In all transformed NiS3A1 cells,
NiS3B1 cells, and NiS7A1 cells, the cytoplasmic fluorescence represents greater than
70% of the total fluorescence, with averages of 87%, 84%, and 88%, respectively
(Figures 32 - 36). All transformed NiO2C3 cells have cytoplasmic fluorescent Ca
+2
ion
staining accounting for more than 80% of the total fluorescence, with an average of 91%
(Figures 35 and 36). When a comparison is made between various cell lines, non-
transformed cells with Ca
+2
ions located mostly in the cytoplasm have cytoplasmic
fluorescence similar to transformed cells (Figure 36). Non-transformed 10T1/2 cells with
Ca
+2
ion staining predominantly in the nucleus have a lower percentage of cytoplasmic
fluorescence out of the total cellular fluorescence (Figure 36). Non-transformed 10T1/2
cells appear capable of shifting their Ca
+2
ion distributions from a state (State 2) where
the cytoplasm may account for almost the entire total cellular fluorescent (Figure 8A) to a
state (State 1) in which the cytoplasm can account for as little as 30% of the total
fluorescence, and the nucleus accounts for more of the Ca
+2
ions (Figure 8B and Figure
36). MCA-transformed and nickel compound-transformed 10T1/2 cells appear to have
38

lost this ability. The nickel ion-transformed and MCA-transformed cells have a much
narrower range of cytoplasmic fluorescence and never achieve cytoplasmic fluorescence
lower than 70% of the total cellular fluorescence (Figures 30-35 and 36).

Figure 29. Non-transformed 10T1/2 cells are capable of altering their Ca
+2
ion
distributions from a state (State 2) where the cytoplasm accounts for almost the entire
total cellular fluorescence to a state (State 1) in which the cytoplasm accounts for as little
as 30% of the total fluorescence. The majority of 10T1/2 cells have cytoplasm that
account for 75% to 100% of the total cellular fluorescence and this subpopulation
represents the 10T1/2 cells with predominantly cytoplasmic Ca
+2
ion staining. The
10T1/2 cells with predominantly nuclear Ca
+2
ion staining represent the subpopulation of
10T1/2 cells with less than 75% of cytoplasmic to total cellular fluorescence.

0
10
20
30
40
0% 25% 50% 75% 100%
Number of Cells (n = 142)
Cytoplasmic Fluorescence
Cytoplasmic Fluorescence of 10T1/2 Cells as
Percentage of Total Fluorescence
39

Figure 30. All MCA Cl 15 cells have cytoplasms representing 75% or more of the total
fluorescence. MCA Cl 15 cells have an average of 89% cytoplasmic to total cellular
fluorescence.

Figure 31. All MCA Cl 16 cells have cytoplasms representing 90% or more of the total
fluorescence. MCA Cl 16 cells have an average of 93% cytoplasmic to total cellular
fluorescence.
0
2
4
6
8
10
0% 25% 50% 75% 100%
Number of Cells (n = 24)
Cytoplasmic Fluorescence
Cytoplasmic Fluorescence of MCA Cl 15 Cells
as Percentage of Total Fluorescence
0
2
4
6
0% 25% 50% 75% 100%
Number of Cells (n = 8)
Cytoplasmic Fluorescence
Cytoplasmic Fluorescence of MCA Cl 16 Cells
as Percentage of Total Fluorescence
40

Figure 32. All NiS3A1 cells have cytoplasms representing 70% or more of the total
fluorescence. NiS3A1 cells have an average of 87% cytoplasmic to total cellular
fluorescence.

Figure 33. All NiS3B1 cells have cytoplasms representing 70% or more of the total
fluorescence. NiS3B1 cells have an average of 84% cytoplasmic to total cellular
fluorescence.

0
10
20
30
40
0% 25% 50% 75% 100%
Number of Cells (n = 97)
Cytoplasmic Fluorescence
Cytoplasmic Fluorescence of NiS3A1 Cells as
Percentage of Total Fluorescence
0
10
20
30
40
0% 25% 50% 75% 100%
Number of Cells (n = 85)
Cytoplasmic Fluorescence
Cytoplasmic Fluorescence of NiS3B1 Cells as
Percentage of Total Fluorescence
41

Figure 34. All NiS7A1 cells have cytoplasms representing 70% or more of the total
fluorescence. NiS7A1 cells have an average of 88% cytoplasmic to total cellular
fluorescence.

Figure 35. All NiO2C3 cells have cytoplasms representing 80% or more of the total
fluorescence. NiO2C3 cells have an average of 91% cytoplasmic to total cellular
fluorescence.

0
6
12
18
24
0% 25% 50% 75% 100%
Number of Cells (n = 62)
Cytoplasmic Fluorescence
Cytoplasmic Fluorescence of NiS7A1 Cells as
Percentage of Total Fluorescence
0
1
2
3
0% 25% 50% 75% 100%
Number of Cells (n = 4)
Cytoplasmic Fluorescence
Cytoplasmic Fluorescence of NiO2C3 Cells as
Percentage of Total Fluorescence
42

Figure 36. (c) The non-transformed cells with Ca
+2
ions mostly in the cytoplasm (c) have
cytoplasmic fluorescence similar to transformed cells. Non-transformed cells with Ca
+2

ions predominantly in the nucleus (n) have a lower percentage of cytoplasmic
fluorescence out of the total fluorescence.

Distribution of Ca
+2
Ion Staining as a Function of Cell Density

To investigate the causes of heterogeneity in Ca
+2
ion distribution in non-
transformed 10T1/2 cells, the effect of cell density on Ca
+2
ion localization was explored.
Varying numbers of 10T1/2 cells, from 500 cells to 64,000 cells, were seeded into
chamber slide wells of 1 ml capacity and incubated for four days. Cells seeded in high
numbers are expected to reach 100% confluence and stop growing by the fourth day,
0%
25%
50%
75%
100%
10T1/2
(all)
10T1/2
(n)
10T1/2
(c)
MCA15 MCA16 NiS3A1 NiS3B1 NiS7A1 NiO2C3
Cytoplasmic Fluorescence
Cell Lines
Cytoplasmic Fluorescence of All Cell Lines as
a Percentage of Total Fluorescence
43

when confocal images are taken. Cells seeded at low numbers are expected to be actively
growing and sub-confluent when confocal images are taken. Thus, cell density is related
to cell growth. In non-transformed 10T1/2 cells, the percentage of cells with
predominantly nuclear Ca
+2
ion staining decreased from 45% down to 14% as cell
density increased (Table 4). This indicated that cells with predominantly nuclear staining
may be actively growing. However, the calcium dynamics did not change with cell
density for transformed cells. The MCA Cl 15 cell line had predominantly cytoplasmic
localization of Ca
+2
ion at all cell densities (Table 5).

44

Table 4. Distribution of Ca
2+
Ion Staining as a Function of density of Cells Seeded

Table 4. Varying numbers of non-transformed 10T1/2 cells were seeded into wells of 1
ml capacity and incubated for four days. Cells with predominantly nuclear staining or
cytoplasmic staining were counted. The percentage of 10T1/2 cells with predominantly
nuclear fluorescence to total number of cells was calculated.

45

Table 5. Distribution of Ca
+2
Ion Staining as a Function of Density of MCA Cl 15 cells
Seeded.

MCA Cl 15
cells/ml
Seeded
Number of Cells
With
Predominantly
Nuclear Staining
Number of Cells
With
Predominantly
Cytoplasmic
Staining Nucleus/Total
500 0 8 0
1000 0 4 0
5000 0 2 0

Table 5. The MCA Cl 15 transformed cell line was seeded into wells of 1 ml capacity
and incubated for four days. Cells with predominantly nuclear fluorescence or
cytoplasmic fluorescence staining were counted. No MCA Cl 15 cells had predominantly
nuclear fluorescence at any cell density.

46

Chapter Four: Discussions

Intracellular Calcium Ion Distributions in Non-Transformed 10T1/2 Cells

Non-transformed 10T1/2 mouse embryo fibroblast cells appear to transport Ca
+2

ions freely between the nucleus and the cytoplasm. Non-transformed 10T1/2 cells have
two states of intracellular Ca
2+
ion distribution. The nuclei of non-transformed 10T1/2
cells can vary from a state where nuclear Ca
+2
ions represent up to 70% of the total
cellular Ca
+2
ions (State 1) to a state of low Ca
+2
ion concentrations in the nucleus (State
2). Non-transformed 10T1/2 cells freely transit between the two states. Similar
observations have been described by other investigators in other biological systems. In a
similar experiment, Bernier et al. used confocal microscopy to determine the
concentration of cytosolic Ca
+2
ions verses the concentration of nuclear Ca
+2
ions in
cultured rat vascular cells (43). They reported some baseline calcium-associated
fluorescence in the cytoplasm but higher Ca
+2
ion levels in the nucleus of some cells.
However, the opposite calcium distribution pattern was seen in other cells (43). Kapur et
al. has also observed heterogeneity in Ca
+2
ion distributions in mouse embryonic stem
cells (mES) (38). Calcium oscillation in mES cells was dependent on the cell cycle
progression of mES cells, where 70% of oscillating cells are in the G1/S phase verses
15% of oscillating cells in the G2/M phase (38). Spontaneous calcium oscillations
occurred in 36% of asynchronous cultured mES cells, which have higher levels of
intracellular calcium than non-oscillating cells (38). The percentage of mES cells
47

undergoing calcium oscillation (36%) closely matches the percentage of mouse embryo
fibroblast cells with predominantly nuclear fluorescence (30%) described in our study.
This suggests a connection between mES cells undergoing calcium oscillation and non-
transformed 10T1/2 cells in State 1.
The majority of asynchronous 10T1/2 cells (68.9% of total cell population) have
greater calcium-associated fluorescence in the cytoplasm than in the nucleus (Figure 8A,
Figure 8B, and Table 3). A smaller population of asynchronous non-transformed 10T1/2
cells (30.3% of total cell population) has higher Ca
+2
ion concentrations in the nucleus
than in the cytoplasm (Figures 8A, Figures 8B, and Table 3). Non-transformed 10T1/2
cells with predominantly nuclear Ca
+2
ions (State 1) are highly variable in terms of
nuclear, cytoplasmic, and total cellular fluorescence. They average twice the
concentration of Ca
+2
ions in the nucleus as 10T1/2 cells containing Ca
+2
ions
predominantly in the cytoplasm. The cells with greater Ca
+2
ions in the nucleus (State 1)
also have higher total cellular fluorescence, suggesting higher levels of total intracellular
Ca
+2
ions. Non-transformed 10T1/2 cells with predominantly cytoplasmic Ca
2+
ion
staining (State 2) make up the majority, 69.7%, of the asynchronous 10T1/2 cell
population. There is less variability in nuclear, cytoplasmic, and total fluorescence in
these cells. Non-transformed 10T1/2 cells in this state (State 2) also resemble MCA-
transformed and nickel compound-transformed cells in terms of Ca
+2
ion levels and Ca
2+

ion localization.

48

Calcium Function in Non-Transformed Cells

The active transport of Ca
+2
ions

between the nucleus and the cytosol is an
integral part of the normal functions of the nucleus. Nuclear Ca
+2
ions are involved in
gene regulation and induction of protein translocation across the nuclear membrane (32).
Many calcium-sensitive enzymes are also present in the nuclei of various cell types (43).
Without oscillations in nuclear calcium levels, the calcium-responsive enzymes, calcium-
regulated genes, and calcium-induced protein translocations would be constitutively
activated or repressed. Therefore, the alternating distribution of Ca
+2
ions in non-
transformed cells between the nucleus and the cytoplasm may be a switch to induce
biological responses critical to the normal functions of the nucleus.
Nuclear Ca
+2
ion mobilization might also be closely correlated with cell cycle
progression. Numerous studies have shown that intracellular Ca
+2
ion signaling is related
to cell growth (12, 33, 38). Ca
+2
ion oscillations accompany the transition from G
0
to G
1

phase and G
1
to S phase in fibroblasts, HeLa, and smooth muscle cells (38). Kapur et al.
reported that spontaneous Ca
2+
ion oscillations occur in 70% of G
1
/S phase mouse
embryonic stem cells but only in 15% of G
2
/M phase cells (38). They proposed that
differential inositol 1,4,5-trisphosphate receptor (IP3R) sensitivity, IP3R nuclear
localization, and IP3R transcription is linked to cell cycle progression (38). IP3R is
expressed in the nuclear envelope where it is involved in nuclear Ca
2+
ion transport (32,
38). Ca
+2
ion signaling would provide a means by which cell growth states can modulate
gene regulation and protein translocation to promote or inhibit cell cycle progression.
49

Cell growth is closely linked to cell density (33). At low cell densities, most cells
are in the proliferative state. At high cell densities (100% confluence), cells are in a non-
proliferative, quiescent state of the cell cycle. Ichikawa et al. reported that intracellular
Ca
+2
ion oscillations in bone marrow stromal cells (BMSC) are influenced by cell density
and cell cycle progression (33). Zhang et al. demonstrated that the IP3R of Madin-Darby
canine kidney (MDCK) cells are localized in the cytoplasm during the sub-confluent,
proliferative state but in the plasma membrane region during the confluent, non-
proliferative state of the cell cycle (12). Koizumi et al. also observed differential IP3R
gene expression in osteoblast cells correlating to cell density (44).
It is possible that cell growth states control Ca
+2
ion mobilization between the
nucleus and the cytoplasm of mouse embryo fibroblast cells as well. We have
investigated the possible relationship between Ca
+2
ion distribution, cell cycle
progression, and cell density in non-transformed, nickel compound-transformed, and
MCA-transformed cells in this study. Images were taken of cells seeded into microscope
chamber slides at varying cell densities. In preliminary studies, we found that at very low
cell densities, approximately 45% of the non-transformed 10T1/2 cells have high levels
of Ca
+2
ion in their nuclei (Table 4). At high cell densities, when the cells are confluent,
only 2% of the cells have high levels of Ca
+2
ions in their nuclei (Table 4). Thus, non-
transformed 10T1/2 cells are more likely to have high Ca
+2
ion levels in the nucleus when
actively proliferating at low cell density.

50

Calcium Distributions in MCA- and Nickel Compound-Transformed Cells

In contrast to non-transformed cells, MCA-transformed and nickel compound-
transformed 10T1/2 cells have consistently higher Ca
+2
ion concentrations in the
cytoplasm than the nucleus. Transformed cells have similar Ca
+2
ion levels and Ca
+2
ion
distributions as non-transformed10T1/2 cells with predominantly cytoplasmic Ca
+2
ion
distributions (State 2). However, the transformed 10T1/2 cells are unable to transition
from State 2 to State 1 (predominantly nuclear Ca
+2
ion staining). Thus, transformed
cells have a much narrower range of nuclear, cytoplasmic, and total fluorescence than
non-transformed 10T1/2 cells. None of the nickel oxide-transformed cells (NiO2C3), the
nickel monosulfide-transformed cells (NiS3A1, NiS3B1, or NiS7A1), or the MCA-
transformed cells (MCA Cl 15 or MCA Cl 16) had nuclear Ca
+2
ion concentrations
exceeding cytoplasmic Ca
2+
ion concentrations. The transformed cell lines generated
from exposure to chemical carcinogens (MCA or nickel compounds) appear to be unable
to sequester Ca
+2
ions in the nucleus to a significant extent. We therefore postulate that
the nickel- and MCA-transformed 10T1/2 cell lines have deficiencies in Ca
+2
ion-induced
nuclear signaling.
It is possible that calcium-regulated gene expression and calcium-induced nuclear
protein translocation would be disconnected from regulation by cell cycle progression
due to deficiencies in Ca
+2
ion-induced nuclear signaling in transformed cells.
Transformed cells have altered Ca
+2
ion distributions under all conditions of cell density.
In preliminary studies, at both low and high cell densities, the transformed MCA Cl 16
cell line consistently has no (0%) cells with heavy nuclear Ca
+2
ion staining (Table 5).
51

This transformed cell line is unable to sequester Ca
+2
ions in the nucleus, even while
actively proliferating under conditions of low cell density. The effect of altered Ca
+2
ion
distribution on cell cycle progression of transformed 10T1/2 cells is unknown.
Other groups have investigated altered calcium homeostasis in cells immediately
after exposure to nickel compounds. Studies have described an initial drop of
intracellular Ca
+2
ion concentrations, which is then followed by an increase in Ca
+2
ion
levels in cells exposed to nickel compounds (45). Denkhaus et al. observed an initial
decrease in the intracellular Ca
+2
ion levels of nickel compound-exposed cells, which
they attribute to the actions of soluble Ni
+2
ions as a calcium channel blockers (45).
Soluble nickel ions compete with calcium for channels thereby reducing the flow of Ca
2+

ions through calcium channels (39). Denkhaus et al. also propose that nickel compounds
may cause aberrant calcium homeostasis by causing calcium release from intracellular
stores. The Ni
+2
ions interact with calcium-sensors or calcium-receptors at the plasma
membrane, induce calcium signaling pathways, and activate Ca
+2
ion release from
intracellular stores (45, 46). Funakoshi et al. also observed an increase of intracellular
calcium concentration in mouse pancreatic cells following nickel exposure (47). Meka et
al. demonstrated that nickel carbonate hydroxide (NiCH) destabilizes calcium
homeostasis in human lymphocytes leading to cell death and induction of DNA single-
stranded breaks (39). Salnikow et al. also observed increases in intracellular calcium
levels following nickel exposure (48). In our study of mouse embryo fibroblast cells,
nickel ion-transformed 10T1/2 cells continue to exhibit altered calcium distributions
many cell generations after exposure to nickel compounds. We propose that nickel
compounds permanently alter calcium homeostasis by causing differential gene
52

expression, including down-regulation of the DRIP-80 gene, and this is perpetuated in the
transformed cell lines. This is the first study detailing the long-term effects of nickel
compound transformation on intracellular Ca
+2
ion gradients.

Calcium Distribution in MCA-Transformed Cells

Particularly puzzling is the aberrant Ca
+2
ion gradient seen in MCA-transformed
cells. Confocal microscopy studies reveal that the normal Ca
+2
ion distribution of 10T1/2
cells is disrupted in MCA-transformed cells and disrupted in nickel compound-
transformed 10T1/2 cells (Figures 8 and 9). However, differential display analyses of
MCA-transformed cells demonstrate that they continue to express the DRIP-80 gene at
levels similar to non-transformed cells but greater than nickel compound-transformed
cells (Figure 2) (2, 8). It is unclear how MCA-transformed cells continue to express the
DRIP-80 gene but nevertheless have aberrant Ca
+2
ion gradients.
One possible explanation is that MCA disrupts DRIP-80 function via mutation
and not gene silencing. MCA is highly mutagenic to bacterial and mammalian cells (49).
Mutational selection by diphtheria toxin of Syrian hamster fetal cells following MCA
exposure showed that MCA associated mutation rates are 17.7 times higher than the
historical control (50). Maddox et al. observed a 15-fold increase in mutation frequency
in transgenic Big Blue rats after MCA exposure (51). In transgenic Big Blue C57/B1
mice, sequence analysis after MCA exposure showed that many G:C to T:A and C:G to
A:T transversions had occurred (49). Therefore, it is possible that MCA-transformed cell
lines continue to express the DRIP-80 gene but in a nonfunctional, mutated form.
53

Nickel Induced DNA Methylation

In contrast to the high mutagenic potential of MCA, carcinogenic metal
compounds are weakly active or entirely inactive when assessed with a wide range of
mammalian and bacterial cell mutagenesis assays (5, 10). Nickel compounds only
induced a weak increase in sister chromatid exchange in Chinese hamster ovary cells (9).
In 10T1/2 mouse embryo fibroblast cells, nickel compounds did not induce mutation to
ouabain resistance (8). The ouabain resistance assay detects stable, specific base
substitution mutations in the gene encoding the large subunit of the (Na, K)-ATPase,
which confer ouabain resistance (8). Thus, the mechanism of DRIP-80 gene inactivation
in nickel compound-transformed cells may be different from that of MCA-transformed
cells.
Since nickel compounds are non-mutagenic or at best weakly mutagenic, nickel-
induced epigenetic effects are likely to be more important to the induction of differential
gene expression during nickel compound-induced neoplastic cell transformation and
carcinogenesis (10). Exposure of a diverse range of mammalian cell lines to nickel
compounds is known to induce extensive DNA hypermethylation and inhibit histone H4
acetylation (10). Nickel ion-generated reactive oxygen species (ROS) are associated with
decreased histone acetylation in human hepatoma cells (10). Nickel ions bind to histones
in heterochromatin regions to cause chromosomal condensation with resultant DNA
methylation of gene promoters, leading to transcriptional quiescence (8, 11, 12). Nickel-
induced silencing of the xanthine-guanine phosphoribosyl transferase (gpt) gene was
54

found to be the result of promoter hypermethylation (52). Aberrant epigenetic regulation
could account for the silencing of the DRIP-80 gene that we observed in nickel
compound-transformed 10T1/2 cells. Promoter methylation might also distinguish
nickel-induced transformation from MCA-induced transformation, because MCA is not
known to cause methylation of DNA. Exploring the hypothesis of nickel compound-
induced methylation of the DRIP-80 gene could lead to a deeper understanding of the
molecular mechanisms of nickel compound-induced neoplastic cell transformation and
how it differs from that of neoplastic cell transformation induced by other carcinogenic
chemicals.

Calcium Transport Mechanisms

At present, the calcium transport mechanism which causes Ca
+2
ion levels to
oscillate between a predominantly nuclear distribution and a cytoplasmic distribution in
non-transformed 10T1/2 cells, but is altered in transformed cells, is unknown. We
propose that nickel ion-induced silencing of the DRIP-80 gene disrupts activity of the
Mediator complex, which in turn disrupts vitamin D-controlled regulation of expression
of specific genes involved in mediating Ca
+2
ion homeostasis in transformed 10T1/2
cells. However, we currently lack a complete understanding of how vitamin D controls
intracellular Ca
+2
ion distribution.
By contrast, the role of vitamin D in bone mineralization, intestinal calcium
absorption, and calcium homeostasis throughout the entire body is well known.
Inactivation of the vitamin D receptor (VDR) gene causes defective intestinal calcium
55

absorption, resulting in hypocalcemia, rickets, and osteomalacia (53, 54). CaBP-D9k
calbindin, a vitamin D-dependent calcium binding protein, is a facilitator of calcium
diffusion across the kidney and intestinal epithelia (53, 54). The vitamin D inducible
TRPV6 calcium channel is located at the brush-border membrane of the intestine, where
it functions in Ca
+2
ion absorption (54). Although these genes are controlled by vitamin
D and modulate Ca
+2
ion distribution, their principal role is the regulation of Ca
+2
ion
homeostasis throughout the body rather than within the cell. Thus, they are unlikely to
account for the intracellular calcium dynamics seen in our work here.
Despite the wealth of knowledge concerning vitamin D-dependent calcium
homeostasis at the organismal level in mammals, little is known about the role of vitamin
D in regulating intracellular Ca
+2
ion levels, particularly levels of Ca
+2
ions in the
nucleus. However, vitamin D is known to induce Ca
+2
ion efflux from intracellular
stores, which activates signal transduction pathways relating to growth inhibition and
programmed cell death (55). Depletion of vitamin D in hepatocytes causes reduced basal
and stimulated intracellular Ca
+2
ion

levels, which also suggests a role for vitamin D in
modulating intracellular calcium concentrations (56).
The nuclear envelope contains all the necessary machinery for regulating nuclear
Ca
+2
ion concentrations independently of the cytoplasm. The nuclear Ca
+2
ion transport
machinery includes a complicated assortment of release channels, calcium pumps, and an
ATP-dependent Ca
+2
ion sequestration system (32, 43). Nuclear Ca
+2
ion signals can be
induced by cyclic ADP-ribose, nicotinic acid-adenine dinucleotide phosphate (NAADP),
and inositol 1,4,5-trisphosphate (IP3) (32). Inositol 1,4,5-trisphosphate receptors (IP3R)
are expressed in the inner and outer nuclear membranes of the nuclear envelop, where
56

they sequester Ca
+2
ions in the nucleoplasm (57). NAADP and cyclic ADP-ribose are
Ca
+2
ion releasing messengers synthesized by ADP-ribosyl cyclase (32). NAADP acts in
conjunction with inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor
(RyR) to generate nuclear Ca
2+
signaling (32). Both IP3R and RyR exist in the inner and
outer membranes of the nuclear envelope (31). IP3R has several isoforms with
distinctive intranuclear distributions and sensitivity to IP3, calcium, and ATP; thereby,
contributing to the versatility of calcium signaling (31).
The existence of three separate Ca
+2
ion release signals (cyclic ADP-ribose,
NAADP, and IP3) as well as multiple IP3R isoforms, provides versatility and complexity
to nuclear Ca
+2
ion transport. This causes difficulty in determining which gene(s) are
differentially expressed by abnormal vitamin D regulation in the nickel compound- and
MCA-transformed cells, and how this would lead to aberrant Ca
+2
ion distributions.
Furthermore, the genes controlling nuclear calcium mobilization are not associated with
known vitamin D regulatory elements (VDRE), so it is unclear whether they are regulated
by vitamin D. However, only a few gene promoters with associated VDREs have been
identified to date, and vitamin D is known to effect intracellular calcium (37, 55, 56).
Therefore, it remains possible for proteins regulating nuclear Ca
+2
ion levels to be
transcriptionally controlled by vitamin D (37). Further investigation is needed to
understand exactly how nuclear calcium transport is disrupted by DRIP-80 gene
silencing.

57

Effects of DRIP-80 Repression on Transcription

Repression of DRIP-80 gene expression would likely have a substantial impact on
regulation of gene expression and cell physiology. DRIP-80 silencing can effect the
regulation of genes directly through Mediator complex inactivation, through alterations in
vitamin D regulation of gene expression, or indirectly through abnormal distribution of
intracellular Ca
+2
ions. The Mediator complex is a binding partner for a broad range of
nuclear receptors and other regulatory factors that modulate the transcriptional activation
or repression of genes (23). Inactivation of the Mediator complex with a DRIP-80
temperature-sensitive mutation is known to reduce CHA1 gene expression and may cause
aberrant expression of many other genes (58). Thus, Mediator regulates basal and
inducible transcription of many genes, and inactivation of Mediator complex will likely
have substantial effects on transcription in general (27).
Inactivation of the Mediator complex would consequently disrupt the vitamin D
genomic response and could likely contribute to the transformed phenotype. In fact,
numerous vitamin D-responsive genes have been associated with cell proliferation and
carcinogenesis. Vitamin D induces apoptosis in breast, colon, and glioma cancer cell
lines via down-regulation of the Bcl-2 gene, which is involved in calcium signaling in the
endoplasmic reticulum (55, 59). Expression of the osteocalcin gene is controlled by a
vitamin D response element (VDRE), and aberrant regulation of this gene is associated
with tumorigenicity and metastasis (60, 61). Vitamin D response elements are also
associated with p21and Mad1 promoters, and their up-regulation prevents cells from
passing through G
0
and G
1
phases of the cell cycle, thereby causing cellular
58

differentiation (37). Vitamin D also modulates expression of the c-fos and c-jun
differentiation-associated proto-oncogenes, which have roles in apoptosis (55, 62).
Finally, vitamin D-induced repression of the c-myc proto-oncogene leads to cells
accumulating the G
2
and G
1
phases of the cell cycle (55). Thus, DRIP-80 gene silencing
potentially disrupts the normal expression of numerous vitamin D-responsive genes
associated with cell cycle control, cell proliferation, and cancer.
The spatiotemporal pattern of Ca
+2
ion oscillation is linked to the regulation of
gene transcription (33). Ca
+2
ions also regulate transcription of certain cancer-associated
genes. Transcription factors like NFAT and CaM kinase II are activated by Ca
+2
ion
oscillation frequencies between 0.66 oscillations/minute and 2 oscillations/minute (32).
NFAT activation is required for chemical carcinogens to induce tumor promotion (10).
The Cap43 gene is over-expressed in colon cancer and nickel compound-transformed
human lung A549 cells (30). Cap43 gene expression is activated by elevations in
intracellular Ca
+2
ions specific to nickel compounds and not other metal carcinogens (30).
Ca
+2
ion signaling also modulates nuclear translocation of FOXO, a transcription factor
and tumor suppressor (63). Since Ca
+2
ions function as important signaling molecules,
the abnormal distribution of Ca
+2
ions in nickel compound-transformed and MCA-
transformed cells would be expected to have severe consequences on cell function,
including effects on regulation of gene expression.
The effect of DRIP-80 gene inactivation on Mediator complex-, vitamin D-, and
calcium-mediated gene regulation is likely to result in aberrant expression of a multitude
of genes. It is estimated that approximately 130 genes are differentially expressed
between nickel compound-transformed cells or MCA-transformed cells and non-
59

transformed 10T1/2 cells (3, 8). How many of these genes are abnormally expressed due
to nonfunctional Mediator complex or inactivated vitamin D and calcium regulatory
pathways remain to be determined. The effects of genome-wide deregulation caused by
DRIP-80 gene repression on cancer development are difficult to predict. We expect to
determine this by utilizing DNA microarrays in the future.

Tissue Specific Effects DRIP-80 Repression

The vitamin D pathway is vital for intestinal calcium absorption. Indeed,
mutations to the VDR cause defective calcium absorption attributed to hereditary vitamin
D-resistant rickets (HVDRR) (29). Considering how indispensible vitamin D is to
intestinal calcium absorption, carcinogenic nickel compounds are particularly toxic if
ingested. Studies have shown that ingestion is one of the main routes of exposure to
nickel compounds (10). Oral intake of nickel compounds may occur through ingestion of
nickel-containing foods such as spinach, cocoa, and nuts (10). Nickel compound-induced
silencing of the DRIP-80 gene and aberrant vitamin D regulation of calcium transport
genes of intestinal cells could render the cells unable to absorb dietary calcium resulting
in hypocalcaemia.
Numerous studies have shown that inhalation of carcinogenic nickel compounds
is associated with lung, pharyngeal, and nasal cancers (8, 10, 64-69). However, the effect
of DRIP-80 gene silencing on lung tissue remains to be elucidated. The N-terminal
domain of VDR has tissue-specific variants, suggesting that vitamin D has different
functions depending on tissue-type (70). The VDR is expressed in 30 different tissues
60

including the brain, liver, breast, cardiac muscle, pancreas, pituitary, skin, placenta, and
immune cells (55, 71). Only the intestine, bone, kidney, and parathyroid have known
roles in calcium handling and calcium homeostasis (71). The function of vitamin D
signaling in the remaining tissues has only begun to be explored (55). This leaves open
the possibility of unexpected roles for vitamin D, such as regulation of intracellular
calcium homeostasis in a variety of tissues. Another ubiquitously expressed vitamin D-
regulated gene is the CaBP-D9k calbindin gene. The CaBP-D9k protein is expressed in
the kidney and intestine where it functions in Ca
+2
ion absorption (72). However, it is
also expressed the brain, uterus, placenta, lung, and pancreas where it has uncertain
functions (72). The heterogeneity in the vitamin D response may be tissue-specific and
cell type-specific as well as controlled by development, aging, and in disease states (37).
Thus, the pleiotropic effects of vitamin D in a variety of tissues should be considered.
Although nickel compound exposure is known to cause lung-specific effects, the
consequences of DRIP-80 gene repression on vitamin D and calcium regulation in lung
tissue, as well as other tissues, remain to be investigated.

61

Chapter Five: Conclusions

The widespread commercial usage of nickel compounds combined with mounting
evidence of their adverse impact on human health makes the investigation of nickel
carcinogenesis an area of intense interest. In particular, the role of DRIP-80 gene
repression in altering intracellular Ca
+2
ion gradients merits further exploration because it
is down-regulated by two nickel compounds, nickel oxide and nickel monosulfide. The
consequence of DRIP-80 gene repression is the inactivation of both calcium and vitamin
D pathways. Both calcium and vitamin D possess a diverse array of cellular functions,
such as regulation of gene expression, and are closely associated with cancer
development. Thus, DRIP-80 gene repression greatly disrupts normal cell function and
contributes to the induction and maintenance of carcinogenesis.
This study examines the intracellular Ca
+2
ion distribution in mouse embryo
fibroblast cells exposed to the chemical carcinogens green nickel oxide, crystalline nickel
monosulfide, and MCA. All transformed cell lines exhibit altered Ca
+2
ion transport.
Non-transformed cells are capable of intracellular Ca
+2
ion fluctuations closely tied to
normal cellular functions. Non-transformed 10T1/2 cells can switch from a state of low
calcium concentration in the nucleus (State 2) to a state of much higher nuclear calcium
concentration (State 1). However, 10T1/2 cells exposed to nickel oxide, nickel
monosulfide, and MCA have lost this ability and never have substantial concentrations of
Ca
+2
ions in the nucleus. The aberrant calcium localization in transformed cells will have
physiological consequences which are only beginning to be understood.
62

This study has made significant progress toward understanding the biological
effects of nickel ion-induced silencing of the DRIP-80 gene in the context of nickel ion-
induced morphological and neoplastic cell transformation. However, many questions still
remain to be answered. Firstly, it is unclear which DRIP-80 regulated calcium signaling
gene(s) is(are) de-regulated in transformed cells. Secondly, the altered Ca
+2
ion
distribution of the MCA-transformed cell lines is difficult to explain considering their
continued expression of the DRIP-80 gene. We hypothesize that in the MCA-
transformed cell lines, there may be a mutational inactivation of the DRIP-80 gene.
Lastly, the molecular switch causing the transition of non-transformed 10T1/2 cells from
a state of higher Ca
+2
ion concentration in the nucleus (State 1) to a state of low nuclear
Ca
2+
ion levels (State 2) remains to be elucidated. We hypothesize that transition of non-
transformed 10T1/2 cells between State 1 and State 2 is regulated by cell cycle
progression.
A deeper understanding of these mechanisms is important for providing insight
into the role of Ca
+2
ion gradients in normal cellular physiology, the effects of nickel ion-
induced silencing of the DRIP-80 gene on disruption of normal Ca
+2
ion gradients, and
the molecular mechanisms of Ni
+2
ion carcinogenesis.

63

Chapter Six: Future Directions

In the future, we plan to analyze DRIP-80 at the DNA, RNA, and protein levels to
provide insight into the mechanism by which expression of the DRIP-80 gene has been
repressed. We currently hypothesize that the promoter of the DRIP-80 gene has been
methylated, rendering it transcriptionally inactive. To test this hypothesis, we will
conduct Southern blot analysis using the isoschizomers, MspI, which is insensitive to
methylation of the DNA site, and methyl-sensitive HpaII (76). The results of this
Southern blotting analysis will determine whether there is DNA methylation at the
promoter region of the DRIP-80 gene. If promoter methylation of the DRIP-80 gene has
occurred, then it is like responsible for silencing of the DRIP-80 gene. Sequential
confirmatory Northern blotting analysis should then reveal an absence of DRIP-80
mRNA in nickel compound-transformed cells but not in non-transformed 10T1/2 cells or
in MCA-transformed 10T1/2 cells, since this is what we found by mRNA differential
display analysis (2). Northern blot analysis is expected to prove that repression of
expression of the DRIP-80 gene occurs at the transcriptional level. Finally, we propose
to conduct Western blotting analysis with the DRIP-80 gene, to determine whether DRIP-
80 protein is present or absent (most likely) in the nickel compound-transformed and
MCA-transformed 10T1/2 cell lines compared to the status of its expression in the non-
transformed 10T1/2 cell lines.
A second hypothesis we will test is that the DRIP-80 gene itself is absent in
transformed cells, perhaps due to chromosomal breakage and loss of the chromosome(s)
bearing this gene. This hypothesis will also be tested by conducting Southern gel
64

analysis and looking for the presence of the DRIP-80 gene in the nickel ion-transformed
and MCA-transformed 10T1/2 cell lines, compared to the presence of this gene in the
non-transformed 10T1/2 cell lines.
We also plan to conduct confocal microscopy studies in conjunction with cell
synchronization to test the hypothesis that intracellular distribution of Ca
2+
ions is a
function of cell cycle progression. Using methods described by McCormick et al., cells
will be synchronized by isoleucine deprivation for 24 hours, and then initiated DNA
synthesis will be initiated 7.5 hours following the release of the isoleucine block (73).
Confocal microscopy images will be taken of the non-transformed and transformed cells
at 7.5 hours after the release of the isoleucine block when the cells are at the G1/S
boundary. Images will also be collected at 16-18 hours after release from isoleucine
deprivation when the cells are in late S phase. By analyzing a homogeneous,
synchronized population of cells, it will be possible to determine whether the cellular
distribution of Ca
2+
ions is regulated by cell cycle progression.
We also plan to quantitatively determine the concentrations of Ca
+2
ions within
the cytosol and nucleus of living transformed and non-transformed 10T1/2 cells. Fluo 3-
AM-associated fluorescence alone is not sufficient for calculating intracellular Ca
+2
ion
concentrations. However, by using the maximal and minimal fluorescence obtained in
the cells, Ca
+2
ion concentrations can be calculated using the equation: [Ca
+2
] = K
d
(F -
F
min
)/(F
max
- F) where K
d
is the dissociation constant for Fluo 3-AM (320 nM) and F is
the observed fluorescence of Fluo 3-AM (43). Using methods described by Burnier et
al., a non-fluorescent calcium ionophore in the presence of extracellular calcium can be
used to saturate the intracellular calcium dye to obtain a maximal fluorescence (43).
65

Minimal fluorescence can be measured using EDTA. A second approach to determining
intracellular calcium concentrations is to use the Fura-2 fluorescent ratiometric calcium
dye. Calcium concentration is calculated based on the ratio of fluorescence intensities at
505 nm when Fura-2 is excited at 340 nm and 380 nm (74).
To confirm that the fluorescence we attributed to nuclear calcium correctly
corresponds with the location of nucleus, Fluo 3-AM calcium dye will be used in
conjunction with a dye that stains nuclei. Counter-staining the nuclei of living cells can
be accomplished with either 4,6-diamidino-2-phenylindole (DAPI) or Hoechst 33342
dyes (75). Co-localization of Fluo 3-AM/calcium associated fluorescence with signals
from DAPI or Hoechst dye would prove that the fluorescence attributed to nuclear
calcium lies within the area of nucleus. To further investigate the localization of Ca
2+
ion
in the cytoplasm, MitoTracker will be used to demonstrate whether cytosolic Ca
2+
ion is
co-localized with mitochondria (77).
Gene transfection and RNA interference studies will also be conducted in the
future by our laboratory to test our hypothesis that the differential Ca
+2
ion distribution
between nickel compound-transformed, MCA-transformed, and non-transformed 10T1/2
cells is a direct consequence of DRIP-80 gene repression. We hypothesize that
transfecting a copy of the wild-type DRIP-80 gene into nickel compound-transformed
and MCA-transformed 10T1/2 cells should restore the calcium mobilization properties of
the transfected, transformed cell lines to those of non-transformed 10T1/2 cells. In
contrast, our other hypothesis is that DRIP-80 gene knockdown with RNA interference
technology in non-transformed 10T1/2 cells is expected to cause the non-transformed
10T1/2 cells to assume the transformed phenotype. These studies should conclusively
66

prove the asserted relationship between DRIP-80 gene silencing and the altered Ca
+2
ion
distribution seen in this experiment.

67

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Abstract (if available)

Abstract 10T1/2 mouse cells were treated with nickel oxide, nickel monosulfide, and 3-methylcholanthrene to establish transformed cell lines. Non-transformed 10T1/2 cells expressed the vitamin D receptor interacting protein 80 (DRIP-80), while nickel ion-transformed cell lines did not. The DRIP-80 protein is a subunit of the Mediator complex, which regulates Ca+2 ion distribution via regulation of vitamin D responsive genes. Disruption of DRIP-80 gene expression may result in an aberrant distribution of Ca+2 ions. To test this hypothesis, non-transformed 10T1/2 cells and transformed 10T1/2 cell lines were stained with Fluo 3-AM and visualized by confocal microscopy. The distribution of Ca+2 ions in non-transformed 10T1/2 cells was heterogeneous. Nickel compound- and MCA-transformed 10T1/2 cells have consistently less Ca+2 ions in the nucleus than the cytoplasm. These results suggest that disruption of normal transport of Ca+2 ions between nucleus and cytoplasm may contribute to the altered phenotypes in transformed 10T1/2 cell lines.

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Creator Mai, Duy (author)

Core Title Silencing of expression of the DRIP-80 gene correlates with aberrations in calcium ion distribution in nickel compound- and 3-methylcholanthrene-transformed C3H/10T1/2 mouse embryo fibroblast cel...

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology

Publication Date 10/15/2009

Defense Date 09/02/2009

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

Tag 3-methylcholanthrene,C3H/10T1/2 mouse embryo fibroblast,calcium ion distribution,DRIP-80,nickel monosulfide,nickel oxide,OAI-PMH Harvest

Language English

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Advisor Landolph, Joseph R., Jr. (committee chair), Schönthal, Axel (committee member), Ying, Shao-Yao (committee member)

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