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Activin-mediated growth inhibition in prostate cancer LNCaP cells by transcriptional regulation of apoptosis-related genes

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Activin-mediated growth inhibition in prostate cancer LNCaP cells by transcriptional regulation of apoptosis-related genes

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ACTIVTN-MEDIATED GROWTH INHIBITION IN PROSTATE
CANCER LNCAP CELLS BY TRANSCRIPTIONAL REGULATION
OF APOPTOSIS-RELATED GENES
by
Hannah Mun-Ly Lui
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
(Cell and Neurobiology)
December 1999
Copyright 1999 Hannah Mun-Ly Lui
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UNIVERSITY O F SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFORNIA S 0 0 0 7
This thesis, written by
___________ Hannah Mun-Ly Lui ___________
under the direction of h^.K.. Thesis Committee,
and approved by all its members, has been pre
sented to and accepted by the Dean of The
Graduate School, in partial fulfillment of the
requirements for the degree of
M aster o f S c ie n c e
Da/E..5.ep.t.eiahfiX..13^...13 9 9
THESIS COMMITTEE
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TABLE OF CONTENTS
Acknowledgements iii
List of Figures iv
List of Abbreviations v
Abstract viii
L Introduction
The prostate and its dependence on hormones and growth factors 1
The LNCaP cell line 3
Activin and activin signal transduction 4
Activin as a cell growth regulator 10
Transcriptional regulation o f cell cycle genes 1 1
Transcriptional regulation o f apoptosis genes 17
n . Methods
Cell culture 22
Northern blot 22
Probe synthesis 23
Single-cell cDNA library amplification (ScLA) 23
Reverse transcription-polymerase chain reaction (RT-PCR) 24
m . Results
Northern blot 23
Single cell cDNA library amplification (ScLA) and reverse transcription-
polymerase chain reaction (RT-PCR) 26
Comparison o f mRNA expression for cell cycle-related genes 28
Activin regulates some apoptosis-related genes 29
Time-dependent expression o f bcl-xi after activin treatment 30
IV. Discussion 31
Summary 36
V. References 38
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Acknowledgements
My most sincere gratitude goes to the individuals who helped me in the process and
completion of this study:
Dr. Shao-Yao Yiag, my thesis advisor and mentor, who offered die opportunity to learn
and study in his laboratory under his bountiful guidance, patience, and enthusiasm;
Mr. Shl-Lung Lin, who taught me numerous technical procedures, including PCR,
ScLA, and subtractive hybridization, thus enabling me to carry out experiments;
Dr. Zhong Zhang, who taught me how to do a Northern blot, which was the first
technique I learned in die lab;
Dr. Charles Haun and Dr. Richard Wood, who generously served on my thesis
committee, offering me insightful advice on how to improve the quality of the study; and
Mr. Dong Jun John Park, for his limidess encouragement and support
I am fortunate to have learned from your collective wisdom, experience, insight, and
generosity. Thankyou.
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List of Figures
1. Activin signal transduction pathway 8
2. Rb tumor suppressor pathway 12
3. Apoptosis pathway 18
4. Northern gel electrophoresis 25
5. cDNA libraries generated by ScLA 26
6. GAPDH PCR product 27
7. Generation o f probes by RT-PCR 27
8. Expression o f cell cycle-related genes 28
9. Expression o f apoptosis-related genes 29
10. Expression o f bcl-xi 30
11. Proposed mechanisms of cell growth inhibition and
suppression of metastasis by activin 36
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List of Abbreviations
Apaf-1 apoptosis activating factor-1
A R I activin receptor type I
A R II activin receptor type Q
ARE activin response element
ARF activin response factor
bcl-2 b*cell lymphoma gene
BMP bone morphogenetic protein
BPH benign prostatic hypertrophy
CDK cyclin-dependent kinase
CKI CDK inhibitor
COS monkey kidney cell line
DHT dihydrotestosterone
ECM extracellular matrix
EGF epidermal growth factor
FAST-1 forkhead activin signal transducer-1
FBE FAST-1 binding element
FGF fibroblast growth factor
FSH follicle stimulating hormone
GAPDH glyceraldehyde-3-phosphate
HaCaT human keratinocyte cell line
HepG2 human hepatoma cell line
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IFN a interferon a
IFNy interferon y
IG F insulin-like growth factor
LHRH luteinizing hormone releasing hormone
LNCaP lymph node carcinoma of the prostate cell line
LOH loss of heterozygocity
MEL murine erythroleukemia cell line
MH1 Mad homology domain-1
MH2 Mad homology domain-2
NCAM neural cell adhesion molecule
PAI-1 plasminogen activator inhibitor-1
PCNA proliferating cell nuclear antigen
PCR polymerase chain reaction
PMA 1 6-phorboI-1 2-myristate 13-acetate
RT-PCR reverse transcription-polymerase chain reaction
SBE Smad binding element
SCID severe combined immunodeficient
ScLA single cell cDNA library amplification
SID Smad interacting domain
PAP prostatic acid phosphatase
Rb retinoblastoma gene
PSA prostate specific antigen
TG Fa transforming growth factor a
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TGFP transfonnng growth factor P
TNFa tumor necrosis factor a
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Abstract
Activin A has been shown to inhibit cell proliferation and enhance apoptosis in a variety
o f cells, including prostate cancer LNCaP cells. However, the mechanisms by which this
growth inhibition occur are not understood. Genes known to be involved in cell cycle
regulation and apoptosis were examined for transcriptional regulation by activin. Cells
treated with activin for 12 h showed decreased bcl-xi mRNA expression and increased
bcl-x, and Fas mRNA expression. However, there was no significant difference in
mRNA expression for other genes studied, including p53, Rb, p21, bcl-2, apoptosin,
TNFa, and Apaf-1. These results indicate that activin-induced cell growth inhibition
may be mediated, at least in part, by transcriptional regulation o f bcl-xi, bcl-x,, and Fas.
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L INTRODUCTION
The Centers for Disease Control and Prevention states:
Prostate cancer is the most commonly diagnosed form of cancer, other
than sldn cancer, among men in the United States and is second only to
lung cancer as a cause of cancer-related death. The American Cancer
Society estimates that 179,300 new cases of prostate cancer will be
diagnosed and that approximately 37,000 men will die of the disease in
1999(1).
The Prostate and its dependence on hormones and growth factors
The prostate, the largest accessory reproductive gland in the male, is an exocrine gland
consisting o f epithelial cells arranged in acini, surrounded by stromal cells, including
fibroblasts and smooth muscle cells. Adenocarcinoma, or carcinoma of die acini,
accounts for 98% of all neoplasms o f the prostate. Histological indications o f
adenocarcinoma include nuclear annplam a, architectural alterations, enlarged round and
vesicular nuclei, crowding and irregularity of die distribution and shape o f acini, and
papillary, ductal, and cribriform growth patterns (2).
Cell division in the prostate is primarily under androgen control (3). Testosterone, die
major circulating human androgen, diffuses into prostatic epithelium, where it is
converted to its more active form, dihydrotestosterone (DHT). DHT binds to die
androgen receptor and the androgen/receptor complex translocates to the nucleus for
transactivation of androgen-responsive genes, including those which control cell division
(4).
1
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Current treatments o f prostate cancer include radical prostatectomy, irradiation, gene
therapy, and hormonal manipulation. Hormonal management of prostate cancer is based
on suppressing androgen production or neutralizing androgenic effects at target cells,
such as by bilateral orchiectomy (castration), administration o f estrogens, LHRH
agonists, or anti-androgens. While initial tumor regression frequently occurs, patients
will relapse into a hormonally unresponsive state. This relapse is thought to be due to
the presence o f androgen-independent cells in the prostate (S). Other growth factors
which contribute to prostatic epithelial cell growth include epidermal growth factor
(EGF), transforming growth factor-a (TGFa), fibroblast growth factors (FGFs), and
insulin-like growth factors (IGFs) (6). Inhibitors of prostatic epithelial cell growth
include transforming growth factor-p (TGFP) and its family member, activin (7).
Because {nostate cancer develops into an androgen-independent state, it is worthwhile to
study growth factors which inhibit prostatic epithelial ceil growth. The goal of this
investigation was to study the molecular mechanisms by which activin exerts its cell
growth inhibition in prostate epithelial cells. Understanding such mechanisms may
advance our current knowledge of hormone/growth factor interaction in the prostate and
mechanisms of cell growth inhibition and apoptosis, possibly leading to the development
of methods to treat or prevent the development of some types o f cancer.
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The LNCaP cell line
LNCaP cells were derived in 1977 from fragments of a biopsied lymph node metastasis
from a 50-year-old man diagnosed one year earlier with a moderately differentiated
prostate cancer. The cells secrete growth factors such as epidermal growth factor (EGF),
transforming growth factor a (TGFa), insulin-like growth factor (IGF), and activin, and
also express their respective receptors. They do not, however, express detectable
amounts of TGFfi or TGFf) receptor. LNCaP cells retain characteristics of their prostatic
epithelial origin, such as the expression of prostate specific antigen (PSA), prostatic
acidic phosphatase (PAP), androgen receptors, and responsiveness to androgen’s
mitogenic effect (8).
Nonmetastasizing tumors formed when LNCaP cells were injected subcutaneously into
athymic nude mice (9). Recently, a metastatic prostate cancer model was developed by
Sato et al. who injected LNCaP cells intp the prostate gland of severe combined
immunodeficient (SCID) mice. These tumors developed grossly evident retroperitoneal
lymph node metastases 3 months after tumor inoculation (10). Metastatic tumor
formation by LNCaP cells is an extremely useful model because LNCaP cells are the
only prostate cell line established with functional androgen receptor and PSA expression
( 10).
LNCaP cells have wild-type p53 and pRb genes, and the concomitant presence of
pl6/CDK4 and cyclin/CDK4 protein complexes, making the cells a suitable model to
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study the G l/S restriction point in cell cycle regulation in cancer cells (11). For
example, a study by Lu et al. determined that androgens, which are mitogenic in LNCaP
cells, caused upregulation o f CDK2 and CDK4 and downregulation of pl6 as detected
by Northern blot and Western blot analyses (12).
Activin and activin signal transduction
Activin A, a member of the transforming growth factor-p (TGFP) superfamily, is a
polypeptide originally isolated from ovarian follicular fluid based on its ability to
enhance the pituitary secretion of follicle stimulating hormone (FSH) (13). Activin
exhibits other activities in different biological systems, including erythrocyte
differentiation, nerve cell survival, induction of embryonic Xenopus laevis mesoderm,
promotion o f bone growth, and somatostatin induction (14,15). Activin expression has
been detected in cell lines derived from various human tissues, including prostate cancer
(16-18), breast cancer (19,20), retinoblastoma (21), retinal pigment epithelium (22),
placenta (23), gut (24), and bone marrow stroma (25), as well as a number of in vivo
human tissues, including the testis (26), ovarian tumor (27), endometrium (28), placenta
(29), oocyte (30), adrenal gland (31), pancreas (32), and bone marrow stroma (33).
Among the diverse biological activities associated with activin are an inhibition of cell
growth and proliferation, and enhancement of apoptosis in some cell types (13,15).
Activins A (Pa Pa ), B (Pb Pb ). and AB (PA Pn) (13) are 28-kDa disulfide-linked dimers of
inhibin PA and/or PB subunits. They have a nine-cysteine distribution pattern which is
4
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similar to TGFP and other members o f the TGFf) superfamily {e.g., inhibin, bone
morphogenetic protein [BMP]) (34). Inhibin, besides having other functions, opposes
activin by inhibiting pituitary FSH secretion (13). BMP induces cartilage and bone
formation during embryonic development and postnatally (33). TGFP acts on a variety
of cell types, affecting a broad range o f cellular responses, including differentiation,
adhesion, and proliferation (34). Between all members of the TGFP superfamily,
homology in overall amino acid sequence within the C-terminal region ranges from 25-
80% (36).
Activin receptors
The receptors for activin are similar to those for other members of the TGFP superfamily
in that there are type II ligand-binding receptors and type I signal-transducing receptors
(37). Activin receptor I (AR I) (30-35 lcDa) and activin receptor n (AR II) (70-75 kDa)
are transmembrane serine-threonine lrinase receptors that are both required for signal
transduction (38). Their structures include a small extracellular ligand-binding domain,
a single membrane-spanning domain, and an intracellular protein kinase domain
adjoining a small serine/threonine-rich region (15). Using immunoprecipitation, AR I
and AR II have been observed to form a noncovalent heteromeric complex; functionally,
AR I was observed to require the presence of AR II to associate with the ligand (15). AR
expression has been detected in cell lines from various tissues, including prostate cancer
(16-18), breast cancer (19), keratinocytes (39), erythroleukemia (40), osteoblasts (41),
5
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and teratocarcinoma (42), as well as in the in vivo human prostate (43), ovary (44), brain
tumor (45), oocyte (30), placenta (46), and pancreatic cancer (47).
Activin signal transduction
In die current model o f activin signal transduction (13), activin binds to AR II at the
plasma membrane and activates its serine-threonine kinase, enabling it to phosphorylate
a glycine- and serine- rich (GS) domain on AR I, in turn activating its kinase activity.
AR I can thereby phosphorylate one or more members o f a family of recently identified
50-60 kDa cytoplasmic factors called Smads, and these Smads carry the signal into the
nucleus.
Nine Smads have been identified thus far (48,49). Smads 1-3 have conserved regions,
in their N-tcrminus Mad-homology-1 (MH1) domain, and C-terminus Mad-homology-2
(MH2) domain, between which lies a dissimilar serine-threonine region (50). In the
current model of activin signal transduction, activated AR I phosphorylates the serine-
rich C-terminus of Smad 2 which then forms a heteromeric complex with Smad 4 (49).
Smad 4 lacks the C terminal serine rich motif and is not phosphorylated; however, its N-
terminal MH1 domain specifically binds DNA (51). This complex translocates to the
nucleus to activate or repress gene transcription (49,50) (Fig. 1). The significance of
Smads as intracellular mediators o f activin has been demonstrated by studies wherein
overexpression of Smad proteins induced activin-like responses (e.g., transcriptional
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activation of the activin-responsive reporter construct p3TP-Lux, induction of Xenopus
dorsal mesoderm) in different cell lines (52,53).
Mutations in Smad 2 and Smad 4 have been identified in human cancers (pancreatic,
colon, and lung), providing an explanation for nonresponsiveness to TGFp/activin in
some cancers and cancer cells (54,55). Interactions between Smad proteins and
transcription factors have been studied in the context of activin response elements
(AREs) present in several gene promoters. One such promoter is that of Mix. 2, an
immediate-early response gene that is expressed in the prospective mesoderm and
endoderm during Xenopus development which is specific to activin-like members o f the
TGFP superfamily (48). In this context, Smad 2 binds to FAST-1 (forkhead activin
signal transducer-1), a member of the winged-helix transcription factor family which
acts as a coactivator at its C-terminal domain, termed Smad-interacting domain (SID)
(37). Along with Smad 4, all are components o f the activin responsive factor (ARF)
which binds to specific repeats in die ARE o f the Mix.2 promoter (56). Several regions
in target gene promoters have been identified as Smad binding elements (SBE) and
FAST-1 binding elements (FBE). Zawel et al. identified the sequence 5*-GTCTAGAC-
3’ as an SBE to which Smad 3 and Smad 4 specifically bind (51). When inserted
upstream of a minimal promoter adjoining a luciferase reporter in human keratinocyte
HaCaT cells, the SBE was induced 20- to 25- fold when treated with TGF0.
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I Ligand binding
activin
»
2 .Smad phosphorylation
.i-'L S W
Smad 2 (
I
&nad2 ?
S m ad 4
3Keteromenc Smad
complex formation
4.Nuclear tnmslocation
P
2 j Sm ad 4
5 .FAST-1 binding
and sequence-
specific DNA
binding
FAST-1
iBE
FAST-1
6.Co-factor
binding and
transcriptional .
activation or I
repression . ▼
▼ Transcription o f genes for cell
proliferation, anti-apoptosis,
angiogenesis
OR
FAST-1
^ Transcription o f genes for tumor
suppression, apoptosis
Fig.1 Activin signal transdncden pathway. l)Activin binds to cell membrane receptors at cell
membrane and activates phosphorylation. 2) Receptor phosphorylates pathway-restricted Smad 2.3)
Phosphorylated Smad 2 binds to common-pathway Smad 4.4) Heteromeric Smad complex translocates
into the nucleus, where it binds FAST-1. 5) Complex binds DNA at specific sequences: SBE=Smad
binding element, FBE=Fast-1 binding element. 6) An additional co-factor binds, either activating or
repressing gene transcription.
An FBE (5 ’-TGT(G/TXT/G)ATT-3 ’) was identified in the ARE of the Mix. 2 promoter to
which human FAST-1, complexed with Smad 2 and Smad 4, binds (57). When inserted
adjacent to an SBE (51) upstream o f a minimal promoter and luciferase reporter and
transfected into HaCaT cells, striking activation was observed that was dependent on the
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presence of TGFp and FAST-1. These elements suggest a model o f transcriptional
regulation in which both the FBE and SBE contribute to the specificity of modulation by
TGFp/activin.
A different SBE, composed of the sequence CAGACA, was identified in the
TGFp/activin-inducible junB promoter in HepG2 hepatoma cells (58). A nuclear
complex containing Smads 3 and 4 bound to the SBEs o f the junB promoter, thereby
activating it Co-overexpression o f Smads 3 and 4 was sufficient to transactivate two
SBEs, and multimerization of SBEs created a powerful TGFp/activin- inducible enhancer
with high Smad-binding affinity (58). However, the junB promoter was not
transactivated by FAST-1 in HepG2 cells, in contrast to the Xenopus Mix. 2 model (58).
A Smad binding site containing a monomeric CAGACA sequence was also identified in
the TGFP-responsive promoter o f the p3TP-Lux construct in murine leukemia MvlLu
cells and the TGFP-inducible PAI-1 promoter in monkey kidney COS cells (59). A
similar CAGA box was identified in the TGFP-inducible plasminogen activator inhibitor-
1 (PAI-1) promoter as well as in many other TGFp-mediated gene promoters (60).
Identification of the SBE as a binding sequence for Smad proteins and the ARE as a
binding sequence for ARF may elucidate investigations on the downstream effectors of
the activin signal. For example, alterations in the SBE or ARE may be involved in
nonresponsiveness to activin or TGFP of some cells or cancers.
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Activin as a cell growth regulator
Activin has been demonstrated to inhibit cell growth in many human cell types,
including prostate cancer (61), breast (20,62), B cell leukemia (63), vascular endothelial
(64), vascular smooth muscle (65), peripheral blood granulocyte-macrophage colony-
forming unit progenitors (66), and fetal adrenal (31), as well as HS-72 mouse B cell
hybridoma (67), mouse plasmacytoma (68), Balb/c 3T3 mouse fibroblasts (69), rat liver
(70), and rodent hepatocytes (71,72). However, the mechanism(s) by which activin
exerts its inhibitory effects are largely unknown.
Activin as a cell growth regulator in the prostate
Activin is expressed in the normal prostate gland, as well as in the hypertrophic, benign
hyperplastic, and cancerous states (73). The concomitant expression of activin and
activin receptors detected by in situ hybridization, RT-PCR, and immunocytochemistry
in the normal rat prostate and in prostate cell lines indicates that activin acts as an
autocrine as well as an endocrine growth factor (38). It was shown in our and other
laboratories that treatment with activin results in decreased cell growth, upregulation of
the prostatic differentiation markers prostate-specific antigen (PSA) and prostate acidic
phosphatase (PAP) (61), and overexpression o f activin inhibits growth and induces
apoptosis in these cells (74). The growth inhibitory response to activin is dose-
dependent and time-dependent, with increasingly effective concentrations ranging from 1
ng/ml (40% decrease) to 100 ng/ml (80% decrease) (61), and results were observed from
10
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24 hours to 5 days after treatment (61,74). Co-incubation with follistatin, an activin-
binding protein, prevented the activin-induced cell growth inhibition (61).
Despite the identification o f activin and activin receptors in LNCaP cells, the
mechanism(s) o f activin-induced cell growth inhibition and apoptosis enhancement
remain unclear. Based on studies in other cell systems, it is possible and probable that
the activin signal is transduced through Smad proteins and that cell cycle inhibitors such
as pl6 and p21 are involved in eliciting activin’s effect on cell growth arrest. The
purpose of this study was to examine the activin signal transduction pathway in
LNCaP prostate cells by determining if activin treatment results in time-dependent,
differential expression of proto-oncogenes or tumor suppressor proteins involved in
cell cycle inhibition and apoptosis.
Transcriptional regulation of cell cycle genes
Cell growth and carcinogenesis
Uncontrolled cell proliferation is one of the hallmarks of cancer. In a current model of
human cancer pathogenesis, overcoming the G1 growth arrest of senescence is an
essential step in the development o f carcinoma (75). The regulatory mechanism for the
G1 checkpoint is centered around the retinoblastoma tumor suppressor protein (pRb)
(76). Suggested to be a point of convergence for possibly all upstream cellular
mitogenic signaling cascades, pRb prevents premature Gl/S phase transition by
functional interaction with transcription factors such as E2F family members, which are
11
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necessary to activate S-phase genes (76). The activity of pRb, in turn, is regulated by
phosphorylation and dephosphorylation, primarily controlled by the cyclin D-dependent
ltinases CDK4 and CDK6. Whereas CDK4/6 phosphorylate pRb, thereby inactivating its
E2F-suppressing function, CDK inhibitors (CKIs) such as pl6 and p21 promote its
inhibitory function, presumably by binding to and inhibiting CDK4/6 (83,84). The
tumor suppressor gene p53 is also implicated in cell cycle arrest (85) (Fig. 2).
CDK4/6
E2F transcription
Fig. 2 Rb tumor suppressor pathway. Cyclin D binds to and activates CDK4 or CDK6.
This active complex phosphorylates Rb, causing it to dissociate from the transcription
factor E2F. E2F is now able to activate transcription o f genes causing Gl/S transition.
1 2
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p l6
The tumor suppressor protein p l6 (pl6m , C 4 A ) is one o f the most direct links between cell
cycle control and cancer. The p l6 gene was isolated as a candidate tumor suppressor
located at chromosomal position 9p21, which is deleted in many human tumors (77,80).
pl6 inactivation occurs in most tumor types with frequencies broadly ranging from 25 to
70%; this is the case with cancers o f the head and neck, esophagus, biliary tract, lung,
bladder, colon, and the breast; leukemias; lymphomas; and glioblastomas. Astoundingly,
98% of the tumors in pancreatic carcinomas have p l6 inactivation (77). Germline
transmission o f mutant pl6 alleles results in hereditary predisposition to the development
of melanomas and cancers o f the pancreas and liver (77). Also, pl6-null mice develop
tumors sporadically at an early age and are very susceptible to carcinogens (77), whereas
transfection o f the pl6 gene into lung cancer cell lines which do not express die pl6
protein resulted in reduced tumorigenicity (81). And application o f a pi 6-specific
ribozyme significantly accelerated cell cycle progression in murine erythroleukemia
(MEL) cells (82). These and other observations define pl6 as a tumor suppressor gene.
Prostate cancer tissues have been evaluated by many groups for pl6 gene structure (83).
Jarrard et al. observed LOH in 12 o f60 (20%) primary tumors and 13 of 28 (46%)
metastases (84). Gao et al. found that 3 of 18 (17%) patients with primary prostate
cancer had mutated pl6 (85). Gu et al. found 2 of 30 (6.7%) of primary prostate tumors
had a p 16 mutation (86). Reports have been made that DU 145 prostate cancer cells
have a mutation in pl6: at codon 84, as reported by Gaddipati et al. (83), and at codon
13
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76, as reported by Tamimi et al. (87). LOH o f pl6 due to raethyladon has been reported
to occur in 60% of prostatic cancer cell lines (88). These studies indicate that pl6
mutations may be important, especially during the later stages of human prostatic cancer.
LNCaP cells, which were used in the present study, have wild type pl6 (84,89),
suggesting that the presence o f plS is not sufficient to suppress tumorgenicity.
p l6 binds to CDK4 and CDK6 and inhibits the ldnase activity o f the CDK4-6/cyclin D
complexes. pl6 could inhibit CDK4 by stabilizing a catalytically inactive conformation
with a lower affinity for cyclin D. Since the only essential function o f the CDK4-6/
cyclin D kinases appears to be die phosphorylation of pRb, overexpression of pl6 in
cells with functional pRb results in G1 arrest (77) (Figure 2).
The main regulation of intracellular levels of p l6 occurs at the transcriptional level. The
level of pl6 RNA is very low in most tissues, but accumulation has been reported in
response to cellular senescence (77). Addition of androgen, which is mitogenic in
LNCaP cells, resulted in pl6 mRNA downregulation in these cells after 24 and 48 hours
(89). The effect of activin on pl6 mRNA expression is unknown.
pS3
The tumor suppressor protein p53 is involved in multiple cellular processes, including
transcription, DNA repair, cell cycle control, and apoptosis. In the prostate, the mutation
frequency of the p53 gene was found to be increased in prostate cancer tissues (26.2%)
14
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compared to benign prostatic hypertrophy (BPH) tissues (19.0%) (90). Mutant p53
expression detected by immunocytochemistry also correlated with Gleason score (91).
In another study, mutant p53 protein was detected in 20% o f benign prostatic glands,
50% of high grade-prostatic intraepithelial neoplasia (HG-PIN), and 72% of prostatic
adenocarcinomas (92).
p53 is hypothesized to have at least two distinct functions throughout the cell cycle: G1
cell cycle arrest and apoptosis induction, discussed in a subsequent section. The two
functions are based on different mechanisms. Cell cycle arrest is primarily mediated by
upregulation o f the CDK inhibitor p21 (79).
P21
p21 (p21W l > n ) was the first cyclin-dependcnt kinase inhibitor to be identified, as a
mediator of p53-induced growth arrest in response to DNA damage (79). It binds to and
inhibits both cyclinD-CDK4 and cyclinE-CDK2 complexes, thereby arresting cell cycle
progression (93), and also binds proliferating cell nuclear antigen (PCNA), thereby
inhibiting elongation by DNA polymerase (94). Overexpression of the p21 gene resulted
in inhibition o f CDK activity and G1 cell cycle arrest in various cell types, including
hamster BHK27 cells (94), HaCaT keratinocytes (95), and hepatocytes (96).
p21 upregulation has also been associated with growth inhibition in prostate cancer cells
lines. Ramsamooj et al. observed that tumorigenic 267B1/D prostate cells exhibited a
15
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p53-independent induction of p21 protein with parallel increase in p l6 protein in
response to ionizing radiation (97). Lovastatin, which induces growth arrest and cell
death, upregulated p21 in PC-3-M human prostate carcinoma cells, which are null in p53
(98). 1 6-phorbol-1 2-myristate 13-acetate (PMA) induces p21 upregulation and growth
inhibition in LNCaP cells (99). Interferon a (IFNa), which also induces growth anest,
upregulates p21 in DU 145 prostate cancer cells, which have mutated p53 (100).
The role o f p21 in human prostate cancer is unclear. A survival study on patients with
prostate carcinoma showed that p53+/p21- phenotype showed poorer prognosis than
p53+/p21+ (101). Gao et al. observed that 3 of 18 (16.7%) patients with primary
prostate tumors had mutations in the p21 gene (85). Facher e t al. found that 9 o f 54
(16.7%) of prostate adenocarcinomas contained p21 mutations (102). In studies in mice
with established subcutaneous prostate tumors, the rate of tumor growth and final tumor
volume were greatly lower in mice that received intratumor injection o f p21 instead of
p53 in a recombinant adenoviral vector (103). In addition, the survival of the p21-
treated mice was significantly extended (103). These studies indicate that p21 mutations
may be important during the pathogenesis of human prostate cancer.
Activin as a regulator o f tumor suppressors/cell cycle inhibitors
Based on studies in our laboratory, the tumor suppressor gene p53 was upregulated in
LNCaP cells 5 days following activin treatment (104). However, it was not known at
what time point after activin treatment the induction began. Activin was observed to
16
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induce p21 expression in mouse B-cell hyfaridoma cells (105) and p53-dependent p21
upregulation and G1 arrest in HepG2 cells (106). However, the effect o f activin on
expression o f die cell cycle inhibitors p l6 and p21 in prostate cells was not known.
Knowing the sequence of expression events following activin treatment would
contribute to onr understanding o f the mechanism of activin-induced cell growth
arrest
Transcriptional regulation of apoptosis genes
Apoptosis
Apoptosis, or programmed cell death, occurs by a pathway separate from the pathways
leading to cell growth arrest, although the two processes may be linked. A variety of
death signals, including extracellular ligands and receptors, removal o f survival factors,
and radiation-induced DNA damage, converge to activate a series o f execution events
referred to as the caspase cascade (107). A common, yet not universal initiator of the
cascade is the release o f cytochrome c from the mitochondria (108). Cytochrome c
activates apoptosis protease-activating factor-1 (Apaf-1), which in turn activates caspase
9, resulting in the caspase activation cascade (108), whereby precursors are cleaved into
mature, active enzymes whose substrates include proteins involved in DNA repair and
replication, RNA splicing, cytoskeletal structure, and cell division (107). Once caspases
are activated, the apoptotic process cannot be stopped or reversed (Fig. 3).
17
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Fig. 3 Apoptosis
pathway. The release of
cytochrome c from
mitochondria, in
association with Apaf-1,
activates caspase 9, which
in turn initiates the caspase
cascade, resulting in the
degradation o f cellular
proteins and cell death.
Bax and bcl-x, are
proposed to facilitate
cytochrome c release,
consequently enhancing
apoptosis, whereas bcl-2
and bcl-x, inhibit the
function of bax and bclx,.
Fas/FasL
An example of death ligand-mediated apoptosis is provided by the immune system.
When the T-cell receptor on a cytotoxic T lymphocyte binds to a foreign peptide present
on die class IMHC o f an antigen-presenting cell, it induces the expression of die protein
Fas ligand (FasL) on the surface of the T lymphocyte. FasL binds to Fas, a cell surface
receptor present on most cells of die body, and activated Fas initiates the caspase
cascade, killing the antigen-presenting cell (107). Although FasL was thought to be only
expressed in T lymphocytes, the ligand is also expressed in Sertoli cells, neurons, and
some epithelial cells, including prostatic epithelial cells (109). LNCaP cells also express
Fas and FasL (110). Adenoviral transfection o f FasL in some prostate cancer cell lines
prevented cell growth and induced apoptosis (111). However, the potential to undergo
Fas-mediated apoptosis was suggested to be decreased in advanced prostate cancer
compared to normal prostatic epithelium and primary prostate cancers (111).
1 8
apoptotk stimuli
v "
mitochondria
cytochrome c
caspase 9 ► caspase 9*
caspase
p j t r w U
I
apoptosis
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p53
The mechanism by which p53 promotes apoptosis in unclear, but it may involve
upregulation of bax, signaling through Fas-related pathways, or caspases, among other
Actors (112). p53 was found indispensable for myc-induced apoptosis, as were Apaf-1
and caspase 9 (112). pS3 induces bax, which contains p53-responsive elements (123),
and, as described above, has been implicated in enhancing apoptosis by facilitating
cytochrome c release (122).
Bcl-2 fam ily
Bcl-2 has been described as an apoptosis protection gene (112). The bcl-2 protein has
been documented to block cell death caused by a variety o f agents, including
chemotherapeutic drugs, gamma and UV radiation, heat shock, some viruses, free
radicals, lipid peroxidation, p53, c-myc, tumor necrosis factor (TNF) (113), growth
factor removal (114,1 IS), TGF0 (116), and activin (117). Evidence for bcl-2’s anti-
apoptosis function is furthered by a study wherein a ribozyme against bcl-2 decreased
bcl-2 mRNA expression and induced apoptosis in prostate cancer LNCaP cells (118).
Bcl-2 is the prototype gene of a family of closely related genes involved in apoptosis,
including bax, bcl-x„ and bcl-x, (112). Whereas bcl-2 and bcl-x, protect cells from
apoptosis induced by a variety of factors including p53 (119), bax and bcl-x, are inducers
of apoptosis (112). The proposed mechanism of bcl-2’s anti-apoptotic action is
inhibition of caspase activity by either directly or indirectly preventing cytochrome c
19
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release (120). Whereas pro-apoptodc factors such as bax trigger cytochrome c release,
possibly due to their ability to form ion channels or small protein channels (108), bcl-2
and bcl-x, may inhibit this process, thereby suppressing cytochrome c release and
subsequent caspase activation (111), hi addition, bax was found to induce apoptosis
independently of the caspase cascade by damaging organelles in yeast lacking caspases
(112). However, this form of cell death is not known to be or not to be extant in
mammals.
Defects in the apoptosis mechanism also play an important role in tumor pathogenesis,
allowing neoplastic cells to survive beyond their normal lifespan, thereby dismissing the
need for exogenous survival factors and allowing opportunity for genetic alterations to
accumulate, resulting in deregulated cell proliferation, promoted angiogenesis, and
increased cell motility and invasiveness during tumor progression (124). The anti-
apoptotic effect of bcl-2 demonstrates its importance in cancer progression. Aberrations
in the bcl-2 gene have been demonstrated in human cancers, including follicular non-
Hodgkin’s B-cell lymphomas (125) and human breast cancer (126, 127). Also, bcl-2
expression was elevated in androgen-independent prostate cancers compared to normal
prostate glands (128, 129). Bcl-2 has been implicated in resistance to therapy, and
reductions in bcl-2 achieved by anti-sense method increased susceptibility of cancer cells
to apoptosis induction by multiple chemotherapeutic drugs (124).
20
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Activin as an inducer o f apoptosis
Activin has been found to induce apoptosis in a variety o f cell types, including prostate
cancer LNCaP cells, HepG3 hepatoma cells, and HS-72 hybridoma cells, (61,63,130)
among others. Activin was found to increase bcl-x, expression and decrease bcl-2
expression in hybridoma cells (130). Previous studies in our laboratory have shown that
p53 mRNA was induced in LNCaP cells after 3 days o f activin treatment (131).
Although these studies suggest that activin-mediated apoptosis involves members o f die
bcl-2 family and/or p53, these are the only apoptotic factors, to our knowledge, which
have been studied in response to activin. It is possible and probable that other factors are
involved in activin-induced apoptosis in LNCaP cells. One purpose of this study was
to identify other possible mediators of activin-induced apoptosis.
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2 1
IL METHODS
Cell culture
Human prostate carcinoma LNCaP cells were obtained from American Type Culture
Collection (ATCC, Rockville, MD) and grown in RPM I1640 medium supplemented
with 10% fetal bovine serum (FBS) and S O pg/ml gentamycin at 37° C under 10% COz .
The medium was replaced every three days; subculture was done every 5 days. Activin
A (25 ng/ml) was added to the medium. Control cells were not treated with activin.
Cells were collected after 12, 24,48, and 68 hours after addition o f activin for RNA
isolation.
Northern blot
Total RNA was isolated from monolayer cell pellets using RNAzol B reagent using the
manufacturer’s protocol (Tel-Test, Friendswood, TX). After RNA concentration was
assessed by use o f a spectrophotometer, 20 pg of total RNA for each sample was
electrophoresed on 1.5% formaldehyde/agarose gels, transferred onto nylon membranes
by pressure blotting, and cross-linked with UV light Specific probes (200-700 bp) were
labeled with [” P]-dATP using the Prime-It II kit (Stratagene, La Jolla, CA), purified
using chromatography columns (BioRad, Hercules, CA), and mixed with 100 pg salmon
sperm DNA to prevent high background. Membranes were prehybridized in 12 ml
QuikHyb (Stratagene) for 1 h at 68°C. Probes were added directly to the hybridization
solution and incubated at 68°C for 4 h. Excess probe was removed by 2 washes in 2%
SSC and 0.1% SDS and 1 wash in 0.1% SSC and 0.1% SDS (25°C, 15 min each). The
22
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membrane was exposed to Kodak X-ray film with an intensifying screen overnight. The
blots were stripped by washing in boiling 0.1% SSC and 0.1% SDS (30 min) and
subsequently reprobed. Housekeeping gene probes, p-actin or glyceraldehyde-3-
phosphate dehydrogenase (Clontech, Palo Alto, CA) were used to verify RNA loading.
Probe synthesis
The polymerase chain reaction was used to obtain most cDNA probes, using primer pairs
and templates from Maxim Biotech, Inc. (So. San Francisco, CA). The probes specific
for the novel gene, apoptosin, were provided by Shi-Lung Lin, who isolated it through
uracil-cDNA subtraction assay (USA) (131).
Single-cell cDNA Library Am plification (ScLA)
Single-cell cDNA Library Amplification was performed as previously described (132).
First-strand cDNA was synthesized from total RNA (2 pg), to which was added S pi
ddH20,4 pi synthesis buffer (5X), 2 pi dNTP mixture (10 mM each), 3 pi oligo(dT)2 4-
T7 primer (30 pmol) and 1.0 pi AMV Reverse Transcriptase (2U) (Roche) at S5°C for 4
h. To the cDNA product, a 3’ poly-A tail was added by using 2.S pi reaction buffer
(10X), 1 pi terminal transferase (10 U) (Roche), and 2.5 pi dATP (2mM), and the
modified cDNA was purified using Microcon filters (Millipore). The 5’ poly-T-tailed,
3 * poly-A-tailed first-strand cDNA was then amplified by PCR using 2 pi cDNA, 1 pi
oligo-dT primer (30 pmol), 1 pi T7 primer (30 pmol), 2 pi dNTP mixture (10 mM each),
5 pi reaction buffer (10X), and 1.0 pi Expand™ PCR system (3.6 U) (Roche). The
23
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reaction underwent 35 cycles o f 94 °C (1 min), 54 °C (1 min), and 72 °C (4 min),
resulting in a full-length cDNA library for each group o f control and activin-treated
cells.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
cDNA libraries obtained by ScLA were used as templates in the polymerase chain
reaction using primers specific for a fragment of the genes o f interest (Maxim Biotech).
After 30 cycles of amplification at reaction times and temperatures specified by die
manufacturer’s protocol, PCR products were run on a 2% agarose gel containing 0.1
mg/ml of ethidium bromide and viewed under UV light (325 nm). The expected sizes o f
the PCR products were: p53,200 bp; bcl-2,235 bp; bcl-x* 181 bp; bcl-x„ 371 bp;
TNFa, 682 bp; Fas, 318 bp; FasL, 251 bp; Apaf-1,498 bp (Maxim Biotech).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
24
DL RESULTS
Northern blot
Total RNA was initially extracted from two groups of cells: those receiving no treatment
and those treated with activin for 12 h. The total RNA was separated by formaldehyde-
agarose gel electrophoresis, and the integrity of die total RNA was confirmed by the
presence of two distinct bands, representing the ribosomal RNAs, and a smear,
representing messenger RNAs (Fig. 4). That RNA was successfully transferred to the
nylon membrane was confirmed upon hybridization with a radiolabeled probe specific
for fi-actin, a ubiquitous housekeeping gene. A band at -1.7 lcb was present on each
membrane in each lane, and served as a control in the densitometric quantification
process.
C A C A
Fig.4 Northern gel electrophoresis. Total R N A
was isolated from activin-treated and control
untreated L N C aP cells. The presence of a smear
and two bands in each lane indicates the integrity
of the messenger R N A and ribosomal R N A s,
respectively.
25
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Single cell cDNA library amplification (ScLA) and reverse transcription-polymerase
chain reaction (RT-PCR)
cDNA libraries were generated by ScLA, separated by agarose gel electrophoresis, and
transferred to a nylon membrane. Under UV light, a smear was detected, spanning from
<200 bp to >7 kb (Fig. S). To confirm the presence of cDNA transcripts, RT-PCR was
performed using primers specific for the ubiquitous housekeeping gene GAPDH
(Maxim). A PCR product o f226 bp, the expected size of the fragment, was detected in
each sample, indicating die presence of intact cDNA libraries generated by ScLA
(Fig. 6). In addition to generating die 226 bp fragment of cDNA, the GAPDH primers
were also specific for a 1.9 kb-fragment spanning part of an intron. The absence of a 1.9
kb PCR product indicated die absence of contamination by genomic DNA.
M 2 kb
3.0 kb
1.0 kb
Z O O bp
C 12 24 48 68
Fig.5 cO N A libraries generated by ScLA. Total
R N A was reverse transcribed into cO N A . The
cD N A I i brari es were amended and am pi j f j ed,
resulting in visible smears spanning from
<2 0 0 bp to >7 kb.
26
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C 12 24 48 68
Fig. 6 G A P O H PC X product. cDNA
libraries gm ratadby ScLA war* uMd
a* templates with primers for PC R
amplification of a fragment of GAPDH
The presence of the band (226 bp)
confirms the presence of G A PD H
transcripts in the cD N A libraries and
indicates that the libraries are intact.
PCR products of expected sizes were also detected for other probes, further confirming
die completeness o f die cDNA libraries (Fig. 7). In addition, one gene was shown for
the first time to be expressed in LNCaP cells. To our knowledge, this was the first time
mRNA transcripts for Apaf-1 were shown to be present in activin-treated and untreated
LNCaP cells by RT-PCR.
< r -
Fas FasLTNF-s p53 bd-2 bcl-x Apaf-1
Fig.7 Generation of probes by RT-PCR. cO N A
libraries from L N C aP cells were used as
templates for P C R amplification using gene-
specific primers. From left to right: Molecular
M arker X (Roche); Fas, 318 bp; FasL, 251 bp;
TNF-alpha, 682bp, p53, 200 bp; bcl-2, 235 bp;
bcl-x (I, 371 bp; s. 181 bp); Apaf-1. 498 bp.
27
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Comparison o f mRNA expression fo r cell cycle-related genes
mRNA for activin-treated and control untreated cells were compared by Northern blot
Because initial hybridizations with p-actin showed that total RNA content was not quite
equal between the two groups, comparisons between mRNA content for the genes o f
interest were made by dividing die density o f die band o f interest by die density of its
corresponding band for P-actin. The ratios revealed no significant difference in mRNA
content between activin-treated cells and control untreated cells for p53, pRb, or p21
(Fig. 8). pl6 mRNA was not detectable in either group (not shown).
P53
M
C A C A C A
■ con trol
□activin
p63 pRb p21
: j t r r ; ■:
P21
Fig. 8 Expression of cell cycle-related genes. Total RNA was extracted from LNCaP
cells treated with activin for 12 hours or LNCaP cells receiving no treatment
C=Control, A=Activin-trcated
28
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Activin regulates some apoptosis-related genes
mRNA content for genes involved in die apoptotic pathway was compared between
activin-treated and control untreated cells. Fas antigen mRNA content was increased by
31.6% in activin-treated cells compared to control untreated cells (Fig. 9). mRNA
content for bcl-x, was increased by 9.6% in activin-treated cells. mRNA levels for bcl-2,
apoptosin, TNFa, and Apaf-1 were relatively unchanged. Hybridization with Fas ligand
did not result in a visible band (not shown).
bcl-2 bd-xs
actin
actin
C A
TNF-alpha
vv
apoptosin
tf
Apaf-1
C A C A C A
Fig. 9 Expression of apoptosis-related genes. Total RNA was extracted from LNCaP
cells treated with activin for 12 h or cells receiving no treatment
29
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Time-dependent expression o f bcl-x, after activin treatment
Because mRNA content for most genes was found to be insignificantly altered by activin
treatment after 12 h, we tested the possibility that transcriptional regulation would occur
at longer time periods after activin treatment Total RNA was isolated from cells after
24,48, and 68 h after activin treatm ent separated by gel electrophoresis, and transferred
to a nylon membrane. After initial hybridization with the housekeeping gene GAPDH to
assess loading, the membrane was hybridized with a radiolabeled probe for bcl-x,.
mRNA content for bcl-x, was found to be decreased by 30% at 12 h with levels gradually
returning toward control levels after 48 and 68 h (Fig. 10).
- i t v l •
GAPDH
Fig. 10 Expression of bcl-x,in LNCaP cells treated with activin for 12,24,48, and 68
hours and cells receiving no treatment.
30
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IV. DISCUSSION
That Rb mRNA was not upregulated after 12 h of activin treatment (Fig. 8) was not
unexpected because of the extensive evidence that pRb’s function in cell cycle regulation
relies on its phosphorylation state (75, 76). Thus, absence of transcriptional
modification o f pRb does not rule out the possibility that pRb was regulated post-
transcriptionally or, more likely, at die level of phosphorylation.
However, that p21 mRNA was also unaltered upon activin treatment (Fig. 8) was
unexpected because of previous evidence in mouse hybridoma cells and HepG2 cells that
p21 mRNA was upregulated after treatment with activin for 3 and 24 hours, respectively
(133, 134), and that TGF-P or Smad2/3 overexpression caused high levels of p21
promoter transactivation (134). Our results suggest that activin-induced p2l expression
is cell-type specific and that activin and TGF0 have distinct mechanisms of inducing
apoptosis. Alternatively, 12 h was not sufficient time to induce observable p21
upregulation in LNCaP cells or was too late for an observable effect
Based on previous studies in our laboratory (131), p53 is upregulated after 3 days of
activin treatment That p53 mRNA was unchanged 12 h after activin treatment (Fig. 8),
suggests that p53 upregulation starts at a time between 12 h and 3 days after activin
treatment. Alternatively, p53 could have been post-transcriptionally modified.
31
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To our knowledge, this is the first time bcl-x,, bcl-x, and Fas have been found to be
regulated in LNCaP cells by activin (Fig. 9 and Fig. 10). The downregulation of bcl-x,
and upregulation o f bcl-x, we observed are consistent with activin’s effect of apoptotic
induction and die proposed effect of bax and bcl-x, to facilitate cytochrome c release and
subsequent caspase activation (108). Thus, one mechanism by which activin mediates
apoptosis may be by decreasing bcl-x, and increasing bcl-x,. That bcl-x, mRNA levels
were only 9.6% higher in activin-treated cells may signify only a slight difference in
expression. The difference would have to be confirmed by further experiments. One
way to test this hypothesis is to construct and insert an anti-bcl-x, oligonucleotide which
would function as a knock-out mechanism to block downstream effects of bcl-x,. If bcl-
x, knock-out, activin-treated cells resulted in delayed or decreased levels of apoptosis, it
would further demonstrate that bcl-x, is one mediator of activin-induced apoptosis.
The significant 32% increase in Fas mRNA expression after activin treatment suggests
that Fas may be a mediator of activin-induced apoptosis. The role o f Fas as a mediator
of growth factor-induced apoptosis is not unprecedented. Ortiz et al. found that TNFa
increased Fas and Fas ligand mRNA and apoptosis in renal fibroblasts (135). Xu et al.
demonstrated that IFNy upregulated Fas and Fas ligand and apoptosis in human colon
adenocarcinoma HT29 cells (136). Whereas these inhibitory growth factors increase Fas
expression, thereby activating caspases and subsequent cell death, stimulatory growth
factors such as IGF-1 downregulated Fas in mouse renal fibroblasts (135) and rabbit
ovarian mesothelial cells (137), which resulted in increased resistance to apoptosis.
32
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Numerous CAGA boxes surrounding a nearly consensus FBE (5’-TGTCTATT-3’) are
present in the Fas gene promoter. A total of 10 CAGA boxes in the promoter were
identifiable (Gcnbanlc Accession D31968). The significance of these elements or
whether these elements bind activin-induced Smad/FAST-1 complexes to increase Fas
transcription are not known. Further experiments to confirm Fas upregulation and assess
Fas function by anti-sense techniques would further define the potential role for Fas in
activin-mediated apoptosis.
Our observations that Apaf-1 mRNA was detected by RT-PCR and Northern blot is the
first time, to our knowledge, that this Apaf-1 gene was found to be expressed in LNCaP
cells (Fig. 9). Its presence in LNCaP cells suggests that die cells may also possess other
components o f the caspase activation pathway and that the activation involves
cytochrome c release. This ties in with the presence and upregulation o f bcl-x*, which is
also proposed to facilitate cytochrome c release.
That bcl-2 mRNA was not altered in activin-treated cells indicates that 12 h of activin
treatment did not affect the gene at the transcriptional level (Fig. 9). This does not
exclude die possibility that activin may have downstream effects on protein translation,
post-translational modification, or protein degradation. Furthermore, it is another
possibility that transcriptional regulation of these genes does not occur until after 12 h of
activin treatment.
33
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This question o f time also seems pertinent to die case of the novel gene apoptosin, which
was identified in our laboratory by Shi-Lung Lin (131). A fragment of apoptosin was
isolated through subtractive hybridization between cDNA libraries of control untreated
LNCaP cells and cells treated with activin for 3 days (131). Whereas apoptosin was
found to be upregulated in cells treated with activin for 3 days, our results indicated no
significant change in mRNA content between untreated cells and cells treated with
activin for 12 h (Fi.g 9). This difference is consistent with die hypothesis that apoptosin
induction is a late-occurring event which acts downstream of caspases to induce the
DNA fragmentation characteristic of apoptosis (oral communication, Shi-Lung Lin).
Further studies using mRNA from cells treated with activin for time points between 12 h
and 3 days would reveal the point at which apoptosin is initially induced and to what
extent
Activin’s upregulation of Fas and bcl-x, and downregulation of bcl-x, are consistent with
its role in enhancing apoptosis. In addition to activin’s roles as an inhibitor of cell
proliferation and inductor of apoptosis, activin possesses other biological functions which
suggest it to be a multi-level inhibitor of carcinogenesis and cancer progression. For
example, as detected by mRNA differential display, activin mRNA expression was
detected in low-metastatic mouse melanoma cells but not in high-metastatic melanoma
cells (138), suggesting that loss of activin expression may contribute to metastasis.
34
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The ability of a subset of cells within a primary tumor to metastasize is determined by a
number of factors, including growth rate, adhesion, motility, secretion o f degradative
enzymes, and angiogenesis factors (139). One of the initial steps of cancer metastasis is
detachment of cells from the primary tumor mass. Development of increased metastatic
capacity in glioma cells has been shown to correlate with decreased NC AM expression
(139). The addition of activin increased NCAM expression in various cell lines (140),
suggesting that this growth factor may help mediate cell-cell adhesion, thereby inhibiting
tumor invasion and migration. In addition, activin decreases matrix degradation by
increasing expression of PAI-1 (141), thereby potentially decreasing metastatic capacity.
Most tumors can induce angiogenesis for purposes of oxygen and nutrient supply and
waste removal. Because angiogenesis also allows tumor cells to metastasize from the
primary or secondary organ to distant sites, inhibition o f angiogenesis may be an
important approach for preventing tumor growth and metastasis (142). One piece of
evidence that activin may inhibit angiogenesis is that activin can inhibit growth in
vascular endothelial cells (143). In addition, die activin-binding protein, follistatin,
induced proliferation of human umbilical vein endothelial cells and resulted in
angiogenesis in the rabbit cornea (144), implicating activin in the inhibition of
angiogenesis in vivo as well as in vitro. The ability of activin to suppress angiogenesis
may imply the ability to inhibit the capacity of endothelial cells to engage in a series of
activities, including proteolytic degradation of the surrounding extracellular matrix and
invasion o f the avascular tissue ( 14S). Although more information is needed on the role
35
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o f activin in tumor invasion and migration, activin’s inhibitory effect on endothelial cell
growth and angiogenesis supports its possible preventive role against cancer progression
and metastasis.
activin
4 cell adhesion ^ caspase activation
t cell proliferation
activin
activin
^ cell growth
^ ECM degradation t angiogenesis
Fig. 1 1 M rrhaahn of[mwth laMMtioa Mil pmriMr upprrnkia ofmcHHsiii l > > si litla
Activin acts on components ofthepRb pathway, apoptosis pathway, cell adhesion factors, E C M
components, and angiogenesis. These attentions may result in decreased tumor invasiveness
and metastasis.
Summary
Activin has a variety of biological functions, including inhibition o f cell proliferation and
enhancement of apoptosis in numerous in vitro and in vivo systems. Examination on the
molecular level has revealed that these mechanisms involve membrane receptors, Smad
proteins, components of the Rb checkpoint pathway, caspase activation, cell adhesion
36
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molecules, and angiogenic potential (Fig. 11). Activin also increases NCAM expression
in various cell lines, suggesting that it has more than one mechanism for decreasing cell
proliferation. Based on our results, activin enhances apoptosis in LNCaP cells by several
mechanisms, including downregulating bcl-x, and upregulating bcl-x, and Fas mRNA
expression. In addition, activin decreases matrix degradation by increasing expression of
PAI-1, thereby possibly decreasing metastatic capacity.
In addition to decreasing cell growth, activin’s ability to increase cell adhesion protein
levels may indicate the capacity to inhibit tumor migration. Activin has been shown to
inhibit angiogenesis in vivo and is associated with low-metastatic capacity.
Understanding the molecular mechanisms o f activin-mediated inhibition of cell growth
and cancer progression may lead to the development o f novel methods to target these
molecules in the treatment and possible prevention of some cancers.
37
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V. REFERENCES
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Creator Lui, Hannah Mun-Ly (author)

Core Title Activin-mediated growth inhibition in prostate cancer LNCaP cells by transcriptional regulation of apoptosis-related genes

School Graduate School

Degree Master of Science

Degree Program Cell and Neurobiology

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