Affiliations of authors: D. B. Agus, W. Fox, D. W. Golde, H. I. Scher (Department of Medicine), C. Cordon-Cardo, M. Drobnjak (Department of Pathology), A. Koff (Sloan-Kettering Institute), Memorial Sloan-Kettering Cancer Center, New York, NY.
Correspondence to: David B. Agus, M.D., Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021 (e-mail: d-agus{at}ski.mskcc.org).
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ABSTRACT |
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INTRODUCTION |
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Reproducible changes in expression of cell cycle regulators were found that could be categorized into early and mid-to-late events. Early events were consistent with a cell stress response in which p53 produces a transient cell cycle arrest without activation of p53-dependent cell death programs. Mid-to-late events included an increase in the expression of the cyclin-dependent kinase inhibitors p27 and p16, which are associated with a further decline and the maintenance of a low proliferative index. Apoptotic changes were not observed after androgen withdrawal. Characterization of the androgen-independent phenotype revealed the overexpression of mdm2, affecting p53 stability, as well as the increased cyclin D1 expression, affecting pRB phosphorylation. These results challenge a commonly held view that the regression of prostate cancers after androgen withdrawal is mediated exclusively by apoptotic mechanisms. Furthermore, the results suggest that therapeutic strategies directed at the cell cycle-arrested prostate cancer cells, after androgen withdrawal, may be clinically important.
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MATERIALS AND METHODS |
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Four- to 6-week-old nude athymic BALB/c male mice were obtained from the National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD, and maintained in pressurized, ventilated caging. Institutional guidelines for the proper and humane use of animals in research were followed. The CWR22 tumor line was propagated in the animals by the injection of a mixture containing reconstituted basement membrane (Matrigel; Collaborative Research, Inc., Bedford, MA) and minced tumor tissue from an established tumor into the subcutaneous tissue of the flanks of athymic nude mice (1,2). For the maintenance of serum androgen levels, mice were administered 12.5 mg of sustained-release testosterone pellets (Innovative Research of America, Sarasota, FL) subcutaneously. Tumors of approximately 1.5 x 1.0 x 1.0 cm grew 3-4 weeks after inoculation. Androgen withdrawal was accomplished by surgical castration under pentobarbital anesthesia and removal of the testosterone pellets. Tumor size was determined by caliper measurements of height, width, and depth. Prostate-specific antigen (PSA) assays were performed on the serum of the mice obtained by tail bleeding and used a Tandem-R PSA immunoradiometric assay (Hybritech, Inc., San Diego, CA). Androgen-independent sublines of the parent CWR22 were obtained by following tumors for regrowth and increases in serum PSA after androgen withdrawal. The androgen-independent sublines regrew 80-400 days after androgen withdrawal. The sublines were serially passaged in three castrated hosts before characterization as a subline. These were duplicates performed at each sampling point in the experiments.
Histopathology, Monoclonal Antibodies, and Immunohistochemistry
Tissues were fixed in 10% buffered formalin and embedded in paraffin, and sections (5 µm) were stained with hematoxylin-eosin. The following well-characterized antibodies were used at the corresponding final working dilutions: anti-Ki67 mouse monoclonal antibody (MAb) MIB1 (Immunotech SA, Marseille, France; 1 : 50 dilution), p53 (MAb clone PAb1801; Calbiochem/Oncogene Sciences, Cambridge, MA; 0.2 µg/mL), p21/WAF1 (MAb clone 2G12; PharMingen, San Diego, CA; 0.5 µg/mL), mdm2 (MAb clone 2A10; gift from Dr. A. Levine, Rockefeller University, New York, NY; 1 : 50 dilution), p27/Kip1 (MAb clone DCS72; Calbiochem/Oncogene Sciences; 0.1 µg/mL), p16 (MAb clone DCS-50.1/H4; Calbiochem/Oncogene Sciences; 2 µg/mL), cyclin D1 (MAb clone DCS-6; Calbiochem/Oncogene Sciences; 1 µg/mL), E2F1 (purified rabbit antiserum, C-20; Santa Cruz Laboratories, Santa Cruz, CA; 0.2 µg/mL), androgen receptor (MAb clone F39.4.1; BioGenex, San Ramon, CA; 2 µg/mL), bcl-2 (MAb clone 124; Dako Corp., Carpinteria, CA; 2.5 µg/mL), and bax (purified rabbit antiserum, 1-19; Santa Cruz Laboratories; 0.05 µg/mL). An MAb (immunoglobulin G [IgG] subclass, clone MIgS-KpI; PharMingen; 1 : 50 dilution) and a preimmune rabbit serum were used as negative controls. Histologic sections were immersed in boiling 0.01% citric acid (pH 6.0) for 15 minutes, allowed to cool, and incubated with primary antibodies overnight at 4 °C. Biotinylated horse anti-mouse IgG antibodies and biotinylated goat anti-rabbit antiserum (both from Vector Laboratories, Inc., Burlingame, CA; 1 : 500 dilution) were applied for 1 hour, followed by avidin-biotin peroxidase complexes for 30 minutes (Vector Laboratories, Inc.; 1 : 25 dilution). Diaminobenzidine was used as the final chromogen, and hematoxylin was used as the nuclear counterstain. Most markers were evaluated for nuclear staining of tumor cells. Exceptions included the evaluation of bcl-2, which renders a punctated mitochondrial pattern, and bax, which produces a diffuse cytoplasmic staining. Positive cells were scored by counting different fields and more than 500 cells. Data were recorded in a continuum as the percentage of cells stained at the specific time evaluated, and the intensity of staining was graded as follows: 0, undetectable; 1+, minimal staining; and 2+, strong staining. Tumors from at least two animals were evaluated at each time. Cell cycle regulators were assessed daily after androgen withdrawal to day 10 and then every 5 days until day 25.
Flow cytometry assays for assessment of S-phase fraction and cell cycle distribution analyses were done on tumor blocks from specific times after androgen withdrawal. Tumor tissue was microdissected to avoid normal tissue contamination and necrotic material. Three consecutive 60-µm-thick sections were cut, and the nuclei were disaggregated, stained with propidium iodide, and analyzed on a Coulter Excel (Beckman Coulter, Inc., Fullerton, CA) with a multicycle soft program for assessment of cell cycle profile (5).
To study the microanatomical distribution of apoptosis, we assayed consecutive sections from the blocks used for immunohistochemistry by a modification of the method of terminal deoxynucleotidyltransferase-mediated uridine triphosphate end labeling (TUNEL) (6), originally described by Gavrieli et al. (7). Briefly, after the exposure of nuclear DNA of histologic sections by proteolytic treatment, terminal deoxynucleotidyltransferase was used to incorporate biotinylated deoxyuridine at sites of DNA breaks. The signal was amplified by avidin-biotin peroxidase complexes and visualized by diaminobenzidine deposition, enabling conventional histochemical identification of reactive nuclei by light microscopy. Nuclear staining was assessed by immunohistochemical scoring, counting different fields, and evaluating more than 500 tumor cells (8).
p27 Degradation and Transcript Quantitative Assays
Extracts were prepared from CWR22 tumors at times before and after androgen withdrawal. The tumor extracts were assayed for p27 degradation activity in vitro as described by Nguyen et al. (9). Digoxigenin-labeled probes were used for in situ hybridization, and 1 µg of recombinant plasmid pCRTMII (Invitrogen Corp., San Diego, CA), containing the full-length human p27KIP1 gene, was linearized by digestion with BamHI and XbaI to generate antisense and sense transcripts. RNA probes were generated, and deparaffinized tissue sections were stained as previously described (10). Each assay was done in duplicate.
pRB Phosphorylation Assays
Western blotting was done with protein extracts from tumors. Proteins were extracted from OCT (optimal cutting temperature) compound-embedded tumors and resolved on polyacrylamide gels for immunoblotting with pRB-specific antibodies as previously described (10). Each assay was done in duplicate.
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RESULTS |
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Early events. Androgen withdrawal was associated with an
abrupt decrease in androgen receptors, detected by nuclear staining,
from 60% (1+) of tumor cells positive on day 0 (baseline) to
10% by days 3, 7, and 10 (Fig. 1, A and B). The
proportion of tumor cells with nuclear staining for p53 increased from
a few scattered nuclei in less than 5% of cells on day 0 to
approximately 30% (1-2+) on day 3 (Fig. 1,
C and D;
maximal staining). The p53 staining decreased to baseline by day 7. After the
increase in p53-positive cells, the number of cells expressing p21/WAF1
increased from approximately 25% (day 0) to a maximum of approximately
50% of the cells (1-2+) by day 5 (Fig. 1,
E and F) and
thereafter gradually decreased. Concurrent with the increase in
p21/WAF1, a substantial increase in p16INK4A was noted. At
days 0-2 after androgen withdrawal, 5%-10% of cells were p16
positive (1-2+); this percentage increased to 30% on day
3 and to 40% on days 5-7 (1-2+). The net effect of these
early changes was a decrease in the Ki67 proliferative index, which
ranged from 60% to 65% on days 0-3 to 20% on day 5 to approximately
5% on day 7 (Fig. 1,
G and H). Levels of cyclin D1 expression also
fell from 10% of tumor cells (pretreatment and day 0) to approximately
2% on day 7 after androgen withdrawal.
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Thus, early cell cycle events after androgen withdrawal are characterized by exit from the cell
cycle (summarized in Fig. 2, A), which is associated with a cellular stress
response that is initiated by an increase in p53, followed by an increase in p21/WAF1 and a
resultant early G1/G0-phase arrest, with the lack of an apoptotic response.
Concurrent with these changes after androgen withdrawal, the level of PSA fell because PSA
transcription is driven by androgen (Fig. 2,
B) (4).
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After androgen withdrawal, the mid-to-late cell cycle events are, thus, characterized by a
progressive and sustained decrease in cyclin D1 expression, followed by changes in pRB
expression (summarized in Fig. 2, A). Growth arrest of the prostate
cancer cells appears to be maintained by steady levels of p27 and p16. The prostate cancers did
not appear to undergo apoptosis after androgen withdrawal but rather underwent a G0/early G1-phase cell cycle arrest.
Cell Cycle Protein Changes Associated With Androgen Independence
Similar to previous observations (2), androgen-independent
proliferation was observed after 80-400 days, with a serum PSA rise
preceding tumor volume changes (Fig. 2, B). The pattern of expression
of cell cycle regulatory proteins in the five androgen-independent
tumors, designated CWR22R, CWRSA1, CWRSA3, CWRSA4, and CWRSA6,
reflected a highly proliferative tumor, similar to the CWR22
xenografts. Androgen-independent tumors had a Ki67 proliferative
index of 60%-70%, a homogeneous and intense staining for androgen
receptor in 60%-80% of CWR22R, CWRSA1, and CWRSA4 cells or 90% of
CWRSA3 and CWRSA6 cells, and, compared with the growth-arrested tumors,
an increase in the proportion of pRB-positive cells with strong nuclear
staining and a decrease in E2F1 (data summarized in Fig.
4,
A). There was a decrease in the proportion of
cells staining for p27, from 80%, when proliferation was low (day 25),
to approximately 5% in the androgen-independent tumors. Consistent
with these findings, there was a decrease in the proportion of cells
staining for p16 from 60% to less than 3%.
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The level of androgen receptor expression was 1+-2+ in
60%-80% of the tumor cells of most androgen-independent sublines. Two sublines,
CWRSA3 and CWRSA6, expressed higher levels of androgen receptor in a greater percentage of
tumor cells (90%, 2+ staining). The growth characteristics of these
sublines were similar to those of the other sublines, except that their growth was repressed, rather
than stimulated, in the presence of physiologic concentrations of androgen. The emergence of
androgen independence in this model system is associated with a return of tumor cells to an active
cell cycle and increased expression of the oncogenic proteins cyclin D1 and mdm2. In addition,
the androgen-repressible growth phenotype is associated with relative androgen receptor
overexpression.
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DISCUSSION |
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The initial cell stress response in this system (an increase in p53 and p21 expression and a block in G1-phase cell cycle progression) is similar to that observed after exposure of cells to high-dose UV radiation (11). Ectopic expression of decorin (12) and treatment of several human cancer cell lines with mimosine and aphidicolin (13) have also been reported to induce increments of p53 and p21 expression, leading to permanent growth arrest. This p21-mediated G1-phase arrest has been shown to be dependent on functional pRB (14).
After androgen withdrawal, tumor regression appears to result from undetectable cell death or, more likely, from changes in cell volume. The increase in p53 staining at day 3, in the absence of apoptosis, suggests a defect in p53-mediated apoptosis. Androgen withdrawal results in the apoptosis of nonmalignant prostate epithelial cells and is associated with a decrease in cell size (15,16). Studies of xenografts and spontaneous rat prostate cancers have been inconclusive. Increased apoptotic indices were observed in PC-82 and LuCaP human prostate cancer xenograft models (17,18), whereas decreased indices were observed in the Dunning R3327PAP rat model after androgen withdrawal (19,20). Conflicting results have been observed in human prostate cancers after androgen withdrawal, since increased levels of apoptosis have been reported by some groups of investigators (21-23) but not by other groups (24-27). The apparently contradicting results may reflect the small proportion of cells actually undergoing apoptosis at any one time, the inability to sample tumors repeatedly at different times, or the small overall contribution of apoptotic cell death to human tumor regression. An alternative explanation is that the cell cycle checkpoint status of the various tumors is different. The integrity of the checkpoint status can change the response to anticancer therapy from cell cycle arrest to cell death in the xenograft model system (28).
Although changes in early cell cycle events were transitory, the mid-to-late responses were progressive and sustained. We found a decrease in cyclin D1 levels followed by changes in pRB expression, changes similar to those described for butyrate-induced G1-phase arrest (29). Butyrate inhibits the mitogen-dependent transcriptional induction of cyclin D1 and phosphorylation of pRB (30). Withdrawing androgen removes a mitogenic signal for cyclin D1, resulting in pRB hypophosphorylation and G1-phase arrest. Additional factors associated with the maintenance of arrested growth are the observed overexpression of p21, p27, and p16.
The inhibition of cell cycle progression and the antiproliferative activity of rapamycin have been reported to be dependent on p27 (31). More recently, it has been reported that the protein kinase inhibitor staurosporine induces a G1-phase arrest in cells expressing functional pRB (32). In staurosporine-treated cells, the levels of p27 increase, preventing activation of cyclin E/cyclin-dependent kinase-2 and contributing to the G1-phase arrest. In hormone-naive primary prostate cancer, low-to-undetectable p27 levels were associated with poor clinical outcomes, independent of tumor stage and grade (10,33-36). However, in one study (10), androgen-independent metastatic bone lesions had low levels of p27, consistent with the xenograft findings. Cellular responses to stress, such as hyperthermia, have been shown to induce p16 and to arrest cells in G0/G1 phase in a p53-independent manner (37). Furthermore, the expression of p16 induces decreased transcription of the RB gene (38).
Androgen independence was marked by the expression of the oncogenic proteins cyclin D1 and mdm2. The increased mdm2 expression increases p53 transcriptional transactivation (39) and also inactivates pRB (40). Cyclin D1 is a pivotal molecule in cell cycle regulation. Its overexpression has been shown to participate in the oncogenesis of many tumors (41,42).
Androgen receptor expression decreased substantially after androgen withdrawal. The mechanism of this decrease is not known, but this observation has been made in other systems (43). The androgen receptor in the CWR22 model has a mutation in the ligand-binding domain at position 874 (44). Similar to the wild-type androgen receptor, the receptor is functional in the presence of testosterone and dihydrotestosterone (44). Androgen receptor expression is present again at days 10-16 after androgen withdrawal and to various amounts in androgen-independent sublines. These results are similar to the human condition, where androgen receptor is expressed in the majority of untreated prostate cancers and in most patients whose cancer recurs during androgen withdrawal therapy (45). Other investigators (46,47) have shown that amplification and the increased expression of a wild-type androgen receptor gene may play an important role in androgen-independent prostate cancer. It is of interest that the two androgen-independent tumors with the highest number of cells staining for androgen receptor and the most intense staining showed androgen-repressed growth. Androgen repression of prostate cancer growth has been described previously (48-50). Similar to the current results, androgen-repressible lines expressed higher basal levels of androgen receptor protein and messenger RNA than did the parental line (50). The overexpression of androgen receptor in androgen-independent prostate cancer may identify a subset of prostate cancers in which androgen treatment may be of clinical benefit.
This study demonstrates that reproducible cell cycle protein regulator changes occur with androgen withdrawal in one xenograft model of human prostate cancer. These changes were associated with a cell stress response and the exit of the prostate cancer cells from the active cell cycle with the absence of observed apoptosis. The cell cycle changes associated with the emergence of androgen independence are more heterogeneous but suggest regulatory pathways for this independence from hormonal stimulation. Therapeutic strategies designed to address the cell cycle-arrested prostate cancer cells after androgen withdrawal are being investigated in an effort to eliminate the emergence of androgen independence.
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NOTES |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 Wainstein MA, He F, Robinson D, Kung HJ, Schwartz S, Giaconia JM, et al. CWR22: androgen-dependent xenograft model derived from a primary human prostatic carcinoma. Cancer Res 1994;54:6049-52.[Abstract]
2 Nagabhushan M, Miller CM, Pretlow TP, Giaconia JM, Edgehouse NL, Schwartz S, et. al. CWR22: the first human prostate cancer xenograft with strongly androgen-dependent and relapsed strains both in vivo and in soft agar. Cancer Res 1996;56:3042-6.[Abstract]
3 Pretlow TG, Wolman SR, Micale MA, Pelley RJ, Kursh ED, Resnick MI, et al. Xenografts of primary human prostatic carcinoma. J Natl Cancer Inst 1993;85:394-8.[Abstract]
4 Agus DB, Golde DW, Sgouros G, Ballangrud A, Cordon-Cardo C, Scher HI. Positron emission tomography of a human prostate cancer xenograft: association of changes in deoxyglucose accumulation with other measures of outcome following androgen withdrawal. Cancer Res 1998;58:3009-14.[Abstract]
5 Bauer KD, Clevenger CV, Endow RK, Murad T, Epstein AL, Scarpelli DG. Simultaneous nuclear antigen and DNA content quantitation using paraffin-embedded colonic tissue and multiparameter flow cytometry. Cancer Res 1986;46:2428-34.[Abstract]
6 Fuks Z, Persaud RS, Alfieri A, McLoughlin M, Ehleiter D, Schwartz JL, et al. Basic fibroblast growth factor protects endothelial cells against radiation-induced programmed cell death in vitro and in vivo. Cancer Res 1994;54:2582-90.[Abstract]
7 Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992;119:493-501.[Abstract]
8 Osman I, Scher HI, Zhang ZF, Pellicer I, Hamza R, Eissa S, et al. Alterations affecting the p53 control pathway in bilharzial-related bladder cancer. Clin Cancer Res 1997;3:531-6.[Abstract]
9
Nguyen H, Gitig DM, Koff A. Cell-free degradation of p27(kip1),
a G1 cyclin-dependent kinase inhibitor, is dependent on CDK2 activity and the proteasome. Mol Cell Biol 1999;19:1190-201.
10
Cordon-Cardo C, Koff A, Drobnjak M, Capodieci P, Osman I,
Millard SS, et al. Distinct altered patterns of p27KIP1 gene expression in benign prostatic
hyperplasia and prostatic carcinoma. J Natl Cancer Inst 1998;90:1284-91.
11 Wu L, Levine AJ. Differential regulation of the p21/WAF-1 and mdm2 genes after high-dose UV irradiation: p53-dependent and p53-independent regulation of the mdm2 gene. Mol Med 1997;3:441-51.[Medline]
12
Santra M, Mann DM, Mercer EW, Skorski T, Calabretta B,
Iozzo RV. Ectopic expression of decorin protein core causes a generalized growth suppression in
neoplastic cells of various histogenetic origin and requires endogenous p21, an inhibitor of
cyclin-dependent kinases. J Clin Invest 1997;100:149-57.
13 Ji C, Marnett LJ, Pietenpol JA. Cell cycle re-entry following chemically-induced cell cycle synchronization leads to elevated p53 and p21 protein levels. Oncogene 1997;15:2749-53.[Medline]
14
Niculescu AB 3rd, Chen X, Smeets M, Hengst L, Prives C, Reed
SI. Effects of p21 (Cip1/Waf1) at both the G1/S and the G2/M cell cycle
transitions: pRb is a critical determinant in blocking DNA replication and in preventing
endoreduplication [published erratum appears in Mol Cell Biol 1998;18:1763]. Mol Cell Biol 1998;18:629-43.
15 Kyprianou N, Isaacs JT. Activation of programmed cell death in the rat ventral prostate after castration. Endocrinology 1988;122:552-62.[Abstract]
16 Kerr JF, Searle J. Deletion of cells by apoptosis during castration-induced involution of the rat prostate. Virchows Arch B Cell Pathol 1973;13:87-102.
17 van Weerden WM, van Kreuningen A, Elissen NM, Vermeij M, de Jong FH, van Steenbrugge GJ, et al. Castration-induced changes in morphology, androgen levels, and proliferative activity of human prostate cancer tissue grown in athymic nude mice. Prostate 1993;23:149-64.[Medline]
18 Bladou F, Vessella RL, Buhler KR, Ellis WJ, True LD, Lange PH. Cell proliferation and apoptosis during prostatic tumor xenograft involution and regrowth after castration. Int J Cancer 1996;67:785-90.[Medline]
19 Brandstrom A, Westin P, Bergh A, Cajander S, Damber JE. Castration induces apoptosis in the ventral prostate but not in an androgen-sensitive prostatic adenocarcinoma in the rat. Cancer Res 1994;54:3594-601.[Abstract]
20 Westin P, Bergh A, Damber JE. Castration rapidly results in a major reduction in epithelial cell numbers in the rat prostate, but not in the highly differentiated Dunning R3327 prostatic adenocarcinoma. Prostate 1993;22:65-74.[Medline]
21 Montironi R, Pomante R, Diamanti L, Magi-Galluzzi C. Apoptosis in prostatic adenocarcinoma following complete androgen ablation. Urol Int 1998;60 Suppl 1:25-9.
22 Denmeade SR, Lin XS, Isaacs JT. Role of programmed (apoptotic) cell death during the progression and therapy for prostate cancer [published erratum appears in Prostate 1996;28:414]. Prostate 1996;28:251-65.[Medline]
23 Reuter VE. Pathological changes in benign and malignant prostatic tissue following androgen deprivation therapy. Urology 1997;49(3A Suppl):16-22.[Medline]
24 Westin P, Stattin P, Damber JE, Bergh A. Castration therapy rapidly induces apoptosis in a minority and decreases cell proliferation in a majority of human prostatic tumors. Am J Pathol 1995;146:1368-75.[Abstract]
25 Murphy WM, Soloway MS, Barrows GH. Pathologic changes associated with androgen deprivation therapy for prostate cancer. Cancer 1991;68:821-8.[Medline]
26 Dhom G, Degro S. Therapy of prostatic cancer and histopathologic follow-up. Prostate 1982;3:531-42.[Medline]
27 Tomic R, Bergman B, Hietala SO, Angstrom T. Prognostic significance of transrectal fine-needle aspiration biopsy findings after orchiectomy for carcinoma of the prostate. Eur Urol 1985;11:378-81.[Medline]
28 Waldman T, Zhang Y, Dillehay L, Yu J, Kinzler K, Vogelstein B, et al. Cell-cycle arrest versus death in cancer therapy. Nat Med 1997;3:1034-6.[Medline]
29 Xiao H, Hasegawa T, Miyaishi O, Ohkusu K, Isobe K. Sodium butyrate induces NIH3T3 cells to senescence-like state and enhances promoter activity of p21WAF/CIP1 in p53-independent manner. Biochem Biophys Res Commun 1997;237:457-60.[Medline]
30 Vaziri C, Stice L, Faller DV. Butyrate-induced G1 arrest results from p21-independent disruption of retinoblastoma protein-mediated signals. Cell Growth Differ 1998;9:465-74.[Abstract]
31 Luo Y, Marx SO, Kiyokawa H, Koff A, Massague J, Marks AR. Rapamycin resistance tied to defective regulation of p27Kip1. Mol Cell Biol 1996;16:6744-51.[Abstract]
32 Juan G, Gruenwald S, Darzynkiewicz Z. Phosphorylation of retinoblastoma susceptibility gene protein assayed in individual lymphocytes during their mitogenic stimulation. Exp Cell Res 1998;239:104-10.[Medline]
33
Cote RJ, Shi Y, Groshen S, Feng AC, Cordon-Cardo C, Skinner
D, et al. Association of p27Kip1 levels with recurrence and survival in patients with stage C
prostate carcinoma. J Natl Cancer Inst 1998;90:916-20.
34 Guo Y, Sklar GN, Borkowski A, Kyprianou N. Loss of the cyclin-dependent kinase inhibitor p27(Kip1) protein in human prostate cancer correlates with tumor grade. Clin Cancer Res 1997;3:2269-74.[Abstract]
35 Cheville JC, Lloyd RV, Sebo TJ, Cheng L, Erickson L, Bostwick DG, et al. Expression of p27kip1 in prostatic adenocarcinoma. Mod Pathol 1998;11:324-8.[Medline]
36 Yang RM, Naitoh J, Murphy M, Wang HJ, Phillipson J, deKernion JB, et al. Low p27 expression predicts poor disease-free survival in patients with prostate cancer. J Urol 1998;159:941-5.[Medline]
37 Valenzuela MT, Nunez MI, Villalobos M, Siles E, McMillan TJ, Pedraza V, et al. A comparison of p53 and p16 expression in human tumor cells treated with hyperthermia or ionizing radiation. Int J Cancer 1997;72:307-12.[Medline]
38 Fang X, Jin X, Xu HJ, Liu L, Peng HQ, Hogg D, et al. Expression of p16 induces transcriptional downregulation of the RB gene. Oncogene 1998;16:1-8.[Medline]
39 Cordon-Cardo C, Latres E, Drobnjak M, Oliva MR, Pollack D, Woodruff JM, et al. Molecular abnormalities of mdm2 and p53 genes in adult soft tissue sarcomas. Cancer Res 1994;54:794-9.[Abstract]
40 Xiao ZX, Chen J, Levine AJ, Modjtahedi N, Xing J, Sellers WR, et al. Interaction between the retinoblastoma protein and the oncoprotein MDM2. Nature 1995;375:694-8.[Medline]
41 Lammie GA, Peters G. Chromosome 11q13 abnormalities in human cancer. Cancer Cells 1991;3:413-20.[Medline]
42 Lebwohl DE, Muise-Helmericks R, Sepp-Lorenzino L, Serve S, Timaul M, Bol R, et al. A truncated cyclin D1 gene encodes a stable mRNA in a human breast cancer cell line. Oncogene 1994;9:1925-9.[Medline]
43 Ruizeveld de Winter JA, van Weerden WM, Faber PW, van Steenbrugge GJ, Trapman J, Brinkmann AO, et al. Regulation of androgen receptor expression in the human heterotransplantable prostate carcinoma PC-82. Endocrinology 1992;131:3045-50.[Abstract]
44
Tan J, Sharief Y, Hamil KG, Gregory CW, Zang DY, Sar M, et
al. Dehydroepiandrosterone activates mutant androgen receptors expressed in the
androgen-dependent human prostate cancer xenograft CWR22 and LNCaP cells. Mol
Endocrinol 1997;11:450-9.
45 Goldenberg SL, Bruchovsky N. Androgen withdrawal therapy: new perspectives in the treatment of prostate cancer. In: Raghavan D, Scher HI, Leibel SA, Lange P, editors. Principles and practice of genitourinary oncology. Philadelphia (PA): Lippincott-Raven; 1997. p. 583-90.
46
Bentel JM, Tilley WD. Androgen receptors in prostate cancer. J Endocrinol 1996;151:1-11.
47 Koivisto P, Kononen J, Palmberg C, Tammela T, Hyytinen E, Isola J, et al. Androgen receptor gene amplification: a possible molecular mechanism for androgen deprivation therapy failure in prostate cancer. Cancer Res 1997;57:314-9.[Abstract]
48
Zhau HY, Chang SM, Chen BQ, Wang Y, Zhang H, Kao C, et
al. Androgen-repressed phenotype in human prostate cancer. Proc Natl Acad Sci U S A 1996;93:15152-7.
49
Kokontis JM, Hay N, Liao S. Progression of LNCaP prostate
tumor cells during androgen deprivation: hormone-independent growth, repression of
proliferation by androgen, and role for p27Kip1 in androgen-induced cell cycle arrest. Mol
Endocrinol 1998;12:941-53.
50 Kokontis J, Takakura K, Hay N, Liao S. Increased androgen receptor activity and altered c-myc expression in prostate cancer cells after long-term androgen deprivation. Cancer Res 1994;54:1566-73.[Abstract]
Manuscript received January 29, 1999; revised August 4, 1999; accepted August 30, 1999.
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