Increased cell growth and tumorigenicity in human prostate LNCaP cells by overexpression to cyclin D1

Deregulated expression of cyclin D1 has been found in several types of human tumors. In order to investigate factors involved in human prostate cancer progression, we studied the effects of cyclin D1 overexpression on human prostate cancer cell proliferation and tumorigenicity by transfecting LNCaP cells with a retroviral vector containing human cyclin D1 cDNA. When compared to the parental and control-vector transfected LNCaP cells, these cyclin D1-transfected cells had more cells in S-phase and lower growth factor requirements. Furthermore, these cells grew more in androgen-free medium. We also detected higher levels of Rb phosphorylation and E2F-1 protein levels in LNCaP/cyclin D1 cells than that in the parental and vector control cells in medium with or without androgen. Cyclin D1 transfected clones formed tumors more rapidly than control and parental cells. These tumors were refractory to the androgen-ablation treatment by castration, whereas tumors from parental and vector-control LNCaP cells regressed within 4 weeks after castration. These results suggest that overexpression of cyclin D1 changes the growth properties, increases tumorigenicity and decreases the requirement for androgen stimulation in LNCaP cells both in vitro and in vivo.     

Idiopathic megacolon

Prostatic adenocarcinoma has a divergent response to androgen ablation and a varied long-term prognosis. BCL-2 is a proto-oncogene that prevents programmed cell death. Since androgen withdrawal induces apoptosis, it has been postulated that BCL-2 may play a role in androgen resistance. Neuroendocrine cells have been demonstrated in prostate cancer and have an adverse influence on long-term prognosis. This study demonstrates a proportional relationship between the tissue levels of BCL-2 and the neuroendocrine marker, neuron-specific enolase in 11 of 13 cases of primary prostate cancer. This relationship does not appear to exist in metastatic prostate cancer or in most nonprostate cancers. Direct immunohistochemical staining confirmed BCL-2 in six of the primary tumors, and these BCL-2-containing cells appeared to be intimately associated with tumor neuroendocrine cells.     

Inhibition of N-linked glycosylation by tunicamycin enhances sensitivity to cisplatin in human head-and-neck carcinoma cells

Using an antibody specific for dually phosphorylated extracellular-regulated kinases 1 and 2, we have examined 82 primary and metastatic prostate tumor specimens for the presence of activated mitogen-activated protein (MAP) kinase. Nonneoplastic prostate tissue showed little or no staining with activated MAP kinase antiserum. In prostate tumors, the level of activated MAP kinase increased with increasing Gleason score and tumor stage. In a separate analysis, tumor samples from two patients showed no activation of MAP kinase before androgen ablation therapy; however, following androgen ablation treatment, high levels of activated MAP kinase were detected in the recurrent tumors. Collectively, these data suggest an increase in the activation of the MAP kinase signal transduction pathway as prostate cancer progresses to a more advanced and androgen-independent disease.     

Reduction in surface urokinase receptor forces malignant cells into a protracted state of dormancy

Considerable evidence links urokinase plasminogen activator (uPA) bound to its surface receptor (uPAR) with enhanced invasiveness of cancer cells. By blocking uPAR expression in human epidermoid carcinoma cells (HEp3), we have now identified an additional and novel in vivo function for this receptor by showing that receptor-deficient cells enter a state of dormancy reminiscent of that observed in human cancer metastasis. Its main characteristic is survival without signs of progressive growth. Five clones transfected with a vector expressing uPAR antisense RNA under the beta-actin promoter were isolated and shown to have uPAR (at the mRNA and protein levels) reduced by 50 to 80%; four clones, transfected with vector alone and having uPAR levels similar to those of parental cells, served as controls. In confirmation of our previous results, reduced uPAR always coincided with a significantly reduced invasiveness. Each of the control clones produced rapidly growing, highly metastatic tumors within 2 wk of inoculation on chorioallantoic membranes (CAMs) of chick embryos. In contrast, each of the clones with low surface uPAR, whose proliferation rate in culture was indistinguishable from controls, remained dormant for up to 5 mo when inoculated on CAMs. Thus, the reduction in uPAR altered the phenotype of HEp3 tumor cells from tumorigenic to dormant. Although protracted, tumor dormancy was not permanent since in spite of maintaining low uPAR levels, each of the in vivo-passaged antisense clones eventually reemerged from dormancy to initiate progressive growth and to form metastases at a level of 20 to 90% of that of fully malignant control. This observation suggested that other factors, whose expression is dependent on cumulative and prolonged in vivo effects, can compensate for the lack of a full complement of surface uPAR required for the expression of malignant properties. These "reemerged," uPAR-deficient clones were easily distinguishable from the vector-transfected controls by the fact that after only 1 wk in culture, the invasion of CAM by all five clones and tumorigenicity of four of the five clones were reduced back to the values observed before in vivo maintenance. In contrast, dissociated and in vitro-grown cells of control tumors were fully invasive and produced large, metastatic tumors when reinoculated on CAMs. Quantitation of the percent of apoptotic and S-phase cells in vivo, in the control and uPAR-deficient, dormant clones, showed that the mechanism responsible for the dormancy was a diminished proliferation.     

Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) in human prostate cancer

Progression of prostate cancer from an androgen sensitive to androgen insensitive tumor has previously been shown to be accompanied by a change in alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) in a rat model of prostate cancer. This change results in loss of the FGF-R2(IIIb) isoform and predominant expression of the FGF-R2(IIIc) isoform. We sought to determine whether this change in FGF-R2 splicing is also associated with androgen insensitivity in human prostate tumors. We analysed three well characterized human prostate cancer cell lines and three metastatic prostate tumors which have been maintained as xenografts in nude mice. One of the cell lines, LNCaP, and two of the xenografts, DUKAP-1 and DUKAP-2, have been characterized as androgen sensitive, whereas two of the cell lines, DU-145 and PC-3, and one of the xenografts, DU9479, display androgen independent growth. Using an RT-PCR based assay, we demonstrated that progressive loss of the FGF-R2(111b) isoform correlated with androgen insensitivity in these human prostate cancer models. These findings lend support to the hypothesis that that loss of FGF-R2(IIIb) may be one step in a series of events which lead to progression of human prostate cancer.     

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

The molecular mechanism of androgen-independent growth of prostate cancer after androgen ablation was explored in LNCaP cells. An androgen-dependent clonal subline of the LNCaP human prostate carcinoma cell line, LNCaP 104-S, progressed to a slow growing stage (104-R1) and then to a faster growing stage (104-R2) during more than 2 yr of continuous culture in the absence of androgen. Androgen-induced proliferation of 104-S cells is inhibited by the antiandrogen Casodex, while proliferation of 104-R1 and 104-R2 cells is unaffected by Casodex. This indicates that proliferation of 104-R1 and 104-R2 cells is not supported by low levels of androgen in the culture medium. Compared with LNCaP 104-S cells, both 104-R1 and 104-R2 cells express higher basal levels of androgen receptor (AR), and proliferation of these two cell lines is paradoxically repressed by androgen. After continuous passage in androgen-containing medium, 104-R1 cells reverted back to an androgen-dependent phenotype. The mechanism of androgenic repression of 104-R1 and 104-R2 sublines was further evaluated by examining the role of critical regulatory factors involved in the control of cell cycle progression. At concentrations that repressed growth, androgen transiently induced the expression of the cyclin-dependent kinase (cdk) inhibitor p21waf1/cip1 in 104-R1 cells, while expression of the cdk inhibitor p27Kip1 was persistently induced by androgen in both 104-R1 and 104-R2 cells. Induced expression of murine p27Kip1 in 104-R2 cells resulted in G1 arrest. Specific immunoprecipitates of Cdk2 but not Cdk4 from androgen-treated 104-R1 cells contained both p21waf1/cip1 and p27Kip1. This observation was confirmed by in vitro assay of histone H1 and Rb (retinoblastoma protein) phosphorylation by the proteins associated with the immune complex. Furthermore, inhibition of Cdk2 activity correlated with the accumulation of p27Kip1 and not p21waf1/cip1. From these results we conclude that androgenic repression of LNCaP 104-R1 and 104-R2 cell proliferation is due to the induction of p27Kip1, which in turn inhibits Cdk2, a factor critical for cell cycle progression and proliferation.     

Inhibition of caspases by cytokine response modifier A blocks androgen ablation-mediated prostate cancer cell death in vivo

Androgen withdrawal is a major therapeutic modality in the treatment of prostate cancer. Although tumors initially respond, they subsequently relapse, and these recurring tumors are androgen independent. To examine possible mechanisms to explain the androgen independence of prostate cancer, we have expressed cytokine response modifier A (CrmA), a competitive inhibitor of caspases, interleukin 1beta-converting enzyme-like proteases, which mediate apoptotic cell death, in the human androgen-dependent prostate cancer cell line LNCaP. LNCaP cells require androgens for continuous growth in culture and to form tumors in nude mice. The expression of CrmA in LNCaP cells prevented the decreased growth rate induced by androgen withdrawal in tissue culture. When CrmA-expressing LNCaP (LNCaP-CrmA) cells were implanted s.c. in nude mice, the tumors grew six times faster than parental cells. Androgen ablation by castration before tumor implantation suppressed the ability of control LNCaP cells expressing nonfunctional CrmA mutant (R291T) to form tumors, but LNCaP-CrmA cells formed tumors similar in size to those formed in normal mice. When orchiectomy was performed 10 days after tumor implantation, control LNCaP cells expressing a nonfunctional CrmA mutant (R291T) regressed, but LNCaP-CrmA tumors continued to grow. Thus, inhibition of caspases prevents androgen withdrawal-induced prostate cancer cell death, suggesting that caspase activation is normally an important part of this process.     

Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo

Normal (nonneoplastic) human prostatic secretory epithelial cells do not express the bcl-2 protein. However, a recent immunohistochemical survey of neoplastic human prostate tissues showed that a fraction of primary untreated prostate adenocarcinoma cells expressed this apoptosis-suppressing oncoprotein at significant levels (Colombel et al., Am. J. Pathol., 143: 390-400, 1993). Additionally, a number of hormone-refractory prostatic adenocarcinomas obtained from hormonally-treated patients (subsequent to surgical or drug castration therapy) were examined and were found to be uniform in their elevated expression of bcl-2 oncoprotein. The results of this preliminary survey imply that bcl-2 expression distinguishes a subgroup of primary human prostate cancers and that the expression of this protein might be a factor enabling prostate cancer cells to survive in an androgen-deprived environment. The current study was undertaken to determine the degree to which overexpression of bcl-2 can protect human prostate cancer cells from apoptotic stimuli in vitro and in vivo. Human prostate cancer cells (LNCaP) were transfected with a neomycin-selectable eucaryotic expression vector containing cDNA encoding human bcl-2. Transfected clonal variants that express bcl-2 protein (LNCaP/bcl-2) were unaltered with regard to their basal growth rate in 10% serum-containing medium, or with regard to their expression of the differentiated human prostate cell gene products prostate-specific antigen or androgen receptor protein. The bcl-2-transfected clones were altered, however, with regard to their growth rate in charcoal-stripped serum lacking dihydrotestosterone. Additionally, in contrast to the parental or control-transfected cell lines, LNCaP/bcl-2 cells were highly resistant to a variety of apoptotic stimuli in vitro including serum starvation and 10 nM phorbol ester (phorbol 12-myristate 13-acetate) supplementation of the medium. Lastly, the overexpression of bcl-2 by these prostate cancer cells altered their tumorigenic potential in a nude mouse assay. s.c. injections of 10(6) LNCaP/bcl-2 cells into male nude mice resulted in earlier and larger tumor formation compared to an equivalent injection of parental or control-transfected LNCaP cells. When these variant cell lines were injected into castrated male nude mice, only the LNCaP/bcl-2-transformed cells gave rise to tumors. Moreover, LNCaP/bcl-2 tumors grown in intact male nude mice were refractory to the growth-inhibiting effects of castration demonstrated by parental LNCaP cells. Data obtained in this study demonstrate that the bcl-2 oncoprotein can protect prostate cancer cells from apoptotic stimuli in vitro and suggest that such protection correlates with the ability to form hormone-refractory prostate tumors in vivo.     

Development of seven new human prostate tumor xenograft models and their histopathological characterization

Seven human prostate tumor models were established by transplanting tumor fragments in NMRI athymic nude mice. Once established, the tumors were serially transplantable in both NMRI and BALB/c nude mice. The xenografts originated from primary prostatic carcinomas (prostatectomy specimens), transurethral resection material, and metastatic lesions (pelvic lymph nodes and scrotal skin). Histological examination revealed that, in the course of several mouse passages (8 to 23), tumors retained their resemblance to the original patient material. The PC-295, PC-310, PC-329, and PC-346 tumors are dependent on androgens for their growth. The PC-324, PC-339, and PC-374 tumors are androgen independent, although growth of PC-374 tumors still seemed androgen sensitive. All tumors are diploid, except for the PC-374, which is tetraploid. The diploid PC-295 tumor has an additional small population of tetraploid cells. All xenografts displayed a heterogeneous expression pattern of the androgen receptor except for the PC-324 and PC-339 tumors in which the androgen receptor could not be detected. Prostatic acid phosphatase and prostate-specific antigen were retained during serial transplantation in all tumors but the PC-324 and PC-339. This panel of permanent human prostate tumor models comprises tumors representing both the androgen-dependent and -independent stages of human prostate cancer with various degrees of differentiation and, therefore, is of great value for the study of many aspects of growth and progression of human prostate cancer.     

Aprotinin inhibits plasmin-induced platelet activation during cardiopulmonary bypass

PURPOSE: We correlated the expression of bcl-2 with accumulation of p53 protein in bone marrow metastases from patients with androgen independent prostate cancer and a history of hormonal ablation therapy. These results were correlated with clinical parameters, including the extent of bone marrow metastases and patient survival. MATERIALS AND METHODS: All 43 patients studied had evidence of prostate cancer progression following androgen deprivation therapy and histologically confirmed bone marrow metastases. Decalcified tissue sections were used for immunohistochemical evaluation of bcl-2 protein and p53 protein accumulation. RESULTS: We previously established that p53 protein accumulation as detected by immunohistochemistry is a reliable indicator of p53 gene mutation in prostate cancer. Immunoreactivity was demonstrated for p53 protein in 22 of 43 cases and for bcl-2 protein in 14. A total of 28 cases (65%) exhibited immunohistochemical evidence of p53 and/or bcl-2 expression, and 15 (35%) were negative for p53 and bcl-2. The expression of bcl-2 and accumulation of p53 were independent events (p < 0.01). The expression of bcl-2 or accumulation of p53 protein in prostate cancer metastases did not significantly influence patient survival or the extent of metastatic disease. CONCLUSIONS: The presence or absence of p53 protein accumulation and/or bcl-2 expression did not correlate with tumor burden or patient survival in stage D androgen independent prostate cancer bone marrow metastases. The expression of bcl-2 protein occurs independently of and is inversely correlated with p53 mutations in advanced prostate cancer.     

Neuroendocrine differentiation in prostatic carcinoma during hormonal treatment

OBJECTIVES: Neuroendocrine differentiation (NED) is a common feature in adenocarcinoma of the prostate. Several studies suggest that NED may have a major impact on cancer progression as neuroendocrine (NE) secretory products have been shown to possess growth stimulatory effects. NED has also been proposed to constitute part of the mechanism by which a prostate cancer cell progresses toward androgen independence as NE tumor cells have been demonstrated to be devoid of androgen receptor immunoreactivity. In this retrospective study, we evaluated NED status in prostate cancer specimens from patients undergoing androgen ablation therapy. METHODS: The degree of NED in transurethral resection of the prostate (TURP) samples from 53 patients with prostate cancer was investigated by immunocytochemistry using polyclonal rabbit immunoglobin G (IgG) against chromogranin A (CgA). Changes in NED with time were determined by a manual semiquantitative cell counting method. RESULTS: During androgen withdrawal therapy, 21 tumors (40%) displayed increased NED concomitant with histopathologic tumor progression, whereas 29 carcinomas (55%) showed no change in NED status. However, a majority of the histopathologically unchanged tumors displayed marked NED at the first TURP and an increase in NED was by definition not possible. In only 3 cases (5%) was a decrease in NED observed with time. CONCLUSIONS: Androgen ablation therapy may be a contributing factor to the increase in NED of prostatic adenocarcinoma with time, and our findings imply that androgen withdrawal therapy enhances the selection and progression of NED, androgen-independent tumor cells.     

G-protein beta3 subunit gene (GNB3) variant in causation of essential hypertension

OBJECTIVES: To evaluate the response of testicular androgen ablation in patients with advanced prostate cancer with a biochemical recurrence after finasteride or combined finasteride and flutamide therapy. METHODS: Eighteen hormone na?ve men with advanced prostate cancer (10 with detectable prostate-specific antigen [PSA] levels after radical prostatectomy, 4 with rising PSA levels after definitive radiation therapy, and 4 with Stage D2 disease) were treated with finasteride (5 mg/day) alone or in combination with flutamide (250 mg three times a day). All men experienced an initial reduction in serum PSA, but later had treatment failure with two consecutive rising PSA measurements. All men were then treated with testicular androgen ablation (bilateral orchiectomy in 15 and luteinizing hormone-releasing hormone analogue in 3). RESULTS: Overall, serum PSA declined by more than 80% in 15 (83%) of 18 and to undetectable levels in 14 (78%) of 18. With a median+/-semi-interquartile range follow-up of 22+/-14.5 months from the initiation of hormone therapy, 12 (67%) of 18 currently have undetectable PSA levels. Two men having rising serum PSA levels above 100 ng/mL and 1 man has died from complications of metastatic prostate cancer. CONCLUSIONS: Testicular androgen ablation effectively lowers serum PSA levels in most men with advanced prostate cancer who have experienced a biochemical recurrence despite initial response and subsequent relapse on finasteride or combined finasteride and flutamide therapy.     

Etoposide sensitivity of human prostatic cancer cell lines PC-3, DU 145 and LNCaP

Metastatic prostatic cancer is typically refractory to androgen ablation therapy due to the presence of androgen-independent clones in the neoplasia. A therapeutical approach which could effectively control androgen-dependent and independent cells is, thus, needed. Maybe the failure of certain cancer cells to engage in apoptosis could explain the inherent drug resistance of many tumors. Anyway, these cells can retain the ability to undergo apoptosis in response to an adequate stimulus. We tested whether etoposide, a topoisomerase II inhibitor, could induce apoptosis in androgen-dependent (LNCaP) as well as independent (PC-3 and DU 145) human prostate cancer cell lines. Morphological examination was performed, as it is regarded as one of the most reliable parameters for the detection of apoptotic changes. Complementarily, biochemical and flow cytometric studies were also used. Characteristical changes of apoptosis were demonstrated in PC-3, Du 145, and LNCaP cancer cells after treatment with etoposide. These cells, thus, retain the ability to undergo apoptosis under adequate conditions, in a promising approach to hormone refractory prostate cancer therapy.     

Inhibition of T-tropic HIV strains by selective antagonization of the chemokine receptor CXCR4

Mutations in the androgen receptor (AR), that alter steroid hormone specificity have been identified in a series of androgen-independent prostate cancers. To address the functional properties of these mutant ARs that may have contributed to their selection in vivo, responses to a series of steroid hormones and antiandrogens were assessed. CV-1 cells were cotransfected with wild-type or mutant ARs and a luciferase reporter plasmid regulated by an androgen-responsive element. Dose-response curves were analyzed for 5alpha-dihydrotestosterone, the most active androgen in normal prostate, and androstenedione, a major androgen derived from the adrenals. Although the mutant ARs responded to both of these steroids, the responses were equivalent to or less than the wild-type AR. In contrast, responses to flutamide, a competitive antagonist of the wild-type AR, were markedly increased by three of the mutations. Similar responses were observed with a second antiandrogen, nilutamide. Bicalutamide, another antiandrogen related to flutamide, remained an antagonist for these mutant ARs. Finally, flutamide was observed to be a weak partial agonist of the wild-type AR in this system. These results indicate that flutamide used in conjunction with androgen ablation therapy for prostate cancer may select for tumor cells with flutamide-inducible ARs.     

Castration-induced apoptosis in the rat ventral prostate is associated with increased expression of genes encoding insulin-like growth factor binding proteins 2,3,4 and 5

Insulin-like growth factor binding proteins (IGFBPs) have recently been demonstrated to act as regulators of apoptosis in vitro in both prostate and breast cancer cell lines. We show here that gene expression of IGFBP-2,-3,-4 and -5 increase rapidly in the rat ventral prostate following castration. Increases in IGFBP mRNA levels were detectable by Northern blotting by 6 hours and reached 5 to 10 fold of control levels at 72 hours after castration. Apoptosis in the ventral prostate, as detected in situ by the TUNEL method, was also induced as early as 6 hours after castration. TRPM-2/clusterin, a gene known to be associated with involution of the prostate, was not detected in sham castrated controls but was expressed by 24 hours following androgen ablation. IGF-I mRNA levels increased to 160% of control values within 6 hours following castration, then decreased gradually over the next 72 hours to 35% of control. Affinity labelling experiments demonstrated that IGF-I receptor levels increased initially after castration with peak binding at 24 hours, then declined to levels lower than control. These results suggest that rapid induction of IGFBPs in the rat ventral prostate following androgen ablation may play a role in apoptosis and involution of the prostate gland.