Prostate Tumor Cell Growth Research Articles

Inhibition of growth and increased expression of insulin-like growth factor-binding protein-3 (IGFBP-3) and -6 in prostate cancer cells stably transfected with antisense IGFBP-4 complementary deoxyribonucleic acid.

Insulin-like growth factor-binding proteins (IGFBPs) both stimulate and inhibit IGF activity, and in the M12 prostate cancer cell line, overexpression of IGFBP-4 was shown to delay tumorigenesis while decreasing the production of IGFBP-2. We have performed the reverse experiment, inhibition of IGFBP-4 expression with antisense complementary DNA, in two prostate tumor cell lines, ALVA-31 and M12. Expression of antisense messenger RNA transcripts was verified by RNase protection assays, and inhibition of mature IGFBP-4 in cell medium was demonstrated by Western blotting. Both transfected lines (ALVA-31asBP4 and M12asBP4) proliferated more slowly in monolayer culture than parental controls. Colony formation in soft agar was strongly inhibited in both cases, and the rate of tumor formation and growth in male athymic nude mice injected with M12asBP4 was markedly reduced relative to that in mice receiving M12 control cells. Apoptosis induced by the topoisomerase inhibitor etoposide was also enhanced in transfected cells. The effects on colony formation in soft agar and tumor formation in mice were maintained for the duration of the experiments, in contrast to the delayed growth observed in the previous study of IGFBP-4 overexpression. A significant difference was found in the patterns of IGFBP expression; production of both messenger RNA and protein for IGFBP-3 and IGFBP-6 was greatly increased in the M12asBP4 and ALVA31asBP4 cell lines. Up-regulation of these binding proteins has been observed in association with actions of 1,25-dihydroxyvitamin D(3) in prostate cancer cells, and the data suggest a role for IGFBP-3 and IGFBP-6 in the suppression of prostate tumor cell growth.

Isolation and Characterization of the Promoter of the Human Prostate Cancer-specific DD3 Gene

Recently, we have described a novel gene, DD3, which is one of the most prostate cancer-specific genes described to date (Bussemakers, M. J. G., van Bokhoven, A., Verhaegh, G. W., Smit, F. P., Karthaus, H. F. M., Schalken, J. A., Debruyne, F. M. J., Ru, N., and Isaacs, W. B. (1999) Cancer Res. 59, 5975-5979). The prostate cancer-specific expression of DD3 indicates that the DD3 gene promoter is a promising tool for the treatment of prostate cancer. To identify the promoter elements that are responsible for the prostate cancer-specific expression of DD3, we have isolated and characterized the DD3 promoter. Sequence analysis of the DD3 5'-flanking region was performed and several promoter-human growth hormone reporter constructs were prepared, which were transiently transfected in the DD3-positive cell line LNCaP and several DD3-negative cell lines. Using a 500-base pair DD3 promoter construct, we could detect promoter activity in LNCaP cells, which was not affected by increasing the size of the constructs. Truncated constructs, however, showed an increased transcriptional activity, suggesting the presence of a silencer that negatively regulates the expression of DD3. DNase-I footprint analysis, using nuclear extracts from LNCaP cells, revealed the presence of three DNase-I-protected areas within the DD3 proximal promoter. We show that the high mobility group I(Y) protein binds to one of the DNase-I-protected areas and recruits another, yet unidentified, protein to the DD3 promoter in LNCaP cells.

Effects of Phytoestrogens on Growth and DNA Integrity in Human Prostate Tumor Cell Lines: PC-3 and LNCaP

Prostate cancer is one of the most common male cancers in Western countries, yet the incidence of this fatal disease remains low in Asian populations. Environmental factors such as diet play an important role in hormone-dependent cancer etiology, and a high phytoestrogen intake may be one factor contributing to the low prostate cancer mortality in Eastern populations. In this study, we investigated the effects of the phytoestrogens genistein, daidzein, coumestrol, and equol on cell growth and DNA damage (strand breakage) in two human prostate tumor cell lines: androgen receptor-positive LNCaP and androgen receptor-negative PC-3. Each compound caused growth inhibition at physiologically relevant concentrations (<10 μM). Genistein induced DNA damage in both cell lines at <10 μM. Daidzein inhibited cell growth at 10-100 μM yet had no effect on DNA damage at up to 500 μM. Thus, despite their structural similarities, different phytoestrogens inhibit prostate tumor cell growth by independent mechanisms.

Synergistic effects of thearubigin and genistein on human prostate tumor cell (PC-3) growth via cell cycle arrest

There is evidence that habitual consumption of green tea by Japanese men is correlated with a reduction in cancers, including prostate; soybean isoflavones are also associated with increased protection. The present study compared the anti-proliferative effect of black tea ( Camellia sinensis) polyphenol, thearubigin (TR), alone or combined with the isoflavone genistein, on human prostate (PC-3) carcinoma cells. TR administered alone did not result in any alteration of cell growth. When combined with genistein, however, TR significantly inhibited cell growth and induced a G2/M phase cell cycle arrest in a dose dependent manner. These findings indicate the potential use of combined phytochemicals to provide protection against prostate cancer.

P16/MTS1/INK4A suppresses prostate cancer by both pRb dependent and independent pathways.

Tumor suppressor gene p16 is a cyclin-dependent kinase inhibitor and an important negative cell cycle regulator. The inactivation of p16 appears to be a common event in prostate cancer. Replacement of p16 inhibits prostate tumor cell growth, but the mechanism is not known. Human prostate cancer cell lines PPC-1, which has an inactivated p16, and DU145, which has a nonfunctional retinoblastoma Rb protein (pRb), were used to determine the possible mechanism of p16 mediated growth inhibition. PPC-1 cells treated with 5-aza-2'-deoxycytidine (5-aza-dC), a demethylating agent, induced p16 expression, inhibited cell growth, and induced senescence. Similarly, PPC-1 cells transduced by an adenoviral vector containing the p16 gene (AdRSVp16) produced a p16 protein that suppressed cellular proliferation and induced senescence. Co-staining of AdRSVp16-transduced PPC-1 cells by p16 immunohistochemistry and by beta-galactosidase substrate X-gal showed that the morphologically enlarged cells expressed both p16 and senescence-associated beta-galactosidase. In contrast, AdRSVp16 did not induce senescence in DU145 cells, but did inhibit its growth. However, when wild-type pRb was introduced in DU145 cells, AdRSVp16 was able to induce senescence. Thus, the mechanism by which p16 suppressed prostate cancer was dependent on the pRb functional status of cells whereby p16 caused pRb+ cells to undergo inhibition by senescence, whereas pRb- cells were also inhibited, but not by senescence.

Characterization of nerve growth factor precursor protein expression by human prostate stromal cells: a role in selective neurotrophin stimulation of prostate epithelial cell growth.

Nerve growth factor (NGF) immunoreactive proteins derived from human prostatic stromal cells (hPS) have been implicated in the paracrine regulation of prostate epithelial cell growth. However, mature NGFbeta does not appear to be expressed by these cells. In order to determine whether NGF precursors are expressed by these cells, we investigated the potential processing and expression of precursor forms of NGF by human prostatic stromal cells, and examined the effects of NGF precursor moieties along with the other members of the neurotrophin family of gene products on soft agar colony formation of prostate epithelial cells. Specific antibodies to the peptide domains defined as N4 and L38, and the NGFbeta moiety of prepro-NGF, were used in immunoblot assays to characterize the molecular weight forms of precursor NGF secreted by human prostatic stromal cells. The potential processing of NGF precursors with two enzymes, NGFgamma and trypsin, was performed by incubation with stromal cell secretory protein containing precursor NGF. The selective effects of the N4, L38, and NGFbeta peptide domains of precursor NGF, along with the remaining members of the neurotrophin family, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), were examined for their ability to stimulate growth of prostate tumor epithelial cells in an assay of soft agar colony formation. Immunoblot analysis of stromal cell secretory protein identified NGF precursors of 35 kDa and 27 kDa, along with the partially processed 22-kDa form of pro-NGF, whereas mature NGFbeta was not observed. Treatment of precursor NGF with NGFgamma and trypsin did not produce the large intermediate forms of pro-NGF, although these two enzymes did appear to cleave the N-terminal peptide from NGFbeta. Of the N4, L38, and NGFbeta peptide domains of precursor NGF, only NGFbeta significantly stimulated the anchorage-independent growth of TSU-pr1 prostate epithelial cells in soft agar. The other members of the neurotrophin family of gene products had no effect on the anchorage-independent growth of prostate tumor cells. Human prostate stromal cells secrete the 35-kDa and 27-kDa precursor forms of NGF arising from alternate start sites, and the partially processed 22-kDa form of pro-NGF. Whereas the N4, L38, and NGFbeta peptide domains present within pro-NGF were previously shown to induce phosphorylation of the high-affinity NGF receptor, tropomyosin receptor kinase (Trk), only the NGFbeta moiety was able to stimulate anchorage-independent growth of prostate tumor cells. Likewise, the other neurotrophin family members did not stimulate anchorage-independent growth of prostate tumor cells. Hence, it would appear that NGF may be the predominant neurotrophic growth factor for prostate growth, albeit via precursor forms of NGF, and that its effect appears to be selectively mediated via the NGFbeta moiety of these NGF precursors.

Androgens decrease and retinoids increase the expression of insulin-like growth factor-binding protein-3 in LNCaP prostatic adenocarcinoma cells

Changes in circulating levels of insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) have been related to prostate cancer, but the nature and the significance of this relationship remains elusive. Recent reports suggest that modulation of the production of IGFBP-3 by retinoids may affect growth of breast and prostate tumor cells. In the present study we explored whether androgens (R1881), retinoids (all- trans- and 9- cis-retinoic acid: atRA and 9cRA), deltanoids (1α,25-dihydroxycholecalciferol: VD 3) and thyroid hormone (triiodothyronine: T3) influence the production of IGFBPs by LNCaP prostatic adenocarcinoma cells and whether the observed changes affect tumor cell growth. Northern blot experiments demonstrated that LNCaP cells express IGFBP-2, -3, -4 and (to a small extent) -5. IGFBP-4 and -5 were not measurably affected by the mentioned agonists. At a growth promoting concentration (10 −10 M), R1881 increased IGFBP-2 transcript levels two- to three-fold and this effect was neutralized by atRA and VD 3. Similar effects could not be demonstrated, however, at the protein level using Western ligand blotting. R1881 decreased and atRA increased the mRNA levels of IGFBP-3 and these effects were confirmed by Western ligand blotting and by radioimmunoassay. The effects of atRA were mimicked by 9cRA and by a specific RAR agonist but not by a RXR agonist. VD 3 and T3 had no significant effect on IGFBP-3 secretion but respectively enhanced or decreased the effect of 9cRA. The effects of retinoids required high concentrations (10 −6–10 −5 M) that also induced growth inhibition. R1881, however, decreased IGFBP-3 at growth promoting (10 −10 M) as well as at growth inhibitory (10 −8 M) concentrations. Moreover, under serum-free conditions, we were unable to demonstrate any growth modulating effect of IGFBP-3. It is concluded that several agonists acting by nuclear receptors affect IGFBP-3 secretion by LNCaP cells but that the functional significance of these changes warrants further investigation.

Involvement of heparin affin regulatory peptide in human prostate cancer.

Heparin affin regulatory peptide (HARP) composes, together with midkine (MK), a new family of heparin-binding growth/differentiation factors. Recently, HARP was incriminated in cancer progression, as an angiogenic factor and as a tumor growth factor. In this study, we analyzed the possible involvement of HARP in human prostate cancer (Pca). The localization of HARP protein and its mRNAs in normal prostate (n = 5), benign prostate hyperplasia (BPH) (n = 7), and prostate cancer (Pca) (n = 9) was analyzed by immunohistochemistry and in situ hybridization. The mitogenic activity of this growth factor for prostate epithelial cells was determined with a thymidine incorporation assay. HARP cDNA was transfected into normal prostate epithelial (PNT-1A) cells, and their growth was evaluated by soft-agar growth assay. We found HARP protein associated with epithelial cells in PCa but not in normal prostate or BPH, while the corresponding mRNAs were located in the stromal compartment. Furthermore, HARP is mitogenic for PNT-1A, LNCaP, and DU-145 cells. Overexpression of the human HARP in PNT-1A transfected cells induced both anchorage-independent growth and growth at low serum concentrations. Our results suggest that HARP may act in a paracrine manner from mesenchymal to tumoral epithelial cells, and may play a role in the molecular mechanisms that regulate prostate tumor cell growth.

The Carboxyl-terminal Region of Biliary Glycoprotein Controls Its Tyrosine Phosphorylation and Association with Protein-tyrosine Phosphatases SHP-1 and SHP-2 in Epithelial Cells

Biliary glycoprotein (Bgp, C-CAM, or CD66a) is an immunoglobulin-like cell adhesion molecule and functions as a tumor suppressor protein. We have previously shown that the Bgp1 isoform responsible for inhibition of colonic, liver, prostate, and breast tumor cell growth contains within its cytoplasmic domain two tyrosine residues positioned in immunoreceptor tyrosine-based inhibition motif (ITIM) consensus sequences. Moreover, we determined that these residues, upon phosphorylation, associate with the protein-tyrosine phosphatase SHP-1. In this report, we have further evaluated the structural bases of the association of Bgp1 with Tyr phosphatases. First, we demonstrate that Bgp1 also associates with the SHP-2 Tyr phosphatase, but not with an unrelated Tyr phosphatase, PTP-PEST. Association of Bgp1 and SHP-2 involves the Tyr residues within the Bgp1 ITIM sequences, Val at position +3 relative to the second Tyr (Tyr-515), and the SHP-2 N-terminal SH2 domain. In addition, our results indicate that residues +4, +5, and +6 relative to Tyr-515 in the Bgp1 cytoplasmic domain play a significant role in these interactions, as their deletion reduced Bgp1 Tyr phosphorylation and association with SHP-1 and SHP-2 by as much as 80%. Together, these results indicate that both SHP-1 and SHP-2 interact with the Bgp1 cytoplasmic domain via ITIM-like sequences. Furthermore, they reveal that the C-terminal amino acids of Bgp1 are critical for these interactions.

THE EFFECT OF RHODAMINE-123 ON 3 PROSTATE TUMORS FROM THE RAT

Purpose Rhodamine-123 (Rh-123) was found to have a specific attraction to the mitochondria of tumor cells. The destruction of rat prostate tumor cells by Rh-123 is described. Materials and Methods Tissue was used from rat prostate studies of Rh-123 treatment of R3327-H, PA III prostate tumor of Pollard and the autochthonous tumor in Lobund-Wistar rats. All tissues were fixed in 10% buffered formalin, paraffin embedded and sectioned at 1 to 3 micro. for good cellular detail. Results Destructive processes were seen in all 3 rat prostate tumor models evaluated. The changes included acinar cell clumping, acinar destruction with scarring, cyst formation within acinar cells and increased stromal cells. Conclusions Since all tumor models were found to respond to Rh-123 in a similar manner, any of them could be used for the evaluation of anticancer agents. These studies demonstrated that Rh-123 was effective in suppressing the growth of hormone sensitive and insensitive rat prostate tumor cells.

THE EFFECT OF RHODAMINE-123 ON 3 PROSTATE TUMORS FROM THE RAT

Rhodamine-123 (Rh-123) was found to have a specific attraction to the mitochondria of tumor cells. The destruction of rat prostate tumor cells by Rh-123 is described. Tissue was used from rat prostate studies of Rh-123 treatment of R3327-H, PA III prostate tumor of Pollard and the autochthonous tumor in Lobund-Wistar rats. All tissues were fixed in 10% buffered formalin, paraffin embedded and sectioned at 1 to 3 micron. for good cellular detail. Destructive processes were seen in all 3 rat prostate tumor models evaluated. The changes included acinar cell clumping, acinar destruction with scarring, cyst formation within acinar cells and increased stromal cells. Since all tumor models were found to respond to Rh-123 in a similar manner, any of them could be used for the evaluation of anticancer agents. These studies demonstrated that Rh-123 was effective in suppressing the growth of hormone sensitive and insensitive rat prostate tumor cells.

Human prostate tumor angiogenesis in nude mice: metalloprotease and plasminogen activator activities during tumor growth and neovascularization of subcutaneously injected matrigel impregnated with human prostate tumor cells.

A critical aspect for growth of solid tumors is the development of a blood supply. Our objective was to establish a model for the study of angiogenesis of human prostate tumors by examining the growth of microvessels into Matrigel containing human prostate tumor cells implanted subcutaneously in nude mice. Human prostate tumor cell lines PC-3 and LNCaP were injected in Matrigel under the abdominal skin of nude mice and were harvested at 4, 8, and 14 days post-injection. The growth of tumor cells and blood vessels was examined histologically and by immunohistochemical localization of von Willibrand Factor VIII (vWF). Since plasminogen activators and matrix metalloproteases are associated with angiogenesis, the activities and molecular forms of these proteases were determined in Matrigel control and Matrigel-tumor cell subcutaneous implants. Blood vessel formation in the Matrigel implants containing LNCaP and PC-3 cells was demonstrable at 8 days post-injection. However, the pattern of blood vessel formation by the two tumor cell lines was different; PC-3 tumors showed a more invasive phenotype and smaller diameter blood vessels, whereas LNCaP tumors grew as large cellular spheroids surrounded by large, dilated blood vessels. Many blood vessels of PC-3 tumors expressed vWF by day 14 of growth, whereas most blood vessels in LNCaP tumors were immunohistochemically negative for this antigen. Mouse skin blood vessels in the same PC-3 and LNCaP tumor histological sections were positive for vWF. Matrigel contained both plasminogen activator and metalloprotease activities. The plasminogen activator activity in Matrigel control implants was totally inhibited by 4 days post-injection, indicating the presence of an inhibitor provided by the host mouse. LNCaP tumor cells injected did not have appreciable plasminogen activator activity, nor did LNCaP tumors develop plasminogen activator activity with tumor growth post-injection. PC-3 cells did have plasminogen activator activities, which were partially negated after subcutaneous injection (4 days), but then increased again by 8 days post-injection. This increase in plasminogen activator activity was due to urokinase (about 54 kDa) produced by the tumor and not by the mouse host (mouse urine urokinase about 44 kDa). Matrigel alone demonstrated gelatinase B (about 95 kDa) activity in zymograms, and gained considerable gelatinase A (about 70 and 74 kDa) activity after subcutaneous implantation. No metalloprotease activity from the tumor cells could be distinguished over that contributed by the mouse host cells in the Matrigel. Matrigel also contains caseinolytic activities of approximately 56, 80, 85, and 89 kDa. After subcutaneous injection of Matrigel, the 89 kDa form increases considerably in activity and the others are diminished. This pattern is also observed in LNCaP and PC-3 tumors post-injection, except the PC-3 tumors demonstrate increased 56 kDa activity. The subcutaneous growth of LNCaP and PC-3 prostate tumor cells in Matrigel in nude mice can be used to study tumor-induced angiogenesis. However, the organization of LNCaP and PC-3 tumor growth and the pattern of microvessels associated with each tumor are different in this system, implying that each tumor has unique influences on the pattern of microvessel development. The mode of action by which this is brought about is not known, but may be due to specific factors produced/ released by the tumor cells.

Human prostate tumor angiogenesis in nude mice: Metalloprotease and plasminogen activator activities during tumor growth and neovascularization of subcutaneously injected matrigel impregnated with human prostate tumor cells

Background A critical aspect for growth of solid tumors is the development of a blood supply. Our objective was to establish a model for the study of angiogenesis of human prostate tumors by examining the growth of microvessels into Matrigel containing human prostate tumor cells implanted subcutaneously in nude mice. Methods Human prostate tumor cell lines PC-3 and LNCaP were injected in Matrigel under the abdominal skin of nude mice and were harvested at 4, 8, and 14 days post-injection. The growth of tumor cells and blood vessels was examined histologically and by immunohistochemical localization of von Willibrand Factor VIII (vWF). Since plasminogen activators and matrix metalloproteases are associated with angiogenesis, the activities and molecular forms of these proteases were determined in Matrigel control and Matrigel-tumor cell subcutaneous implants. Results Blood vessel formation in the Matrigel implants containing LNCaP and PC-3 cells was demonstrable at 8 days post-injection. However, the pattern of blood vessel formation by the two tumor cell lines was different; PC-3 tumors showed a more invasive phenotype and smaller diameter blood vessels, whereas LNCaP tumors grew as large cellular spheroids surrounded by large, dilated blood vessels. Many blood vessels of PC-3 tumors expressed vWF by day 14 of growth, whereas most blood vessels in LNCaP tumors were immunohistochemically negative for this antigen. Mouse skin blood vessels in the same PC-3 and LNCaP tumor histological sections were positive for vWF. Matrigel contained both plasminogen activator and metalloprotease activities. The plasminogen activator activity in Matrigel control implants was totally inhibited by 4 days post-injection, indicating the presence of an inhibitor provided by the host mouse. LNCaP tumor cells injected did not have appreciable plasminogen activator activity, nor did LNCaP tumors develop plasminogen activator activity with tumor growth post-injection. PC-3 cells did have plasminogen activator activities, which were partially negated after subcutaneous injection (4 days), but then increased again by 8 days post-injection. This increase in plasminogen activator activity was due to urokinase (about 54 kDa) produced by the tumor and not by the mouse host (mouse urine urokinase about 44 kDa). Matrigel alone demonstrated gelatinase B (about 95 kDa) activity in zymograms, and gained considerable gelatinase A (about 70 and 74 kDa) activity after subcutaneous implantation. No metalloprotease activity from the tumor cells could be distinguished over that contributed by the mouse host cells in the Matrigel. Matrigel also contains caseinolytic activities of approximately 56, 80, 85, and 89 kDa. After subcutaneous injection of Matrigel, the 89 kDa form increases considerably in activity and the others are diminished. This pattern is also observed in LNCaP and PC-3 tumors post-injection, except the PC-3 tumors demonstrate increased 56 kDa activity. Conclusion The subcutaneous growth of LNCaP and PC-3 prostate tumor cells in Matrigel in nude mice can be used to study tumor-induced angiogenesis. However, the organization of LNCaP and PC-3 tumor growth and the pattern of microvessels associated with each tumor are different in this system, implying that each tumor has unique influences on the pattern of microvessel development. The mode of action by which this is brought about is not known, but may be due to specific factors produced/released by the tumor cells. Anat. Rec. 249:63–73, 1997. © Wiley-Liss, Inc.

Response of cell growth and retinoic acid receptor expression to retinoic acid in neoplastic and non-neoplastic prostate cell lines.

Retinoic acid (RA) is recognized as an inhibitor of tumorigenesis, but conversely, has also been shown to act as a tumor enhancer, therefore its role in prostate tumor cell growth was investigated. The response of two human prostate tumor cell lines (PC-3 and DU-145), and cell lines derived from a well-differentiated canine prostate adenocarcinoma (CPA) and normal canine prostate epithelium (CAPE) to all-trans RA was determined using growth curve analysis. Additionally, the constitutive expression and RA-challenged expression of retinoic acid receptors (RARs) -alpha, -beta, and -gamma mRNA was examined using Northern blotting techniques. In response to all-trans RA, the PC-3 and DU-145 cell lines showed considerable growth promotion, while CAPE and CPA cell growth was dramatically inhibited. Each cell line expressed RAR alpha and RAR gamma, with either negligible or no RAR beta transcripts being detected. RAR alpha and -gamma mRNAs detected in the four cell lines were variably regulated in response to RA, and no distinct patterns of RAR regulation that could be related to cell growth responses were observed. The data indicates that no simple association exists between the expression or regulation of RAR subtype mRNAs and the divergent growth responses to RA displayed by the prostate cell lines.

Human androgen receptor expression in prostate cancer following androgen ablation.

Metastatic prostate cancer kills patients because their tumor cells fail to respond to combined androgen blockade (CAB) or respond and then relapse. To understand the molecular basis of androgen-insensitive growth of prostate tumor cells, we evaluated changes in human androgen receptor gene (hAR) mRNA levels in patients with prostate cancer treated with CAB. The study was carried out using quantitative reverse-transcriptase polymerase chain reaction analysis. The level of hAR mRNA were compared to serum prostate-specific antigen and the mutant status of p53 in the tumor. hAR was expressed in 44 of 46 tumors from untreated patients, as opposed to 30 of 45 from those who had received CAB (p = 0.001). These 30 were from 8 of 9 stage D patients and from 22 of 36 patients on downsizing CAB therapy prior to radical prostatectomy. Expression was most often seen in high stages (56% of stage B vs. 89% of stage D) and high grades (52% of Gleason 3-7 vs. 92% of Gleason 8-10, p = 0.015). No tumor with a missense p53 mutation had hAR expression following CAB. Twenty-two patients following CAB were found to have undetectable serum prostate-specific antigen levels, while their tumor expressed hAR. hAR expression after CAB is seen preferentially in high-grade, high-stage tumors, the type of prostate carcinomas that fail to have a durable remission. Undetectable serum prostate-specific antigen from tumors that remain hAR positive may predict relapse after hormonal ablative therapy.

Anti-proliferative effect of the kinase inhibitor K252a on human prostatic carcinoma cell lines.

Members of the K252 family of kinase inhibitors have been demonstrated to inhibit a number of neurotrophin-mediated cellular responses, and to preferentially inhibit the activity of neurotrophin receptors. In this study, we examined the effects of K252a and K252b on the growth of human prostate carcinoma cells, whose growth is, in part, mediated by a prostatic nerve growth factor(NGF)-like protein(s). K252a inhibited [3H]thymidine incorporation by three androgen-independent prostate tumor cell lines (TSU-pr1, DU-145, and PC-3), under basal growth conditions, and in response to growth stimulation by human prostatic stromal (hPS) cell proteins and serum. K252b, which does not readily penetrate cell membranes, had no significant effect on [3H]thymidine incorporation by the prostate tumor cell lines. The decrease in [3H]thymidine incorporation by the cell lines in response to K252a did not appear to be the result of K252a cytotoxicity at concentrations as high as 100 nM, as measured by the Trypan blue assay for cell viability. Treatment of cells for 25 hours with 100 nM K252a resulted in accumulation of TSU-pr1, DU-145, and PC-3 cells in G0/G1, concurrent with a substantial decrease in cells synthesizing DNA. Treatment of androgen-responsive LNCaP prostatic carcinoma cells for 25 hours with 100 nM K252a also resulted in a significant decrease in DNA synthesis. Human recombinant NGF-mediated phosphorylation of a 140-kDa Trk NGF receptor in the TSU-pr1 cell line was inhibited by treatment with 100 nM K252a. Hence, K252a inhibition of Trk phosphorylation most probably contributed, in part, to the inhibition of prostate tumor cell growth in vitro. These results suggest that the mechanism of K252a action may be useful in the design of potential therapies for prostate cancer treatment.

Chemical and hormonal induction of prostate cancer in animal models

38. Loop SM, Rozanski TA, Ostenson RC. Human primary prostate tumor cell line, ALVA-31: A new model for studying the hormonal regulation of prostate tumor cell growth. Prostate 1993; 22:93-108. 39. Nakhla AM, Rosner W. Characterization of ALVA-41 cells, a new human prostatic cancer cell line. Steroids 1994;59:5869. 40. Liu X-H, Wiley HS, Meikle AW. Androgens regulate oroliferation of hum& prostate cancer cells in culturi by c77: 1472-8. 41. Turner T, Bern HA, Young P, et al. Serum-free culture of enriched mouse anterior and ventral prostatic epithelial cells in collagen gel. In Vitro Cell Develop f%ol 1990;28:722-30. 42. Geller J, Sionit L, Connors K, et al. Exoression of orostatespecific antigen in human prostate specimens in in vitro threedimensional histoculture. In Vo29A: 523-4. 43. Pantel K, Dickmanns A, Zippelius A, et al. Establishment of micrometastatic carcinoma cell lines: A novel source of tumor cell vaccines. J Nat1 Cancer Inst 1995;87:1162-8. 44. Yamazaki K, Pienta K.J. Establishment of an immortalized Copenhagen rat prostate epithelial cell line. In Vivo 1996;9:427432.

Effect of epidermal growth factor on prostate cancer cell line PC3 growth and invasion

Elevated levels of epidermal growth factor (EGF) and epidermal growth factor receptor (EGF-R) have been demonstrated in prostate cancer cell lines and clinical specimens suggesting a role for polypeptide growth factors in prostate tumor cell growth and invasion. To more clearly define the role of EGF in prostate cancer invasion, we undertook a series of studies utilizing the PC3 prostate cancer cell line, an aggressive, hormone-independent cell line derived from a metastatic lesion. No statistical differences were noted in the growth of PC3 cells under serum-free conditions when EGF (10(-10) M-10(-8) M) or monoclonal anti-EGF-R antibody (10(-11) M-10(-8) M) were added. Utilizing the Boyden chamber microinvasion assay, EGF supplemented cells demonstrated a statistically significant augmentation in invasion (P < 0.05) when compared to control cells at each time point in the study. With increasing length of exposure to EGF, the number of concentrations that produced significant invasion increased: day 1 (10(-8) M), day 3 (10(-8), 10(-9) M), and day 5 (10(-7), 10(-8), 10(-10) M). Northern blot analysis of EGF supplemented cells revealed an increase in expression of urokinase plasminogen activator (uPA) RNA, a serine protease involved in the regulation of pericellular proteolysis and membrane degradation. Protein analysis confirmed these findings. Statistically significant inhibition of invasion by anti-uPA antibodies was demonstrated for EGF-stimulated and PC3 control cells. Our results demonstrate that certain concentrations of EGF augment invasion in the PC3 cell line. This enhancement of invasion occurs in part by an overproduction of uPA, an extracellular protease. These findings suggest that the autocrine production of EGF may potentiate tumor cell invasion.

Basis for hormonal management of advanced prostate cancer

In the early 1940s, when it was established that most prostatic cancers were androgen dependent and could be controlled by androgen withdrawal, little was known about the mechanism of androgen action. Measurements of hormones, both in the circulation and in the tissue, were not available, nor were measurements of androgen receptors known at that time. Since that time, a large body of information has been published regarding the mechanism of androgen-mediated action. With the understanding of androgen-mediated action has come the opportunity to develop drugs targeted to block specific steps in the sequence of androgen action, beginning in the hypothalamus-pituitary area and extending down to the intracellular processes of enzymatic reduction, receptor binding, and nuclear translocation of the hormone receptor complexes. The major focus in prostate cancer therapy currently is the role of the adrenal androgens. It was established in the 1970s that, after castration, there was a 75% reduction in the dihydrotestosterone (DHT) present in prostate tissue. This observation contrasted with the finding that there was a greater than 90% reduction in circulating testosterone levels in the plasma after castration. Based on this important observation regarding tissue DHT concentrations after castration, attempts were made in the 1980s to block androgen totally using simultaneous gonadal and adrenal suppression. Dramatic results were reported after this type of therapy in the early uncontrolled studies. A luteinizing hormone-releasing hormone agonist plus flutamide was used for total androgen blockade. Other techniques for such blockade were available using megestrol acetate in combination with 17-beta-estradiol. One of the key issues has been whether the 25% residual DHT after castration provides a sufficient stimulus to growth of residual prostate tumor cells. The best evidence for the importance of the role of adrenal androgens came from clinical studies in which objective clinical responses were found in patients treated with various inhibitors of androgen action after relapse and castration. If "objectively stable" is included as a category after treatment, then approximately 33% of patients who have relapses after castration can be shown to have an additional response, albeit short, to adrenal androgen withdrawal. Thus, the control of the relatively small amounts of DHT remaining after castration became a major focus for therapy in metastatic prostate cancer.

Human primary prostate tumor cell line, ALVA‐31: A new model for studying the hormonal regulation of prostate tumor cell growth

A new human prostate tumor cell line (ALVA-31) has been established from a biopsy specimen of primary tumor obtained during prostatectomy. The cell line has been maintained for more than 48 months in stable growth. The in vitro doubling time was determined to be approximately 26 hr. The chromosome number ranged from 24-112, with a modal number of 59 tested over several time points throughout continuous culture. Karyotypic analysis of late-passaged cells demonstrated approximately 70 human chromosomes, 8-14 markers, and two X chromosomes without a Y chromosome. Prostatic origin was confirmed by the expression of both prostate specific antigen and prostatic acid phosphatase, using specific antisera and immunoradiolabelling techniques. Prostate tumor xenografts were grown in intact male, castrate male, and female athymic mice; however, the rate of tumor growth was clearly dependent upon serum testosterone levels.