Prostate-specific membrane antigen: a novel folate hydrolase in human prostatic carcinoma cells

A novel monoclonal antibody has been developed that reacts strongly with human prostatic cancer, especially tumors of high grade. This antibody (7E11C-5) is currently in Phase 3 trials as an imaging agent for metastatic disease. We have cloned the gene that encodes the antigen that is recognized by the 7E11C-5 monoclonal antibody and have designated this unique protein prostate-specific membrane (PSM) antigen. PSM antigen is a putative class II transmembranous glycoprotein exhibiting a molecular size of Mr 94,000. Functionally, class II membrane proteins serve as transport or binding proteins or have hydrolytic activity. Preliminary studies have demonstrated binding of pteroylmonoglutamate (folate) to membrane fractions that also cross-reacted with the PSM monoclonal antibody. We observed substantial carboxypeptidase activity as folate hydrolase associated with PSM antigen. The purpose of our study was to demonstrate that human prostatic carcinoma cells expressing PSM antigen exhibit folate hydrolase activity using methotrexate triglutamate (MTXGlu3) and pteroylpentaglutamate (PteGlu5) as substrates. Isolated membrane fractions from four human prostate cancer cell lines (LNCaP, PC-3, TSU-Prl, and Duke-145) were examined for folate hydrolase activity using capillary electrophoresis. After timed incubations at various pH ranges and in the presence and absence of thiol reagents, separation of pteroyl(glutamate)n derivatives was achieved with an electrolyte of sodium borate and SDS, while absorbance was monitored at 300 nm. The results demonstrate clearly that LNCaP cells, which highly express PSM, hydrolyze gamma-glutamyl linkages of MTXGlu3. The membrane-bound enzyme is an exopeptidase, because it progressively liberates glutamates from MTXGlu3 and PteGlu5 with accumulation of MTX and PteGlu1, respectively. The semipurified enzyme has a broad activity from pH 2.5 to 9.5 and exhibits activity maxima at pH 5 and 8. Enzymatic activity is maintained in the presence of reduced glutathione, homocysteine, and p-hydroxymercuribenzoate (0.05-0.5 mm) but was inhibited weakly by DTT (>/=0.2 mm). By contrast to LNCaP cell membranes, membranes isolated from other human prostate adenocarcinoma cells (PC-3, Duke-145, and TSU-Pr1) did not exhibit comparable hydrolase activity, nor did they react with 7E11-C5 monoclonal antibody. After transfection of PC-3 cells with a full-length 2.65-kb PSM cDNA subcloned into a pREP7 eukaryotic expression vector, non-PSM antigen-expressing PC-3 cells developed immunoreactivity to 7E11-C5 monoclonal antibody and demonstrated folate hydrolase activities and optimum pH activity profiles identical to those of LNCaP cells. The membrane-bound enzymes from both LNCaP- and PC-3-transfected cells also have a capacity to hydrolyze an alpha-linked glutamyl moiety from N-acetyl-alpha-aspartylglutamate. We have identified that PSM antigen is a pteroyl poly-gamma-glutamyl carboxypeptidase (folate hydrolase) and is expressed strongly in human prostate cancer. Cancer cells that express this enzyme are resistant to methotrexate therapy. Those developing future therapeutic strategies in the treatment of prostate cancer that utilize folate antagonists need to consider this mechanism of resistance.     

HTEX4, a new human gene in the MHC class I region, undergoes alternative splicing and polyadenylation processes in testis

PURPOSE: Jasplakinolide is a novel natural product anticancer agent which acts by inducing overpolymerization of actin. The aim of the current study was to explore the activity of jasplakinolide with hyperthermia and radiation. METHODS: The response of human PC-3 and DU-145 prostate carcinoma cells and DU-145 xenografts and the response of the Lewis lung carcinoma to jasplakinolide were studied. RESULTS: Jasplakinolide was cytotoxic toward human prostate carcinoma cells, DU-145, PC-3 and LNCaP in culture, killing 1 log of cells with 0.8, 0.3 and 0.07 microM of drug in 24 h, respectively. The combination of jasplakinolide and hyperthermia resulted in primarily additive cell killing by the two modalities in the three prostate carcinoma lines. In combination with radiation, jasplakinolide produced some diminution in the shoulder of the survival curve of normally oxygenated PC-3 cells and was a radiation sensitizer of hypoxic DU-145 cells and hypoxic PC-3 cells. In vivo, jasplakinolide was an active antitumor agent against the Lewis lung carcinoma and the DU-145 prostate carcinoma xenograft. Jasplakinolide was a radiation sensitizer in the Lewis lung carcinoma. Jasplakinolide was also effective against the systemic Lewis lung carcinoma, decreasing lung metastases. Lung metastases were further decreased when jasplakinolide was administered along with radiation to the subcutaneous primary tumor. In the DU-145 tumor, the effects of jasplakinolide and fractionated radiation for 1 or 2 weeks appeared to be primarily additive. CONCLUSION: Jasplakinolide is an interesting new anticancer agent for which further study both as an anticancer agent and in combined modality regimens is warranted.     

Tumor-targeted apoptosis by a novel spermine analogue, 1,12-diaziridinyl-4,9-diazadodecane, results in therapeutic efficacy and enhanced radiosensitivity of human prostate cancer

Interference with polyamine transport and biosynthesis has emerged as an important anticancer strategy involving polyamine analogues and specific inhibitors of key biosynthetic enzymes. Because the prostate gland has a high polyamine content, by using the polyamine transporter for selective uptake into cancer cells, alkylating polyamines are likely to be highly effective against prostatic tumors. We have recently synthesized a novel class of spermine analogues, the lead compound of which has efficacy against human cancer cells (P. S. Callery et al., U. S. patent, 5,612,239, Issued March 17, 1997.). In this study, to investigate the potential therapeutic efficacy of the lead spermine analogue 1,12-diaziridinyl-4, 9-diazadodecane (BIS), against advanced prostate cancer, we examined the in vitro effect and in vivo efficacy of the compound in two androgen-independent human prostate cancer cell lines, PC-3 and DU-145. BIS exhibited a dose-dependent cytotoxic effect against prostate cancer cells via induction of apoptosis. Treatment of cells with BIS (1 microM) for 24 h resulted in a significant induction of apoptosis (24%). Exposure of BIS-treated PC-3 prostate cancer cells to gamma-irradiation resulted in a significant increase in the number of cells undergoing apoptosis and a subsequent decrease in the IC50. Furthermore, BIS treatment led to a significant enhancement of loss of clonogenic survival in irradiated prostate cancer cells (both PC-3 and DU-145). In vivo efficacy trials demonstrated a significant antitumor effect of BIS against both PC-3 and DU-145 tumor xenografts in severe combined immunodeficient mice in a dose-dependent pattern at maximally tolerated doses. Terminal transferase end-labeling analysis indicated that BIS-mediated tumor regression in vivo occurs via induction of apoptosis among prostatic tumor cells. These results suggest that the novel spermine analogue BIS: (a) has a potent antitumor effect against prostatic tumors via induction of apoptosis; and (b) increases the radiosensitivity of human prostate cancer cells by decreasing the apoptotic threshold to radiation. This study may have important clinical implications for the manipulation of this antitumor activity of the polyamine analogue for the optimization of the therapeutic efficacy of radiation in patients with advanced prostate cancer.     

Adenovirus-mediated expression of PML suppresses growth and tumorigenicity of prostate cancer cells

Our previous studies demonstrated that the promyelocytic leukemia gene, PML, encodes a growth and transformation suppressor. Overexpression of PML inhibits cancer cell growth in vitro and in vivo. In this study, we further explored the possibility of applying PML as a potential agent for developing prostate cancer gene therapy using an adenovirus delivery system. We have constructed and produced the recombinant PML-adenovirus, Ad-PML, in which the full-length PML cDNA is driven by the strong cytomegalovirus promoter. In LNCaP, DU145, and PC-3 prostate cancer cell lines, an infection efficiency of 90% can be achieved at a concentration of 2, 10, and 100 multiplicity of infection (MOI), respectively. Western blotting and immunofluorescence staining demonstrated that the AD-PML-infected cells expressed a high level of PML protein. The protein expression peaked at days 3-4 postinfection, and a detectable level of PML was found at day 18 after viral infection. To test the effect of Ad-PML on the growth of prostate cancer cells, the DU145 and LNCaP cells were infected with 10 and 2 MOI of Ad-PML. We found that the growth rate of the Ad-PML-infected DU145 and LNCaP cells were significantly inhibited. A tumorigenicity test in nude mice showed that the Ad-PML-treated DU145 cells failed to form tumors. Most importantly, direct injection of Ad-PML into DU145-induced tumors was able to repress tumor growth in nude mice by 64%. Taken together, these data indicate that PML is a tumor growth suppressor in prostate cancer and that Ad-PML may be a potential candidate for human prostate cancer therapy.     

Expression of retinoic acid receptor-beta sensitizes prostate cancer cells to growth inhibition mediated by combinations of retinoids and a 19-nor hexafluoride vitamin D3 analog

Retinoids and analogs of vitamin D3 may achieve greater in vivo applications if the toxic side effects encountered at pharmacologically active doses could be alleviated. These seco-steroid hormones often act in concert, and therefore, we attempted to dissect these interactions by isolating combinations of receptor-selective retinoids and a potent vitamin D3 analog [1alpha,25(OH)2-16ene-23-yne-26,27,F6-19nor-D3, code name LH] that were potent inhibitors of prostate cancer cell growth at low, physiologically safer doses. Using a panel of prostate cancer cell lines representing progressively more transformed phenotypes, we found that the LNCaP cell line (least transformed) was either additively or synergistically inhibited in its clonal growth by LH and various naturally occurring and receptor-selective retinoids, the most potent combination being with a retinoic acid receptor (RAR)betagamma-selective retinoid (SR11262). The effect was not found with either PC-3 (intermediate transformation) or DU-145 (most transformed). We also undertook RT-PCR to examine the subtypes of RARs present, and we found that PC-3 and DU-145 did not express RARbeta. Stable expression of RARbeta into the RARbeta-negative PC-3 cells resulted in increased sensitivity to SR11262 and LH proportional to the amount of RARbeta expressed. This study indicates that RARbeta may play an important role in synergistically controlling cell proliferation, and expression is lost with increased prostate cancer cell transformation. Simultaneous administration of a potent vitamin D3 analog and receptor-selective retinoids may have therapeutic potential for the treatment of androgen-dependent and -independent prostate cancer.     

Differential inhibition by hyperammonemia of the electron transport chain enzymes in synaptosomes and non-synaptic mitochondria in ornithine transcarbamylase-deficient spf-mice: restoration by acetyl-L-carnitine

These studies were undertaken to assess the relative expression and autocrine activation of the epidermal growth factor receptor (EGFR) in normal and transformed prostatic epithelial cells and to determine whether EGFR activation plays a functional role in androgen-stimulated growth of prostate cancer cells in vitro. EGFR expression was determined by Western blot analysis and ELISA immunoassays. Immunoprecipitation of radiophosphorylated EGFR and evaluation of tyrosine phosphorylation was used to assess EGFR activation. The human androgen-independent prostate cancer cell lines PC3 and DU145 exhibited higher levels of EGFR expression and autocrine phosphorylation than normal human prostatic epithelial cells or the human androgen-responsive prostate cancer cell line LNCaP. PC3 and DU145 cells also showed higher levels of autonomous growth under serum-free defined conditions. Normal prostatic epithelial cells expressed EGFR but did not exhibit detectable levels of EGFR phosphorylation when cultured in the absence of exogenous EGF. Addition of EGF stimulated EGFR phosphorylation and induced proliferation of normal cells. LNCaP cells exhibited autocrine phosphorylation of EGFR but did not undergo significant proliferation when cultured in the absence of exogenous growth factors. A biphasic growth curve was observed when LNCaP cells were cultured with dihydrotestosterone (DHT). Maximum proliferation occurred at 1 nM DHT with regression of the growth response at DHT concentrations greater than 1 nM. However, neither EGFR expression nor phosphorylation was altered in LNCaP cells after androgen stimulation. In addition, DHT-stimulated growth of LNCaP cells was not inhibited by anti-EGFR. These studies show that autocrine activation of EGFR is a common feature of prostatic carcinoma cells in contrast to normal epithelial cells. However, EGFR activation does not appear to play a functional role in androgen-stimulated growth of LNCaP cells in vitro.     

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.     

Insulin-like growth factor I: action and receptor characterization in human prostate cancer cell lines

The role of insulin-like growth factor I (IGF-I) in the growth and development of prostate cancer was studied using established human prostate cancer cell lines. Under steroid and growth factor-free culture conditions, IGF-I significantly stimulated the androgen-independent cell lines PC-3 and DU-145 to incorporate [3H]thymidine into DNA, while the androgen-dependent cell line, LNCaP, was not affected. However, in the presence of dihydrotestosterone (DHT), DNA synthesis of LNCaP cells was stimulated by IGF-I in a dose-dependent manner. None of the cell lines tested secreted an immunoreactive level of IGF-I into their conditioned medium. Characterization of receptors by ligand binding assays revealed that all prostate cancer cell lines tested express specific binding sites for IGF-I with similar dissociation constants (0.23-0.39 nM). Crosslinking studies supported the suggestion that 125I-IGF-I was bound to a receptor on these cells. The IGF-I receptor concentrations of androgen-independent cell lines were significantly higher than those of the androgen-dependent cell line. Androgen appeared to affect neither the expression of IGF-I receptors nor the secretion of IGF-I. The results suggest that IGF-I may play an important role in stimulating the growth and progression of prostate cancer.     

Characterization of insulin-like growth factor-binding protein-related protein-1 in prostate cells

Insulin-like growth factor-binding protein-related protein-1 (IGFBP-rP1; also known as Mac25, TAF, and PSF) is a member of the IGFBP superfamily. It is a cysteine-rich protein that shares structural and functional similarities with the conventional IGFBPs. In situ hybridization of prostate tissue sections show intense IGFBP-rP1 messenger ribonucleic acid (mRNA) expression in normal stroma and glandular epithelium. There was a significant loss of detectable IGFBP-rP1 mRNA in metastatic prostate tissue. IGFBP-rP1 mRNA (Northern blots) and protein (immunoblots) were detectable in primary cultures ofprostatic stromal and epithelial cells as well as in the immortalized nonmalignant prostatic human epithelial cells, P69, and in the P69 metastatic subline, M12. IGFBP-rP1 expression was not detectable in the prostatic cancer cell lines PC-3, DU145, and LNCaP. IGFBP-rP1 expression was regulated in P69 cells but not in M12 cells. Protein and mRNA expression was up-regulated by IGF-I, transforming growth factor-beta, and retinoic acid. The observations that IGFBP-rP1 expression is significantly diminished in prostate tumorigenesis and is regulated in nonmalignant prostate cells suggest IGFBP-rP1 is important in normal prostatic cell growth.     

Gland atrophy following retrograde injection of methyl violet as a treatment in chronic obstructive parotitis

BACKGROUND: The growth and progression of prostate cancer depends on the stromal-epithelial interaction which is under paracrine control. Hepatocyte growth factor (HGF), produced by mesenchymal cells, is a multifunctional growth factor stimulating the movement and growth of epithelial cells including cancer cells. We therefore assessed the relationship between the invasive potential of prostate cancer and HGF in vitro. METHODS: Three human prostate cancer cell lines were used including PC-3 and DU145 (androgen-independent), and LNCaP (androgen-dependent). We studied the expression of the HGF receptor c-met proto-oncogene (c-met) by Western blot analysis, and also determined the effects of HGF on cell scattering, and the mechanisms of invasion and proliferation, by microscopic observation, the matrigel invasion chamber assay, and the MTT assay. RESULTS: c-met was detected in PC-3 and DU145 cells, but not in the LNCaP cells. There was increased cell motility in the scatter assay and an increased cell invasive potential in the matrigel invasion chamber assay by stimulation with HGF only with DU145 cells. CONCLUSION: HGF plays an important role in the invasion and metastasis of the DU145 cell line through a paracrine mechanism mediated by the c-metreceptor. In the PC-3 cell line, the lack of downstream signal transduction after the c-met receptor is suggested.     

Fas ligand is constitutively secreted by prostate cancer cells in vitro

LNCaP, DU145, and PC3 prostate carcinoma cells secrete the 27-kDa soluble Fas ligand (sFasL) into their local environment. sFasL arises from the 40-kDa membrane-bound form (mFasL), which can be found on the cell surface in the LNCaP line, as demonstrated by monoclonal antibody staining. mFasL was also found in extracts of all three cell lines, as demonstrated by Western blotting. FasL mRNA was detected not only in the cell lines, but in the normal prostate as well. sFasL protein could also be detected immunohistochemically in prostate secretions and in human semen. Cleavage of mFasL to sFasL could be inhibited by several matrix metalloprotease inhibitors without a change in the cellular levels of FasL. Prostate-derived sFasL is biologically active, as demonstrated by its induction of apoptosis in Fas-positive Ramos cells, which was detected by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Mitoxantrone induces cellular apoptosis in all three prostate cancer cell lines. Mitoxantrone treatment and doxorubicin treatment also cause up-regulation of Fas, the cell surface receptor for FasL, in LNCaP cells, but not in DU145 or PC3 cells. Furthermore, the up-regulation of Fas expression by mitoxantrone at a high concentration was potentiated by hydrocortisone. When FasL interacts with its Fas, the Fas-bearing cell undergoes apoptosis. When LNCaP cells were treated with mitoxantrone and incubated with an anti-FasL monoclonal antibody, apoptosis was partially blocked. This not only further suggests that the sFasL is biologically active, but that the up-regulation of Fas in the presence of sFasL accounts, in part, for the cytotoxicity of mitoxantrone.     

Chemosensitization of human prostate carcinoma cell lines to anti-fas-mediated cytotoxicity and apoptosis

Androgen ablation has been an effective treatment in patients with advanced prostate cancer. However, most treated patients develop hormonally resistant disease and do not respond to conventional chemotherapy. Immunotherapy against prostate cancer is an alternative approach in overcoming hormonal/drug-resistant prostate cancer. Cytotoxic immune lymphocytes kill target cells via the perforin/granzyme and the Fas-ligand (Fas-L) pathways. We hypothesize that tumor cells respond poorly to immunotherapy by developing resistance to killing by the Fas-L mechanism. This study investigated whether prostate tumor cells are sensitive to Fas-mediated killing. The human prostate carcinoma cell lines DU145, PC-3, and LnCAP were examined for their sensitivity to killing and apoptosis by the Fas-L agonist anti-Fas antibody and CTLs. All three lines moderately expressed the Fas antigen on the cell surface; however, all three lines were relatively resistant to cytotoxicity mediated by anti-Fas (CH-11) antibody. Pretreatment of DU145 and PC-3 with subtoxic concentrations of drugs followed by anti-Fas antibody resulted in synergistic cytotoxicity and apoptosis, whereas only an additive effect was obtained with LnCAP. Chemosensitization with drugs and anti-Fas was completely blocked by the addition of neutralizing anti-Fas antibody. The murine CTL hybridoma, PMMI, which kills only via the Fas-L pathway, was able to kill chemosensitized PC-3 and DU145 but not LnCAP cells. Furthermore, this cytotoxicity was blocked by anti-Fas neutralizing antibody. Chemosensitization of PC-3 and DU145 prostate tumor cells was not due to up-regulation of Fas-receptor antigen expression. Treatment of tumor cells with cisplatin did not down-regulate the antiapoptotic genes bcl-2, FAP-1, and c-myc. Further, there was no induction by cisplatin of Fas-L on the tumor cells, thus ruling out Fas/Fas-L-mediated autologous killing. These findings demonstrate that pretreatment of drug-resistant/CTL-resistant prostate DU145 and PC-3 tumor cells with subtoxic concentrations of certain chemotherapeutic drugs sensitizes the tumor cells to Fas-mediated cytotoxicity. These findings suggest that chemosensitization of tumor cells should optimize the response to immunotherapeutic interventions in the treatment of hormone-resistant/drug-resistant prostate cancer.     

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.     

Persistent membrane translocation of protein kinase C alpha during 12-0-tetradecanoylphorbol-13-acetate-induced apoptosis of LNCaP human prostate cancer cells

Others have reported that the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA), an activator and down-regulator of most protein kinase C (PKC) isozymes, can induce apoptotic cell death of androgen-sensitive LNCaP but not androgen-insensitive PC-3 or DU 145 human prostate cancer cells. As a first step toward uncovering the mechanism by which TPA induces apoptosis of LNCaP cells, we quantified expression of PKC isozyme mRNAs in unmodified and TPA-resistant LNCaP cells and in naturally TPA-resistant PC-3, PC-3M, and DU 145 cells. All of the cell lines and normal prostate expressed RNAs for PKC alpha, delta, epsilon, eta, and mu; only DU 145 cells and normal prostate expressed PKC beta and theta RNAs, and none expressed PKC gamma. The amount of PKC alpha RNA and protein was 6- to 38-fold lower, and PKC mu RNA was 4.5- to 16.5-fold higher in unmodified and TPA-resistant LNCaP cells than in the androgen-independent cells. We examined the effects of TPA on PKC alpha and mu mRNA levels and on membrane translocation of PKC alpha. Incubation with TPA for 6 h or more induced 95% inhibition of cell growth, a transient 12-fold increase and 5-fold decrease in PKC alpha and mu mRNA levels, respectively, and prolonged translocation of PKC alpha to non-nuclear membranes in unmodified LNCaP cells and in TPA-resistant LNCaP cells from which TPA had been removed for 10 days. TPA-resistant LNCaP cells in the continuous presence of TPA, or 24 h after removal of TPA, had down-regulated PKC alpha and remained resistant to re-addition of TPA. These data demonstrate a strong correlation of the presence and absence of membrane PKC alpha with apoptosis and resistance to apoptosis, respectively.     

Sensitivity of human prostatic carcinoma cell lines to low dose rate radiation exposure

PURPOSE: Low dose rate radioemitters, such as 125I, 103Pd, and 89Sr, have been used both for local and systemic treatment of prostate cancer. Most normal cells exposed to ionizing radiation characteristically activate cell cycle checkpoints, resulting in cell cycle arrest at the G1/S and G2/M transition points. Cancer cells are typically quite sensitive to radiation killing late in the G2 phase of the replicative cell cycle. Furthermore, most cancer cells accumulating at the G2/M transition point as a result of low dose rate radiation exposure appear to become sensitive to further low dose rate irradiation. For this reason, protracted exposure of cancer cells to low dose rate radiation has been proposed to result in increased cancer cell killing as compared with brief exposures of cancer cells to high dose rate radiation. Since many human prostatic carcinomas contain somatic genome alterations targeting genes which affect the cell cycle and radiation-associated cell cycle checkpoints, we evaluated the effects of low dose rate radiation exposure on the cell cycle and on clonogenic survival for various human prostatic carcinoma cell lines. MATERIALS AND METHODS: Human prostatic carcinoma cells from the LNCaP, DU 145, PC-3, PPC-1, and TSU-Pr1 cell lines were exposed to low dose rate (0.25 Gy/hour) or high dose rate (60 Gy/hour) radiation in vitro and then assessed for radiation cytotoxicity by clonogenic survival assay. Cell cycle perturbations following protracted exposure to low dose rate radiation were evaluated using flow cytometry. RESULTS: For LNCaP cells, low dose rate radiation exposure resulted in an accumulation of cells at both the G1/S and the G2/M cell cycle transition points. For DU 145, PC-3, PPC-1, and TSU-Pr1 cells, treatment with low dose rate radiation triggered G2/M cell cycle arrest, but not G1/S arrest. Unexpectedly, the cell cycle redistribution pattern phenotypes observed, G1/S and G2/M cell cycle arrest versus G2/M arrest alone, appeared to have little effect on low dose rate radiation survival. Furthermore, while PC-3, PPC-1, and TSU-Pr1 cells exhibited increased cytotoxic sensitivity to low dose rate versus fractionated high dose rate radiation treatment, DU 145 and LNCaP cells did not. CONCLUSIONS: Radiation-associated pertubations in replicative cell cycle progression were not dominant determinants of low dose rate radiation killing efficacy in human prostate cancer cell lines in vitro.     

Expression of human prostatic acid phosphatase correlates with androgen-stimulated cell proliferation in prostate cancer cell lines

Androgen plays a critical role in regulating the growth and differentiation of normal prostate epithelia, as well as the initial growth of prostate cancer cells. Nevertheless, prostate carcinomas eventually become androgen-unresponsive, and the cancer is refractory to hormonal therapy. To gain insight into the mechanism involved in this hormone-refractory phenomenon, we have examined the potential role of the androgen receptor (AR) in that process. We have investigated the expression of AR and two prostate-specific androgen-responsive antigens, prostatic acid phosphatase (PAcP) and prostate-specific antigen (PSA), for the functional activity of AR in LNCaP and PC-3 human prostate carcinoma cells. Our results are as follows. (i) Clone 33 LNCaP cells express AR, PAcP, and PSA, and cell growth is stimulated by 5alpha-dihydrotestosterone (DHT). Stimulation of cell growth correlates with decreased cellular PAcP activity. (ii) In clone 81 LNCaP cells, the expression of PAcP decreases with a concurrent decrease in the degree of androgen stimulation of cell growth, whereas the expression of PSA mRNA level is up-regulated by DHT, as in clone 33 cells. Conversely, in PAcP cDNA-transfected clone 81 cells, an additional expression of cellular PAcP correlates with an increased stimulation by androgen, higher than the corresponding control cells. (iii) PC-3 cells express a low level of functional AR with no detectable PAcP or PSA, and the growth of PC-3 cells is not affected by DHT treatment. Nevertheless, in two PAcP cDNA-transfected PC-3 sublines, the expression of exogenous cellular PAcP correlates with androgen stimulation. This androgen stimulation of cell growth concurs with an increased tyrosine phosphorylation of a phosphoprotein of 185 kDa. In summary, the data indicate that the expression of AR alone is not sufficient for androgen stimulation of cell growth. Furthermore, in AR-expressing prostate cancer cells, the expression of cellular PAcP correlates with androgen stimulation of cell proliferation.