Molecular characterization of the loss of p75(NTR) expression in human prostate tumor cells.

The low-affinity nerve growth factor receptor p75(NTR) is a 75-kDa glycoprotein that belongs to the tumor necrosis factor receptor superfamily and has been implicated in the induction of apoptosis in various tissues and cell lines. Immunohistochemistry on tissue sections from radical prostatectomies has shown that expression of p75(NTR) is limited to the epithelial cells. Western blot and immunohistochemical analyses have also shown a progressive loss of p75(NTR) expression in prostate epithelial cells during the malignant progression of organ-confined adenocarcinomas, with complete loss of expression in the naturally occurring prostate tumor cell lines DU-145, PC-3, LNCaP, and TSU-pr1, which were derived from metastases. Reintroduction of p75(NTR) expression into the TSU-pr1 tumor cell line was shown to reestablish the ability of these cells to undergo p75(NTR)-mediated apoptosis. It is not known whether this loss of expression is due to deletion of part or the entire p75(NTR) gene or to other factors. Through the use of southern blotting and polymerase chain reaction (PCR), we showed that loss of p75(NTR) protein expression was not due to deletion or loss of the gene. Furthermore, through reverse transcription-PCR, RNase protection, and the chromatin immunoprecipitation assay, we showed that transcription of the p75(NTR) gene occurred in these prostate tumor cell lines. Finally, through transient transfection using two constructs of p75(NTR), one containing the full 2-kb 3' untranslated region and one that contains only a few hundred bases of the 3' untranslated region (UTR), we showed that the 3' UTR may have a role in the loss of p75(NTR) expression in prostate cancer.

Epidermal growth factor modulates the expression of vascular endothelial growth factor in the human prostate.

The growth and dissemination of tumors in the body has been associated with angiogenesis. Vascular endothelial growth factor (VEGF) is an angiogenic factor that stimulates endothelial cell growth and enhances vascular permeability. VEGF exerts its action by binding to specific cell surface receptors. Three receptors, VEGFR-1 (flt-1), VEGFR-2 (flk-1), and VEGFR-3 (flt-4) have been identified. Very little information on the coordinated expression of VEGF and its receptors in normal prostate, benign prostatic hyperplasia (BPH), and prostate carcinoma is available. Therefore, we examined the immunohistochemical localization of VEGF and its receptors in tissues derived from normal human prostate, BPH, and prostatic carcinoma. Immunostaining for VEGF was absent in the normal prostate. Epithelium lining the glands of prostate derived from patients with BPH exhibited strong immunostaining. The intensity of staining was relatively less in prostate carcinoma. It is interesting that VEGFR-1 and VEGFR-3 were strongly expressed in both stromal and epithelial tissues in normal prostate, BPH, and carcinoma. In comparison, VEGFR-2 was not localized to normal prostate and its expression in the stroma of BPH and epithelium of carcinoma was very weak. Because progression of prostate cancer is accompanied by altered expression of epidermal growth factor (EGF) and its receptor (EGFR) in malignant cells, we investigated the effect of EGF on VEGF gene expression by Northern blot analysis in 2 human prostate cancer cell lines that express EGFR. EGF greatly enhanced the expression of VEGF messenger RNA in DU145 and PC3 cell lines in a dose-dependent manner. The EGF induction of VEGF gene expression suggests a mechanism by which angiogenesis could be accelerated in BPH and prostate carcinoma.

The neurotrophin receptor p75NTR is a tumor suppressor in human prostate cancer.

The loss of tumor suppressor gene function contributes to the transformation of human prostate epithelial cells to a malignant pathology. Previous studies have demonstrated a loss of protein expression of the neurotrophin receptor, the p75NTR, from the pathological human prostate. Through cell cycle analysis, we demonstrate that a dose-dependent increase in the expression of p75NTR protein induces increased quiescence of a series of prostate tumor cells in vitro. When the same series of tumor cells were injected into the flanks of SCID mice, the growth of prostate tumors was suppressed in proportion to increased p75NTR expression. Tumor analysis showed that a dose-dependent increase in p75NTR expression was associated with a dose-dependent increase in the proportion of apoptotic cells, as determined by TUNEL assay, and a dose-dependent decrease in the proportion of cells committed to proliferation, as determined by proliferating cell nuclear antigen assay. These results demonstrate that the neurotrophin receptor p75NTR is a tumor suppressor of prostate growth by enhancing cell cycle quiescence, reducing proliferation and increasing apoptosis of the tumor cells.

Human spermatogenesis as a model to examine gene potentiation.

The first tier of control over the expression of genic domains utilizes chromatin structure. Before the onset of transcription, the chromatin domain that encompasses the gene(s) must assume an open conformation. This renders large segments of the genome available to the tissue-specific and ubiquitous trans-factors necessary for proper expression of the genes present. This process has been termed potentiation. It is a necessary obligate, but alone it is not sufficient for gene expression. Spermatogenesis, the development of a viable fertile male gamete, provides a unique model to begin to address the underlying mechanism(s) governing differentiation and tissue-specific gene expression. Male gametogenesis is typified by the activation of numerous genes whose products have novel functions, as well as testis-specific forms of constitutively expressed somatic genes. We have shown that mouse spermatogenesis represents a selective potentiative process (Kramer et al., 1998: Development 125:4749-4655), but little is known about its human counterpart. To fill this void we have examined the potentiative state of several spermatid-expressed genes during the latter stages of human spermatogenesis. We have shown that spermatidexpressed genes are potentiated by the pachytene stage of differentiation. Furthermore, we establish that a chromatin domain functions as a discrete structural unit during differentiation. Interestingly, some of these open structures are maintained in the mature spermatozoon.

Epidermal growth factor promotes MDA-MB-231 breast cancer cell migration through a phosphatidylinositol 3'-kinase and phospholipase C-dependent mechanism.

Epidermal growth factor receptor (EGFR) levels predict a poor outcome in human breast cancer and are most commonly associated with proliferative effects of epidermal growth factor (EGF), with little emphasis placed on motogenic responses to EGF. We found that MDA-MB-231 human breast cancer cells elicited a potent chemotactic response despite their complete lack of a proliferative response to EGF. Antagonists of EGFR ligation, the EGFR kinase, phosphatidylinositol 3'-kinase, and phospholipase C, but not the mitogen-activated protein kinases (extracellular signal-regulated protein kinase 1 and 2), blocked MDA-MB-231 chemotaxis. These findings suggest that EGF may influence human breast cancer progression via migratory pathways, the signaling for which appears to be dissociated, at least in part, from the proliferative pathways.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Differentiation: the selective potentiation of chromatin domains.

Potentiation is requisite for the expression of our genome. It is the mechanism of opening chromatin domains to render genes accessible to tissue-specific and ubiquitous transacting-factors that enables transcription. The results presented in this study demonstrate that modulation of stage- and cell-type-specific gene expression during mammalian spermatogenesis involves selective potentiation of testis-expressed genes that reverses their repressive state when present in the spermatogonial stem cell. This directly contrasts hematopoiesis, which acts to selectively restrict lineage potential during differentiation from its permissive stem cell. These results are key to understanding how differentiative pathways are controlled and cellular phenotypes determined. A window to their modulation is presented.

Expression of p75NTR in a human prostate epithelial tumor cell line reduces nerve growth factor-induced cell growth by activation of programmed cell death.

Epithelial expression of the 75-kDa low-affinity neurotrophin receptor (p75NTR) is inversely associated with the malignant progression of the human prostate. To elucidate the function of p75NTR in the prostate, the human prostate epithelial tumor cell line TSU-pr1, which does not express p75NTR, was stably and transiently transfected with the cDNA for the receptor. The stably transfected cells were assessed for levels of p75NTR expression and categorized into low, intermediate, and high receptor-expressing clones by immunocytochemical and immunoblot analyses. Incorporation of [3H]thymidine was used to assess nerve growth factor (NGF)-induced changes in cell proliferation. TSU-pr1 epithelial cells transfected with a neomycin-resistance vector alone demonstrated a dose-dependent increase in the rate of NGF-stimulated [3H]thymidine uptake. Expression of p75NTR decreased the dose-dependent NGF-mediated proliferation of the TSU-pr1 prostate epithelial cells. The greater the degree of expression of p75NTR in the transfected clones, the less the stimulatory effect of exogenous NGF on cell proliferation. Furthermore, the ratio of p75NTR to tropomyosin receptor kinase for each clone was inversely correlated with the ability of NGF to stimulate growth of the TSU-pr1 transfectants. To determine whether p75NTR-mediated growth inhibition of prostate epithelia occurs by induction of programmed cell death, transiently transfected clones were analyzed by an in situ DNA nick-translation assay. NGF deprivation and anti-NGF treatment of transiently transfected TSU-pr1 cells significantly increased the proportion of epithelial cells undergoing programmed cell death by approximately fourfold above control levels. Conversely, addition of NGF was able to rescue p75NTR-expressing clones from undergoing programmed cell death at levels not significantly different from those of mock-transfected clones. These results demonstrate that p75NTR is a negative regulator of human prostate epithelial cell growth by induction of programmed cell death. Hence, loss of p75NTR expression in human prostate epithelia eliminates a growth-inhibitory pathway, thereby contributing to the malignant progression of the prostate.

Cell cycle-independent death of prostate adenocarcinoma is induced by the trk tyrosine kinase inhibitor CEP-751 (KT6587).

Advanced prostate cancer remains largely incurable, primarily because the very low growth fraction present in these tumors makes them generally resistant to treatment with standard chemotherapeutic agents that target cell division. Effective therapies should therefore induce death of prostate cancer cells, independent of their growth rate. trkA, the high-affinity tyrosine kinase-linked receptor for nerve growth factor, has been implicated in prostatic cancer growth and may represent a molecular target for therapeutic agents. At low mg/kg doses, the trk tyrosine kinase inhibitor CEP-751 (KT6587) inhibits prostatic cancer growth in nine different animal models independent of the tumor growth rate, androgen sensitivity, metastatic ability, or state of tumor differentiation. CEP-751 is selective for cancerous versus normal prostate cells and affects the growth of only a limited number of nonprostate tumors. Importantly, CEP-751 induces cell death of prostate cancer cells in a cell cycle-independent fashion and, therefore, represents a novel therapeutic approach to the management of both hormone-dependent and hormone-independent prostate cancer.

Molecular characterization of neurotrophin expression and the corresponding tropomyosin receptor kinases (trks) in epithelial and stromal cells of the human prostate.

The prostate is one of the most abundant sources of nerve growth factor (NGF) outside of the nervous system. NGF is a member of the neurotrophin family of growth factors which in mammals also includes brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). These neurotrophins can bind with high affinity to a family of tropomyosin receptor kinases (trks). These receptors are trkA, which binds NGF; trkB, which binds both BDNF and NT-4/5; and trkC, which binds NT-3. In order to characterize the molecular expression of the neurotrophins and their corresponding trk receptors in the prostate we performed Northern blot analysis for the neurotrophins and reverse transcription-polymerase chain reaction (RT-PCR) coupled with Southern blot analysis for the trk family of receptors on smooth muscle stromal cells from the prostate, the androgen responsive LNCaP prostate tumor cell line and the androgen refractory TSU-pr1 prostate tumor cell line. The results show that smooth muscle stromal cells expressed NGF, BDNF and trkC, whereas both epithelial cell lines expressed trkA, trkB and trkC to various degrees. NT-3 was not detected in either the smooth muscle stromal cells or in both epithelial cell lines. This suggests that the stromal cell derived NGF and BDNF may interact via paracrine mechanisms with trkA and trkB receptors, respectively, on the adjacent epithelial cells. Interestingly, the androgen responsive LNCaP cell line did not express any of the neurotrophins, whereas the androgen refractory TSU-pr1 cell line expressed NGF, BDNF and NT-4/5. This suggests that the autocrine expression of NGF, BDNF and NT-4/5 is up-regulated in prostate epithelial cells following their transformation to an androgen refractory pathology. Hence, the malignant transformation of prostate epithelial tumor cells may facilitate their escape from a paracrine dependence on stromal cell derived neurotrophins by the acquisition of the autocrine expression of neurotrophins. Since the pathology of malignant cell migration within the prostate is predominantly by direct extension around prostatic nerves the upregulation of autocrine neurotrophin expression within prostate epithelial tumor cells may be concomitant with transformation to a malignant phenotype capable of invasion along the perineural space and extracapsular metastasis to distant sites of tumor formation.

Loss of telomerase activity during male germ cell differentiation.

Although the activity of telomerase, an enzyme which synthesizes telomeres de novo and stabilizes telomere length has been demonstrated in the testis, the precise expression of activity in different germ cell types is not known. We examined telomerase activity using a PCR-based telomeric repeat amplification protocol during development of the rat testis from birth to adulthood. Telomerase activity was relatively high from birth to the 4th week of age, and then low between the 5th to 10th week, suggesting that the type A spermatogonial stem cells may be the population which is expressing the highest levels of telomerase activity. To ascertain which germ cells expresses the telomerase activity, purified populations of type A spermatogonia from 9-day old rats, and pachytene spermatocytes, round spermatids and epididymal spermatozoa from adult rats were isolated. While type A spermatogonia expressed very strong telomerase activity, the fractions containing pachytene spermatocytes and round spermatids also expressed telomerase activity, but at comparatively lower levels. Telomerase activity was totally absent in epididymal spermatozoa. Thus, it appears that the telomerase activity is expressed at high levels in the type A spermatogonial stem cells, is down-regulated during spermatogenesis, and is absent in the differentiated spermatozoa.

Loss of low-affinity nerve growth factor receptor during malignant transformation of the human prostate.

BACKGROUND: The low-affinity nerve growth factor receptor (LNGFR) exhibits an inverse association of epithelial expression with the degree of differentiation of prostate adenocarcinoma tissue. However, the stage at which loss of LNGFR expression is first manifested in the malignant prostate has not been determined. METHODS: In order to characterize loss of LNGFR expression in the clinically localized malignant prostate of untreated patients, the pattern of expression of the LNGFR was examined in nonmalignant tissues, consisting of normal and prostatic intraepithelial neoplastic tissues, and in malignant tissues that had been graded by Gleason's scores and categorized into well, moderately, and poorly differentiated adenocarcinomas. In order to determine whether there was an inverse correlation between LNGFR expression and prostate-specific antigen (PSA) secretion, preoperative concentrations of PSA in the serum were also analyzed in relation to the differentiative state of the adenocarcinomas. RESULTS: Premalignant prostate tissues exhibited expression of the LNGFR on all epithelia, whereas in malignant prostate tissues a proportion of epithelia exhibited loss of expression of the LNGFR. An increase in Gleason score of the adenocarcinoma tissue was associated with an increase in the proportion of epithelia that exhibited loss of expression of the LNGFR. Moreover, the proportion of epithelia expressing the LNGFR was inversely correlated with an increase in the concentration of serum PSA. The loss of LNGFR expression was first manifested in epithelia that occurred toward the center of adenocarcinoma tissue. Furthermore, extensive loss of expression of the LNGFR of approximately 63% occurred in well-differentiated adenocarcinomas. CONCLUSIONS: These observations demonstrate that loss of LNGFR expression is first observed in well-differentiated malignant epithelia. Hence, loss of the LNGFR may be indicative of the initial stages of malignant transformation of prostate epithelia, as well as all subsequent stages of prostate adenocarcinomas.

Characterization of nerve growth factor precursor protein expression in rat round spermatids and the trophic effects of nerve growth factor in the maintenance of Sertoli cell viability.

Nerve growth factor (NGF) is expressed by rat round spermatids and is thought to participate in the paracrine regulation of spermatogenesis. In order to elucidate the role of NGF in the rat testis, we further characterized the NGF immunoreactive protein secreted by round spermatids and examined the effect of NGF beta and related neurotrophin family members on the maintenance of Sertoli cell viability. Round spermatids were isolated from rat testes by centrifugal elutriation and the conditioned medium dialyzed/concentrated for the preparation of round spermatid protein (RSP). Immunoblot analysis of RSP with anti-NGF beta antibody identified two immunoreactive bands of 31 and 22 kD, whereas the 13 kD mature form of NGF beta was not observed. Similarly, immunoblot analysis of RSP with an antibody raised against a synthetic peptide (L38) corresponding to the -3 to -40 sequence of proNGF also recognized two immunoreactive bands of 31 and 22 kD. These results are consistent with the identification of two NGF precursors. Interestingly, immunoblot analysis of RSP with an antibody raised against a synthetic peptide (N4) corresponding to the -71 to -46 sequence of proNGF only recognized one immunoreactive band of 31 kD, consistent with the larger NGF precursor observed with the L38 antibody. In a bioactivity test of PC-12 neurite outgrowth, the 31 kD NGF precursor induced flattening and neurite outgrowth of PC-12 cells, consistent with NGF bioactivity. The 22 kD NGF precursor induced modest and inconsistent neurite outgrowth. These results suggest that round spermatids express the 31 and 22 kD precursor forms of the NGF gene product, and that processing of this gene product is incomplete such that the 13 kD mature form of NGF beta is not observed. In view of the role of round spermatids in the paracrine regulation of spermatogenesis, we examined the effect of RSP on the rescue of the viability of Sertoli cells cultured under serum deprived conditions. In the absence of serum, RSP was able to extend the viability of Sertoli cells, and the elimination of this activity by anti-NGF antibody immunoprecipitation of RSP suggests that the NGF precursors in RSP support the maintenance of Sertoli cell viability. In addition, treatment with exogenous NGF beta was able to rescue Sertoli cell viability, whereas L38 peptide, N4 peptide, or the neurotrophins, brain derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 were unable to rescue Sertoli cell viability above control levels. Hence, it appears that round spermatids express the precursor forms of the NGF gene product, but not the mature form of NGF beta, and that the NGF beta moiety of the NGF precursor proteins exhibits trophic activity in the rescue of Sertoli cell viability, consistent with the paracrine regulation of spermatogenesis.

Two peptides derived from the nerve growth factor precursor are biologically active.

This report provides evidence that the proregion of the NGF precursor protein contains two novel bioactive peptides. The presence of pairs of basic amino acid (aa) residues in the NGF proregion suggests that two or three peptides other than NGF may be generated by proteolytic cleavage. Synthetic peptides of 29 aa (LIP1) and 38aa (LIP2) corresponding to the sequences -71 to -43 and -40 to -3 of the proNGF, respectively, were used in this study. ELISA specific for these two peptides revealed their presence in the rat intestine. LIP1 was localized by immunohistochemistry in endocrine cells of the intestinal epithelium, and LIP2 was immunoprecipitated from an intestinal extract. We also provide evidence for the presence of specific receptors for LIP2 in several cell lines. Scatchard analysis indicated the presence of a low affinity binding site with a Kd of approximately 10(-7) M and a high affinity binding site of 10(-9) M. Cross-linking studies revealed receptor forms of about 140 kD and 93 kD in a prostatic adenocarcinoma cell line. LIP1 and LIP2 induced rapid F-actin redistribution in PC12 cells within 2 min of incubation, which suggests a role of LIP1 and LIP2 in the process of neurite outgrowth. Furthermore, both propeptides induced rapid tyrosine phosphorylation of the Trk protein in both prostatic adenocarcinoma cells and PC12 cells, thus implicating trk in their mechanism of action. These results support our hypothesis that two peptides within the NGF precursor protein are biologically active.

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.

Inhibition of chemomigration of a human prostatic carcinoma cell (TSU-pr1) line by inhibition of epidermal growth factor receptor function.

Chemoattractants expressed at bony sites and pelvic lymph nodes are thought to promote the preferential metastasis of human prostate tumor cells to these organs. Epidermal growth factor (EGF) is a potent chemoattractant for several human metastatic prostate tumor cell lines, including the TSU-pr1 cell line, and EGF has been localized to the stroma of both bony sites and pelvic lymph nodes in humans. Hence, we investigated whether the TSU-pr1 cell line expresses a functional EGF receptor (EGFR), which when antagonized reduces EGF-mediated chemomigration of this cell line. In this context, the EGFR immunoprecipitated from cell lysates of TSU-pr1 cells comigrated with the EGFR from A431 cells at a molecular weight of 170 kD. Addition of human EGF (hEGF) to the TSU-pr1 cells for 5 min stimulated the dose-dependent biphasic phosphorylation of the EGFR, with maximal stimulation of EGFR phosphorylation occurring at 2 ng/ml hEGF. In addition, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with 0.5 microgram/ml anti-hEGF monoclonal antibody or 100 nM staurosporine inhibited EGFR phosphorylation. Conversely, as negative controls, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with K252a or dimethyl sulfoxide (DMSO) vehicle did not inhibit EGFR phosphorylation. TSU-pr1 cells were stimulated to migration in 4 hr across Boyden chambers in response to 10 ng/ml hEGF. Treatment of the TSU-pr1 cells with anti-hEGFR monoclonal antibody inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. Similarly, treatment of the TSU-pr1 cells with staurosporine inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. These results demonstrate that antagonists of hEGF-mediated hEGFR phosphorylation also antagonize chemomigration of the TSU-pr1 cells across Boyden chambers, suggesting that antagonists of the EGFR in prostate cancer may be useful in the treatment of metastatic disease.

Epidermal growth factor (EGF) promotes chemomigration of a human prostate tumor cell line, and EGF immunoreactive proteins are present at sites of metastasis in the stroma of lymph nodes and medullary bone.

Prostate tumor cells preferentially metastasize to bony sites and lymph nodes at a frequency in excess of that which would be predicted by random tumor cell dissemination. In order to determine whether chemoattractants in these organs promote organ-specific metastasis, we utilized human cell lines derived from and/or related to these organs as sources of potential chemoattractants. Secretory proteins derived from the cell lines MG-63 (osteosarcoma), SK-ES-1 (Ewing's sarcoma), and KG-1 (leukemia) stimulated chemomigration of the TSU-pr1 prostate tumor cells in a dose-dependent manner in Boyden chambers. In addition, secretory proteins from a human prostatic stromal cell line (hPS) and from the TSU-Pr1 prostate tumor cell line were also able to stimulate chemomigration of the TSU-pr1 cells through Boyden chambers. Since lymph nodes and bony sites represent organs of hematopoietic/lymphoid proliferation and activation, we undertook identification of specific cytokines present at these sites which may promote the chemomigration of prostate tumor cells. In this context, the cytokines interleukin-1 alpha, interleukin-2, interleukin-6, tumor necrosis factor-beta, transforming growth factor-beta, interferon alpha 2-a, and granulocyte-macrophage colony-stimulating factor did not stimulate chemomigration of the TSU-pr1 prostate tumor cell line. In contrast, the cytokine epidermal growth factor (EGF) stimulated chemomigration of the TSU-pr1 prostate tumor cells through the Boyden chambers in a dose-dependent manner. Western blot analysis of secretory proteins from the cell lines KG-1, SK-ES-1, MG-63, hPS, and TSU-pr1 identified EGF-immunoreactive proteins in all cases. In addition, EGF immunoreactivity was localized to the stroma of the human prostate, the osteogenic stroma of pelvic medullary bone, and the stroma within the capsule and trabeculae of pelvic lymph nodes. Hence, these results demonstrate that the cytokine EGF promotes the chemomigration of the TSU-pr1 prostate tumor cell line, and that EGF within the stroma of pelvic lymph nodes and medullary bone may act as a chemoattractant for prostate tumor cells, thereby facilitating the preferential formation of metastatic foci within these organs.

Coordinate expression of the PRM1, PRM2, and TNP2 multigene locus in human testis.

Maintenance of the transcriptionally inert state of the mature human spermatozoon requires the expression of the various members of the human protamine gene cluster prior to the final stages of spermatogenesis. During this process, known as spermiogenesis, round spermatids morphologically differentiate into mature spermatozoa. The expression of the PRM1, PRM2, and TNP2 genes facilitates the compaction and condensation of the genetic material within the developing spermatid. To understand better the coordinate control governing this transformation, we have examined the localization and distribution of the human protamines PRM1 and PRM2 and transition protein TNP2 transcripts during human spermatogenesis. The stage-specific expression of these transcripts was determined by in situ hybridization analysis using [alpha-35S]-labeled cRNA probes. PRM1, PRM2, and TNP2 transcripts were abundant in association with round and elongating spermatids, located in the adluminal region of the seminiferous epithelium. They were not observed in association with spermatogonia, spermatocytes, Sertoli cells, or interstitial cells. These data indicate that the human PRM1, PRM2, and TNP2 transcripts are expressed postmeiotically in round and elongating spermatids. The quantitative evaluation of each transcript was determined as a function of the relative optical density per unit area. In all cases examined, the relative level of each transcript was consistent with the following pattern, PRM2 > PRM1 congruent to TNP2.

Expression of a Trk high affinity nerve growth factor receptor in the human prostate.

Nerve growth factor-beta (NGF beta) and a NGF beta-immunoreactive protein derived from human prostatic stromal cell secretory protein (hPS) have been shown to stimulate the growth of prostate epithelial cells. An NGF beta-immunoreactive protein has been localized to the stroma of human prostate tissues, and a low affinity NGF receptor (gp75NGFR) has been localized to the adjacent epithelia, consistent with the paracrine regulation of prostate growth. Interestingly, gp75NGFR is progressively lost during neoplastic progression of the human prostate. In this report we have characterized the expression of the signal-transducing component of the NGF receptor, the Trk tyrosine receptor kinase, in prostate epithelial cells that bind exogenous NGF beta and an endogenous NGF beta-immunoreactive protein in hPS. In this context, a pan-Trk antibody that recognizes all of the members of the Trk receptor family (TrkA, TrkB, and TrkC) specifically localized expression of the Trk receptor to the epithelial component of normal prostate tissue, benign prostatic hyperplasia, and adenocarcinoma tissue. The binding of [125I]NGF beta to the surface of primary cultures of human prostate epithelia and the TSU-pr1 human metastatic prostate tumor cell line was displaced with either excess cold NGF beta or hPS, whereas binding was not displaced by epidermal growth factor or platelet-derived growth factor. Scatchard plot analysis of [125I]NGF beta binding to these cells identified a low affinity binding site (Kd = 1.9 x 10(-9) M) and a high affinity binding site (Kd = 1.8 x 10(-11) M) on the primary prostate epithelia, whereas only a high affinity binding site (Kd = 1.3 x 10(-11) M) was observed on the TSU-pr1 tumor cells. Stimulation of TSU-pr1 cells with either NGF beta or hPS induced tyrosine phosphorylation of Trk proteins, whereas no phosphorylation was evident in untreated cells, cells treated with hPS immunoprecipitated with anti-NGF beta antibody, or brain-derived neurotrophic factor- and neurotrophin-3-treated cells. The Trk protein was also observed in these cells by immunoblot analysis with pan-Trk antibody. These results demonstrate a functional Trk receptor in the epithelia of the human prostate that is responsive to exogenous NGF beta and an endogenous NGF beta-immunoreactive protein in hPS, thereby supporting the concept of the paracrine regulation of growth in the human prostate via a stromal neurotrophin-epithelial Trk receptor interaction.