TSU-Pr1 Cells Research Articles

Tributyrin induces differentiation, growth arrest and apoptosis in androgen‐sensitive and androgen‐resistant human prostate cancer cell lines

Progression to androgen independence remains the main problem that impacts on survival and quality of life in prostate cancer patients. We have investigated the potency of tributyrin, an orally available prodrug of butyrate, to induce growth arrest, differentiation and apoptosis in LNCaP, PC-3 and TSU-PR1 human prostate cancer cell lines. Cells were treated with 0.1 to 5 mM tributyrin or sodium butyrate. Growth inhibition, cell cycle arrest and apoptosis induction was assessed using standard methods. Both agents induced a more differentiated, fibroblast-like phenotype in androgen-sensitive as well as androgen-resistant cell lines. Expression of prostate-specific antigen was increased in LNCaP cells by tributyrin as a indicator of differentiation. The IC50 for sodium butyrate was 2.5 mM in PC-3 and TSU-PR1 cells. LNCaP cells exhibited <50% growth inhibition at 5 mM sodium butyrate. However, the IC50 for tributyrin was 0.8 mM in PC-3 cells, 1.2 mM in TSU-PR1 cells and 3.1 mM in LNCaP cells. Flow cytometry revealed a strong G1-arrest after exposure to tributyrin or sodium butyrate. Both agents resulted in a strong increase of apoptosis rates compared with mock-treated cells. Overall, tributyrin had a 2.5- to 3-fold growth inhibitory and apoptosis-inducing potency compared with equimolar concentrations of sodium butyrate. Our results demonstrate that tributyrin is more potent than butyrate in regard to cell growth inhibition and apoptosis induction at pharmacologically relevant concentrations. Hence, tributyrin may be a promising candidate for clinical protocols in prostate cancer. Int. J. Cancer 88:245–251, 2000. © 2000 Wiley-Liss, Inc.

Cloning and Characterization of Human Estrogen Receptor β Promoter

Estrogen receptors beta (ERβ) belong to the nuclear receptor superfamily of ligand-dependent transcription factors that play critical roles in regulating genes involved in a wide array of biological processes. To investigate regulation of tissue-specific expression of ERβ, we cloned and characterized a 2.1-kilobase 5′-flanking region of the human ERβ gene. Two major transcription start sites were identified by primer extension and rapid amplication of 5′-cDNA end. The human ERβ proximal promoter contains both TATA box and initiator element (Inr) and is GC-rich with a GC content of 65%. An Alu repeat sequence containing an ER-dependent transcription enhancer exists between −1416 and −1703. The full-length 5′-flanking sequence of ERβ fused to a luciferase reporter exhibited functional promoter activity in ERβ-positive TSUPr1 cell, but not in ERβ-negative DU145 cells. In addition, DNase I protection assays of the proximal promoter showed unique protection patterns with nuclear extracts from TSUPr1 cells and ERβ negative HeLa cells, suggesting presence of cell-specific trans-acting factors that mediate tissue/cell-specific ERβ expression. Serial deletion analysis revealed that a 293-bp region encompassing the TATA box and Inr element possesses basal promoter activity.

Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) on prostate cancer cell lines

Membrane-type metalloproteinase-1 (MT1-MMP) is a transmembrane metalloproteinase, which activates proMMP-2 and expressed on the cell surface in many invasive cancer cells. We investigated the expression of MT1-MMP in prostate cancer cell lines. MT1-MMP protein and mRNA were expressed in PC-3, DU-145 and TSU-pr1 cells (androgen-independent prostate cancer cell lines), but in LNCaP cells (androgen-dependent prostate cancer cell line). MT1-MMP protein was negative and mRNA was low to detect by RT-PCR. Cell lysate of PC-3 cleaved proMMP-2 to the active form. In addition, both hepatocyte growth factor (HGF) and gastrin-releasing peptide (GRP) increased Matrigel invasion and induced the expression of MT1-MMP protein in DU-145 prostate cancer cells. These results suggest that MT1-MMP is indeed the tumor-specific activator of proMMP-2 in androgen-independent prostate cancer cells and plays an important role in the invasive properties of prostate cancer cells.

Effects of androgens on telomerase activity in normal and malignant prostate cells in vitro.

Recent studies have shown that sex hormones regulate telomerase activity in endometrium and breast tissues. The present study was designed to clarify the effects of androgen on telomerase activity in normal and malignant prostate cells. Androgen-sensitive (LNCaP) and -independent (TSU-Pr1 and DU145) prostate cancer cell lines and normal prostate cells including basal cells were cultured in the presence or absence of 5alpha-dihydrotestosterone (DHT). Prostate cancer cell lines exhibited high telomerase activity, and normal prostate cells showed low activity. Short or prolonged androgen-deprivation reduced telomerase activity in LNCaP cells, and DHT induced telomerase activity at the G(1) phase of the cell cycle. DHT did not modulate telomerase activity in TSU-Pr1, DU145, and normal cells. LNCaP cells had an androgen-dependent pathway to activate telomerase, whereas TSU-Pr1 and DU145 cells as well as normal prostate cells had an androgen-independent pathway. These findings suggest that the regulatory mechanism of telomerase varies during the progression of prostate cancers.

Low-dose staurosporine suppresses proliferation and induces neurites in human prostatic cancer TSU-Pr1 cells.

Despite the beneficial effects of androgen ablation therapy in patients with prostate carcinoma, advancing prostate cancer usually becomes hormone-refractory. We attempted to establish a new prostate cancer therapy by controlling the malignancy of tumor cells through the induction of differentiation in vitro. We examined the ability of staurosporine to induce differentiation of human prostate cancer TSU-Pr1 cells into the cells with neuronal characteristics. At low concentrations, staurosporine remarkably suppressed proliferation of human prostate cancer TSU-Pr1 cells without increasing dead cell number. TSU-Pr1 cells treated with 10(-8) M staurosporine began to extend neurites within 1 day, and approximately 80% of cells were changed to a neuronal morphology at 3 days. The expression of mRNA of tau, a microtubule-associated protein that is one of the essential components of neurite outgrowth, time-dependently increased in the cells treated with 10(-8) M staurosporine. Similarly, the amount of acetylcholinesterase increased. Colony-forming activity of TSU-Pr1 cells treated with 10(-8) M staurosporine for 7 days was 40% that of control cells. The invasive ability of TSU-Pr1 cells treated with staurosporine to penetrate through a reconstituted basement membrane of Matrigel was 20% that of untreated cells. These results suggest that staurosporine might induce differentiation of human prostate cancer TSU-Pr1 cells to cells with neuronal characteristics.

Tributyrin induces differentiation, growth arrest and apoptosis in androgen-sensitive and androgen-resistant human prostate cancer cell lines

Progression to androgen independence remains the main problem that impacts on survival and quality of life in prostate cancer patients. We have investigated the potency of tributyrin, an orally available prodrug of butyrate, to induce growth arrest, differentiation and apoptosis in LNCaP, PC-3 and TSU-PR1 human prostate cancer cell lines. Cells were treated with 0.1 to 5 mM tributyrin or sodium butyrate. Growth inhibition, cell cycle arrest and apoptosis induction was assessed using standard methods. Both agents induced a more differentiated, fibroblast-like phenotype in androgen-sensitive as well as androgen-resistant cell lines. Expression of prostate-specific antigen was increased in LNCaP cells by tributyrin as a indicator of differentiation. The IC(50) for sodium butyrate was 2.5 mM in PC-3 and TSU-PR1 cells. LNCaP cells exhibited <50% growth inhibition at 5 mM sodium butyrate. However, the IC(50) for tributyrin was 0.8 mM in PC-3 cells, 1.2 mM in TSU-PR1 cells and 3.1 mM in LNCaP cells. Flow cytometry revealed a strong G1-arrest after exposure to tributyrin or sodium butyrate. Both agents resulted in a strong increase of apoptosis rates compared with mock-treated cells. Overall, tributyrin had a 2.5- to 3-fold growth inhibitory and apoptosis-inducing potency compared with equimolar concentrations of sodium butyrate. Our results demonstrate that tributyrin is more potent than butyrate in regard to cell growth inhibition and apoptosis induction at pharmacologically relevant concentrations. Hence, tributyrin may be a promising candidate for clinical protocols in prostate cancer.

Transforming growth factor-beta1-induced proliferation of the prostate cancer cell line, TSU-Pr1: the role of platelet-derived growth factor.

The results of our previous study revealed that transforming growth factor-beta1 (TGFbeta1) stimulated proliferation of the prostate cancer cell line, TSU-Pr1. This observation is unexpected, for TGFbeta usually inhibits proliferation in prostate cancer cells. The present study examines possible mechanisms through which TGFbeta1 induces this proliferation. We postulate that TGFbeta1 action is mediated through an indirect mechanism by inducing the expression of platelet-derived growth factor (PDGF), which, in turn, stimulates proliferation. The TGFbeta1-induced proliferation can be abrogated by treatment with a PDGF-neutralizing antibody. Treatment with exogenous PDGF significantly increased TSU-Pr1 proliferation. Finally, treatment of TSU-Pr1 cells with TGFbeta1 resulted in an increase in PDGF secretion. These results indicate that TGFbeta1-induced proliferation in TSU-Pr1 cells is at least mediated through an increased secretion of PDGF.

Synthesis of procaspases-3 and -7 during apoptosis in prostate cancer cells.

Cells differ in the time required to execute cell death after receipt of a death signal. One reason may be the requirement for de novo synthesis of components of the death pathway. TSU-Pr1 prostate cancer cells treated with okadaic acid demonstrated activation of caspase-3, PARP cleavage, and nuclear fragmentation by 24 h and apoptosis by 72 h. Levels of procaspase-3 and procaspase-7, the precursor molecules of two effector caspases, were not depleted during apoptosis. Levels of procaspase-3 and -7 mRNA increased steadily in TSU-Pr1 cells up to 72 h after exposure to okadaic acid. Nuclear run-off experiments showed that the increase in mRNA was not due to transcriptional activation of caspase-3 and -7 mRNA. Antisense caspase-3 and caspase-7 oligodeoxynucleotides caused a depletion of procaspases-3 and -7 and a delay in apoptosis of TSU-Pr1 cells. Caspase antisense oligodeoxynucleotides inhibited apoptosis to a similar extent as peptide inhibitors of cysteine proteases. Synthesis of procaspases-3 and -7 was necessary to sustain programmed cell death in TSU-Pr1 prostate cancer cells.

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.

A proliferative effect of transforming growth factor-beta1 on a human prostate cancer cell line, TSU-Pr1.

Transforming growth factor -beta (TGF-beta) is growth inhibitory to many malignant cells, including prostate cancer cells. The present study reports an unusual observation in that TGF-beta is growth stimulatory to a human prostate cancer cell line, TSU-Pr1. The TSU-Pr1 line is highly aggressive and exhibits a rapid rate of proliferation in culture. These cells underwent further proliferation in response to TGF-beta1. Both type I and II receptors to TGF-beta (TPR-I, TPR-II) are expressed in TSU-Pr1 cells. Activation of a luciferase reporter gene, which contains a TGF-beta response element, confirmed that the TGF-beta receptors in TSU-Pr1 cells were functional. RT-PCR analysis and an ELISA assay determined that TSU-Pr1 cells secreted TGF-beta. In conclusion, TSU-Pr1 cells contain functional TGF-beta receptors but instead of the usual growth inhibition by TGF-beta1, these cells undergo proliferation. The present observation provides a proliferative role of TGF-beta in TSU-Pr1 cells, which may play a part in the aggressive phenotype of these cells and, perhaps other prostate cancer cells.

SENSITIVITY OF HUMAN PROSTATIC CARCINOMA CELL LINES TO LOW DOSE RATE RADIATION EXPOSURE

Purpose Low dose rate radioemitters, such as 125 I, 103 Pd, and sup 89 Sr, 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 G 1/S and G 2/M transition points. Cancer cells are typically quite sensitive to radiation killing late in the G 2 phase of the replicative cell cycle. Furthermore, most cancer cells accumulating at the G 2/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 G 1/S and the G 2/M cell cycle transition points. For DU 145, PC-3, PPC-1, and TSU-Pr1 cells, treatment with low dose rate radiation triggered G 2/M cell cycle arrest, but not G 1/S arrest. Unexpectedly, the cell cycle redistribution pattern phenotypes observed, G 1/S and G 2/M cell cycle arrest versus G 2/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.

Evaluation of the tetracycline-repressible transactivator system for inducible gene expression in human prostate cancer cell lines.

Studies of genes that may inhibit growth or induce death of cells are facilitated greatly by tightly controlled expression of those genes. A promising system for control of transgene expression over a wide range is the tetracycline-repressible transactivator (tTA) system developed by Gossen and Bujard [Proc Natl Acad Sci USA 1992;89:5547-5551]. We investigated the effectiveness of this system in three well-established human prostate cancer cell lines. LNCaP, PC-3, and Tsu-Pr1 cells were transfected with a vector coding for the tTA protein and/or a luciferase reporter vector, and luciferase activity was measured in the presence and absence of tetracycline or the tTA protein. In the absence of tetracycline, the tTA system yielded high levels of luciferase activity in all three cell lines. Background luciferase activity in the presence of tetracycline was nearly undetectable in LNCaP cells, moderate in Tsu-Pr1 cells, and more than 20-fold higher in PC-3 than in Tsu-Pr1 cells. Similar background activity was observed in Tsu-Pr1 and PC-3 cells, even in the absence of the transactivator protein. The tTA system should be useful for stable transfection of cytotoxic transgenes in LNCaP cells and for control of transgene expression over a wide range in Tsu-Pr1 and PC-3 cells.

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.

Expression of homeobox gene-GBX2 in human prostatic cancer cells.

Homeobox genes encode transcription factors involved in the genetic control of normal development and differentiation, as well as in malignant transformation. To begin to assess the possible role of homeobox genes in prostatic cell carcinogenesis, we surveyed initially for expression of homeobox-containing genes in the TSU-PR1 cell line. This was performed by RT-PCR using degenerate oligodeoxyribonucleotide primers to the homeobox-binding sequence to generate partial cDNAs which were cloned and sequenced. Using this method, expression of 14 members of homeobox-containing genes were detected in TSU-PR1 cells. All of these expressed genes correspond to previously identified homeobox genes located within the HOXA, B, C, and D clusters. We further examined the expression of these homeobox genes in different human prostatic cell lines by using whole cDNA slot blot and Northern blot analysis. One of the sequences corresponding to the human GBX2 homeobox gene is overexpressed in TSU-PR1, LNCaP, PC-3, and DU145 metastatic prostate cell lines relative to the normal prostate. Our results suggest that the homeobox gene GBX2 may participate in metastatic progression in prostatic cancer.

Expression of homeobox gene‐GBX2 in human prostatic cancer cells

BACKGROUND Homeobox genes encode transcription factors involved in the genetic control of normal development and differentiation, as well as in malignant transformation. To begin to assess the possible role of homeobox genes in prostatic cell carcinogenesis, we surveyed initially for expression of homeobox-containing genes in the TSU-PR1 cell line. METHODS This was performed by RT-PCR using degenerate oligodeoxyribonucleotide primers to the homeobox-binding sequence to generate partial cDNAs which were cloned and sequenced. RESULTS Using this method, expression of 14 members of homeobox-containing genes were detected in TSU-PR1 cells. All of these expressed genes correspond to previously identified homeobox genes located within the HOXA, B, C, and D clusters. We further examined the expression of these homeobox genes in different human prostatic cell lines by using whole cDNA slot blot and Northern blot analysis. One of the sequences corresponding to the human GBX2 homeobox gene is overexpressed in TSU-PR1, LNCaP, PC-3, and DU145 metastatic prostate cell lines relative to the normal prostate. CONCLUSIONS Our results suggest that the homeobox gene GBX2 may participate in metastatic progression in prostatic cancer. © 1996 Wiley-Liss, Inc.

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.

Expression and sequence analysis of the SDI1/WAF1/CIP1/p21 tumor suppressor gene in prostate cancer cell lines

The SDI1/WAF1/CIP1 gene encodes a M(r) 21,000 protein (p21) that can arrest cell growth by associating with and inhibiting cyclin-dependent kinase complexes necessary for cells to exit G(1). It is a critical downstream effector in the p53 growth control pathway and can be transcriptionally upregulated by increasing levels of wild-type p53 protein. Somatic mutations in the p21 gene have been detected in 17% of primary prostate cancers. In the current study, we examined four prostate cancer cell lines for expression of and mutations in the p21 gene. Transcripts for p21 mRNA were present in all PC cell lines; p21 protein was detected in androgen-dependent LNCaP cells, as well as in androgen-independent DU-145 and TSU-Pr1 cells but not in androgen-independent PC-3 cells. Examination of the entire coding region of the p21 gene by SSCP analysis and direct DNA sequencing did not detect mutations in the coding domains of the p21 gene. These data indicate that mutations of the SDI1/WAF1/CIP1/p21 gene are not present in cultured PC cells and suggest that defects in the p21 gene are infrequent in prostate cancers.

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 μg/ml anti-hEGFR 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. © 1996 Wiley-Liss, Inc.

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.