Heparin-binding growth factor gene expression and receptor characteristics in normal rat prostate and two transplantable rat prostate tumors

Heparin-binding polypeptide growth factors (HBGF) are essential mitogens for isolated prostate cells. HBGF type one (HBGF-1) mRNA was expressed specifically in the epithelial cells of prostates from normal 6- to 8-week-old rats. Expression declined significantly at 14 weeks and was undetectable in 35-week-old animals. Slow-growing, androgen-responsive, nonmetastatic Dunning R3327PAP tumors, which are composed of a well-defined epithelium and stroma, expressed HBGF-1 mRNA constitutively in specifically the mesenchymal cells. A rapid-growing, androgen-independent, metastatic variant (Dunning R3327AT-3), which was composed of a single clonogenic cell type, expressed both HBGF-1 and HBGF type two (HBGF-2) mRNA. HBGF activity in the extracts of normal and tumor tissues correlated with mRNA levels. Epithelial cells from the R3327PAP tumor and the single cell type that composed the R3327AT-3 tumor exhibited alterations in HBGF receptor characteristics that correlated with increased sensitivity to mitogenic effects of HBGF. The results suggest that alterations in HBGF gene expression in both prostate epithelial and mesenchymal cells and in properties of the receptor in specifically epithelial cells may contribute to differential growth rates and malignancy of different prostatic tumors.     

Apoptosis in rat prostatic adenocarcinoma is associated with rapid infiltration of cytotoxic T-cells and activated macrophages

Rats transplanted with the androgen-sensitive, syngeneic Dunning R3327 PAP prostatic tumor were castrated and treated with estrogen or vehicle for 4, 12 and 24 hr and for 6 weeks. Tumor growth was retarded by castration and further inhibited by estrogen. Immediately after castration, an increased number of activated macrophages and T-cells were found in parallel with increasing apoptotic tumor cells. Administration of an immunosuppressive drug, FK 506, abolished the growth-inhibitory effects of castration and estrogen. The tumor growth rate correlated negatively with the number of R73- and OX8-positive T-cells and NK cells and with the percentage of ED3-positive macrophages. There was a positive correlation between the percentage of TdT-mediated- dUTP nick end labeling (TUNEL)-positive apoptotic cells and that of ED3-positive cells. Our results suggest that apoptosis of prostatic carcinoma cells induced by endocrine treatment in vivo is partly due to a rapid infiltration by immunocompetent cells. Int. J. Cancer 71:451-455, 1997. © 1997 Wiley-Liss Inc.     

Intracellular visualization of prostate cancer using magnetic resonance imaging

The term "molecular imaging" can be broadly defined as the in vivo characterization and measurement of biological processes at the cellular and molecular level. Is a gene expression magnetic resonance imaging (MRI) possible? Therefore, we have developed a novel intravital and intracellular MRI contrast agent composed of a gadolinium complex, an oligonucleotide sequence [peptide nucleic acid (PNA)], and a transmembrane carrier peptide that is composed of a peptide sequence similar to that of the homeodomain of the Antennapedia protein. The goal of our study was to determine whether this contrast agent could be accumulated in tumor cells in vitro (HeLa cells) and in vivo (Dunning R3327 AT1 rat prostate adenocarcinoma) and whether the specificity of the PNA for the up-regulated c-myc mRNA in the cell's cytoplasm would have an effect on contrast agent retention in the tumor cells. Using the c-myc-specific and a c-myc-nonspecific control PNA, an increase in signal intensity in the tumor cells was observed after 10 min in vitro and in vivo (maximum was reached in HeLa cells in vitro in 60 min, in Dunning R3327 AT1 rat prostate adenocarcinoma cells in vivo in 30 min). This increase of signal intensity could be maintained in vitro in HeLa cells for only 4 h and in Dunning R3327 AT1 rat prostate adenocarcinoma cells in vivo at least for 5 h by using the c-myc mRNA-specific PNA as a "retention" agent.     

A comparative study on expression of prostatic inhibin peptide, prostate acid phosphatase and prostate specific antigen in androgen independent human and rat prostate carcinoma cell lines

Prostatic inhibin peptide (PIP), consisting of 94 amino-acid residues is synthesized and secreted by the prostate gland. Previous studies on immuno-histochemical localization of PIP in primary prostatic tumor and their metastasis, have documented the value of this peptide as a tumor marker for diagnosis of prostate cancer (PCa). The present study was undertaken to compare the expression of PIP with that of prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) in androgen independent human PCa cell lines (PC-3, DU-145 and TSU-Prl) by immunoperoxidase technique. The results of the study indicated that the staining for PIP was more intense than that of PSA and PAP. The PSA staining was either weakly positive (PC-3) or totally absent (TSU-Prl and DU-145) while PAP staining was intense in PC-3 and moderate in the other two human cell lines. The intense staining observed for PIP in all of the androgen independent cell lines suggests that the synthesis and secretion of PIP is not primarily dependent on androgens. Furthermore, expression of these markers in Dunning rat cultured adenocarcinoma cell lines and tumors were studied. Positive staining for all three human tumor associated antigens (PIP, PSA and PAP) cross-reacting with the Dunning rat PCa cell lines and the tumors, suggest the suitability of this model for preclinical screening of various therapeutic agents.     

A novel immunological model for the study of prostate cancer.

The Dunning R-3327 rat prostatic adenocarcinoma is a widely accepted model for in vivo experimental studies of prostate cancer. We have previously derived phenotypically distinct cell lines from a s.c. tumor resulting from the inoculation of the R-3327-5 subclone into Copenhagen rats. In this study, we report studies using a gelatin sponge model for the delivery of tumor cells and the retrieval of tumor-specific leukocytes responsive to different prostatic cell lines. S.c. preimplanted sponges were inoculated with tumor cells previously selected for differential properties of tumor formation and metastasis and examined for leukocyte content at time points of 1, 3, and 5 weeks after tumor cell inoculation. Cytospin and flow cytometric analyses revealed fewer tumor-associated leukocytes present in sponges inoculated with tumorigenic R-3327-5' and R-3327-5'B lines, with lesser sponge degradation, than in experiments with the nontumorigenic R-3327-5'A line, suggestive of a tumor cell-induced immunomodulatory mechanism. Morphological studies indicate an intermittent tumor growth pattern that gradually disappears in sponges inoculated with the nontumorigenic R-3327-5'A cells but a robust growth pattern in sponges inoculated with the tumorigenic cell lines. Cytokine analyses show the secretion of higher levels of active transforming growth factor-beta by the more invasive and metastatic lines. Total transforming growth factor-beta levels are higher in the epithelial, tumorigenic R-3327-5'B line. Additionally, the more tumorigenic lines secrete interleukin 10, a potent immunosuppressive molecule. In this report, we demonstrate the ability to retrieve viable leukocyte populations from a prostate tumor line bearing sponges, which offers an important model for further in vitro and in vivo manipulations and holds promise for testing adoptive immunotherapeutic strategies.     

Flow cytometric analysis of R3327 rat prostate adenocarcinoma grown in vivo and in vitro

The Dunning R3327 transplantable prostate adenocarcinoma in the Copenhagen rat is an acceptable model for the human disease. The G-subline (a rapidly growing carcinoma) and the H-subline (a slow-growing, well-differentiated adenocarcinoma) represent the extremes of differentiation and growth rate of this tumor. Both sublines were found to have one population that was diploid and a second aneuploid population that was hyperdiploid in DNA content. The percentage of hyperdiploid cells was significantly higher in R3327-G tumors than in R3327-H tumors. The tumor cell population ratios were stable in vivo, but the in vitro culture conditions supported only cells with diploid DNA content following four to five subcultures. These predominantly diploid cultured cells, when injected into intact male rats, resulted in tumors that had both diploid and aneuploid cells.     

Detection of mRNA expression of gonadotropin-releasing hormone and its receptor in normal and neoplastic rat prostates.

Hypothalamic gonadotropin-releasing hormone (GnRH) plays a central role in the regulation of the mammalian reproductive systems as a releasing hormone of pituitary gonadotropins. However, a number of studies have shown that GnRH or its receptor are also expressed in some reproductive organs including prostate gland, mammary gland, ovary and placenta, tumors and tumor cell lines derived from these organs, suggesting that this peptide hormone may have other extrapituitary functions in addition to its role as a gonadotropin-releasing hormone. Moreover, it has been demonstrated that GnRH analogs exert some direct inhibitory effects on the proliferation of human and rat prostate cancer cells, probably mediated by its own specific receptors expressed in these tumor cells. In the present study, we investigated the mRNA expression of GnRH and its receptor in normal Noble rat prostate gland, and in three rat models of prostate cancer including the sex hormone-induced Noble rat model, an androgen-independent Noble rat prostatic tumor (AIT) and Dunning rat prostatic adenocarcinomas by RT-PCR and Southern blot analyses. The results showed that GnRH mRNA was expressed in the normal, hormone-treated and neoplastic rat prostates, in addition to its positive control expression in the hypothalamus, whereas its receptor was only detected in the androgen-dependent Dunning R3327H tumor. The detection of both GnRH and its receptor in the androgen-dependent Dunning R3327H tumor tissue suggests that this peptide hormone may have some autocrine and paracrine regulatory functions in this tumor. However, the gene expression of GnRH receptor was not detected in two androgen-independent Dunning tumor sublines and the Noble rat prostatic tumor, AIT, suggesting that the expression of GnRH receptor is lost or down-regulated in the prostatic tumors during the progression to a hormone-independent phenotype.     

Isolation and characterization of a cloned cell line R3327H-G8-A1 derived from the dunning R3327H rat adenocarcinoma

A cloned cell line (R3327H-G8-A1) has been isolated from the Dunning R3327H adenocarcinoma. Light and electron microscopic studies showed that the cell line possessed features common to secretory epithelial cells. These cells, which grow in monolayer culture, produced s.c. hind flank tumors when inoculated inoculated into Copenhagen X Fischer F1 rats.l Chromosomal karyotype analysis confirmed that the cell line is distinctly that of the Rattus norvegicus genus and species. The cells specifically bind testosterone and dexamethasone with equilibrium dissociation constants (Kd) of 0.49 and 0.8 nM, respectively. The numbers of saturable binding sites per cell are 10,000 for testosterone and 60,000 for dexamethasone. The cells also have 5 alpha-reductase activity. These properties are characteristic of the prostate and of the Dunning tumor from which the cells are derived. Cell growth in vitro was stimulated by androgens and inhibited by glucocorticoids at concentrations of 10(-8) M. An int riguing finding was that estradiol and progestins dramatically stimulated growth in the apparent absence of receptors for these hormones. Finally, comparisons between the G8-A1 cells and the tumor induced by the G8-A1 clone and a second generation of cells from this G8-A1-induced tumor showed that the cloned cells retained their properties following passaging in the animal.     

Growth-related elevations of DNA topoisomerase II levels found in Dunning R3327 rat prostatic adenocarcinomas

The relationship between DNA topoisomerase II expression and mammalian cell proliferation has been evaluated by determining enzyme levels in normal and neoplastic rat prostate tissues. By activity assay and by immunoblot analysis using anti-topoisomerase II antiserum, topoisomerase II levels were found to be elevated in both the Dunning R3327-H and the Dunning R3327-G rat prostatic adenocarcinomas over levels assayed in the normal rat dorsal prostate. Immunohistochemical studies using the antitopoisomerase II antiserum revealed that a greater fraction of nuclei contained detectable levels of topoisomerase II in tissue sections prepared from each of the Dunning tumors than in rat dorsal prostate tissue sections. The Dunning R3327-H and R3327-G tumors grow at different rates in vivo (J. T. Isaacs and D. S. Coffey, Clin. Oncol., 2: 479-498, 1983). When measured topoisomerase II levels were compared to known growth parameters for each of the tissues studied, topoisomerase II expression was found to be correlated with tissue growth rate.     

Radiation sensitivity of Copenhagen rat prostatic carcinoma (R3327-AT and R3327-MATLyLu)

The radiosensitivity of two variants of the Dunning Copenhagen rat prostatic tumor R3327 was investigated. The R3327-AT variant, which is a poorly differentiated anaplastic, fast-growing tumor, was irradiated both in vivo and in vitro. Following irradiation, monodispersed cells were plated in vitro and colonies were counted after 7 days. The survival curve of R3327-AT cells irradiated in vivo showed an initial shoulder (Dq-value 0.97 Gy), followed by two exponential parts. The D0-value for the first part of the curve (0-10 Gy) was 2.76 Gy and for the second part of the curve (greater than 10 gy) 9.05 Gy. Extrapolation of the second part of the curve to the Y-axis indicated that the proportion of more radioresistant cells was about 10%. The survival curve for R3327-AT cells irradiated in vitro also suggested the presence of a radioresistant subpopulation, although the proportion was lower (about 3%). This difference might be due to the presence of an hypoxic fraction in the tumors irradiated in vivo, but not in vitro. Tumor cells from the R3327 tumor variant metastatic to lymph nodes and lungs (R3327-MATLyLu), were irradiated in vitro. The radiation effect was evaluated by in vitro colony formation in agar and by in vivo lung colony assay. The colony formation in agar yielded a D0-value of 1.09 Gy. No radioresistant subpopulation was identified in this variant. A similar radiosensitivity was observed by the in vivo lung colony assay (D0 1.39 Gy). The mean inactivation dose calculated for R3327-AT cells (3.45 Gy) was significantly higher than for the metastatic variant (2.00 Gy).     

Estrogen induces apoptosis in a rat prostatic adenocarcinoma: Association with an increased expression of TGF- β1 and its type-I and type-II receptors

Rats transplanted with the androgen-sensitive Dunning R3327 PAP prostatic adenocarcinoma were castrated and treated with either estrogen or vehicle alone for short periods (4 hr, 12 hr, 24 hr) and for 6 weeks. In these tumors the expression of TGF-β1, TGF-β type-I and type-II receptors (TGF-β RI, TGF-β RII) was examined by immunohistochemistry. Apoptotic cells were identified by in situ nick end labelling (TUNEL). Tumor growth was retarded by castration and even more by additive estrogen treatment. The epithelium of the untreated tumors stained weakly for TGF-β1 and TGF-β RI, but TGF-β RII was not detected. Castration induced moderate TGF-β1 immunoreactivity in a major part of the glandular epithelium after 24 hr. After 12 hr already, castration plus estrogen resulted in an intense staining for TGF-β1 in the basal epithelial cells, some of which also showed an apoptotic appearance. The percentage of cells having stained positive for TGF-β1 was significantly higher in the estrogen-treated groups than in the castrated group after 12 hr, and its elevated TGF-β1 level remained at 6 weeks. Notably, the increased immunoexpression of TGF-β1 occurred before the onset of induction of apoptosis. In parallel with the upregulation of TGF-β1 after castration, the expression of its receptors, TGF-β RI and RII, was induced and was further enhanced by the additive estrogen treatment. The number of intensely stained TGF-β1 tumor cells showed a strong correlation with the number of apoptotic tumor cells identified by TUNEL in the whole material. Furthermore, TGF-β1 immunoreactivity co-localized with the presence of apoptotic cells in the estrogen-treated tumors at 6 weeks after castration. © 1996 Wiley-Liss, Inc.     

Establishment and in vivo characterization of multidrug-resistant dunning R3327 rat prostate-carcinoma cell-lines

We describe the selection of 3 new multidrug-resistant cell lines derived from tumor cells of different metastatic phenotypes within the Dunning R3327 model of rat prostatic carcinoma. Cell lines of weak (AT2) and strong (AT3 and MAT-LyLu) metastatic behavior were cultured in vitro and challenged with doxorubicin at progressively increasing concentrations. Chemosensitivity was determined colorimetrically by release of precipitated formazan pigment (MTT assay). Expression of the multidrug-resistance glycoprotein (P-170) was monitored immunocytochemically and by Western blotting using monoclonal antibody C219. The behavior of the parental and resultant drug-resistant cells was assessed by their growth in syngeneic rats. Doxorubicin challenge of the initially drug-sensitive parental prostatic carcinoma cell lines resulted in the rapid development of multidrug resistance together with simultaneous expression of P-glycoprotein. While lung and lymph-node metastases developed in host animals inoculated with parental AT3 and MAT-LyLu cells, no metastases developed in the multidrug-resistant progeny of these cell lines. This study has shown that Dunning rat prostate-carcinoma cell lines, previously sensitive to different cytotoxic agents, rapidly become multidrug-resistant and express P-glycoprotein following exposure to doxorubicin. Further more, development of multidrug resistance is associated with a less aggressive tumor phenotype and loss of metastatic potential. Nevertheless, it is unlikely that the non-metastatic phenotype of Dunning rat prostatic carcinoma cells is solely associated with expression of P-glycoprotein. These new multidrug-resistant cell lines exhibiting an altered behavioral phenotype will provide a valuable mode with which to analyze the relationship between expression of P-glycoprotein and the metastatic phenotype of prostatic carcinoma cells.     

Basal and estrogen-stimulated hormone receptor profiles in four R3327 rat prostatic carcinoma sublines in relation to histopathology and androgen sensitivity

Four sublines (H, HI, G, and AT) of the R3327 (Dunning) rat prostatic carcinoma have different androgen sensitivities and histopathological characteristics. In order to investigate whether these characteristics were associated with differences in the hormone receptor profile and its response to estrogen, we carried out Scatchard analysis on the cytosolic (C) and high-salt nuclear-associated (N) androgen (AR), estrogen (ER), and progesterone (PgR) receptor in each line, carried in control and diethylstilbestrol (DES)-treated animals. In the H line (androgen sensitive, well differentiated) DES treatment resulted in significant increases in total cellular AR and ER, in redistribution of both receptors between the C and N fractions, and in a marked increased of PgR (greater than 10-fold). The hormone receptor profile and its response to DES was similar in the HI line (androgen insensitive, well differentiated), except that total cellular ER was not increased after treatment. The G line (androgen sensitive, poorly differentiated) contained higher basal concentrations of AR and PgR than the H line, but the concentrations were not increased by DES treatment, although treatment promoted association of ER with the nuclear fraction. The AT line (androgen insensitive, anaplastic) contained no ER and negligible PgR, but AR was present, although in lower concentrations than in the other lines. Diethylstilbestrol treatment had no effect on the concentration, although redistribution of AR between C and N fractions did occur. Some characteristics of the AR in the AT line differed qualitatively from that in the H line, but injection of testosterone into castrated animals bearing the AT tumor promoted association of AR with the nuclear fraction, indicating normal activation. The data suggest that the ability of DES treatment to increase AR and PgR concentrations is associated with differentiation and/or the presence of stroma and that it is unrelated to androgen sensitivity.     

Energy intake and prostate tumor growth, angiogenesis, and vascular endothelial growth factor expression

BACKGROUND: A sedentary lifestyle coupled with excessive energy intake is speculated to be a factor associated with increased incidence of prostate cancer. We have investigated the effects of energy intake on prostate tumor growth in experimental animals. METHODS: Two transplantable prostate tumor models, i.e., the androgen-dependent Dunning R3327-H adenocarcinoma in rats and the androgen-sensitive LNCaP human carcinoma in severe combined immunodeficient mice, were studied. R3327-H tumor growth and relevant tumor biomarkers (proliferation index, apoptosis [programmed cell death], microvessel density, and vascular endothelial growth factor [VEGF] expression) were compared in ad libitum fed control rats, ad libitum fed castrated rats, and groups restricted in energy intake by 20% or 40%. A second set of experiments involving both tumor models examined tumor growth in ad libitum fed rats or in animals whose energy intake was restricted by 30% using three different methods, i.e., total diet restriction, carbohydrate restriction, or lipid restriction. All P values are two-sided. RESULTS: R3327-H tumors were smaller in energy-restricted or castrated rats than in control rats (P<.001). Tumors from energy-restricted rats exhibited changes in tumor architecture characterized by increased stroma and more homogeneous and smaller glands. In castrated rats, the tumor proliferation index was reduced (P<.0001), whereas apoptosis was increased in both energy-restricted (P<.001) and castrated (P<.001) rats. Tumor microvessel density and VEGF expression were reduced by energy restriction and castration (P<.003 versus control). Restriction of energy intake by reduction of carbohydrate intake, lipid intake, or total diet produced a similar inhibition of growth of R3327-H or LNCaP tumors. These effects were associated with reduced circulating insulin-like growth factor-I. CONCLUSIONS: Our observations are consistent with the hypothesis that energy restriction reduces prostate tumor growth by inhibiting tumor angiogenesis. Furthermore, dietary fat concentration does not influence prostate tumor growth when energy intake is reduced.     

Tumor progression in serial passages of the Dunning R3327-G rat prostatic adenocarcinoma: growth rate response to endocrine manipulation

Serial passages of the poorly differentiated, androgen-sensitive R3327-G prostatic adenocarcinoma were used to study the progressive changes that occur in tumor growth rate and androgen sensitivity. Different in vivo transplant generations (21st to 28th) were compared. The tumor doubling and animal survival times resulting from the implantation of the 21st to 22nd generation (21-22G) tumor cells in intact male rats were significantly greater than those resulting from the implantation of 23-28G tumor cells. The most dramatic difference between early (21-23G) and late (26-28G) tumor generations, however, was in androgen sensitivity. The 26-28G tumors displayed androgen sensitivity only when implanted into animals castrated 2 to 7 days previously. Tumors grown in the pretreated castrates grew at a significantly slower rate than those in intact rats and the pretreated castrates had longer survival times than the intact rats. When 26-28G tumors were allowed to grow in intact rats to approximately 1 cu cm and then the rats were castrated, no significant difference in the growth rate between these tumors and tumors grown in intact rats was observed. In contrast, the androgen sensitivity of 21-23G tumors could be demonstrated, regardless of whether treatment was started before or after implantation. The fact that androgen sensitivity was still evident under certain conditions in late-generation R3327-G tumors demonstrates that the basic mechanism involving androgen response was still present, although functioning at a much reduced level.     

Melatonin and beta-glucan alone or in combination inhibit the growth of dunning prostatic adenocarcinoma

In this study, the effects of melatonin or beta-glucan treatments on tumor growth, pro-oxidant, and antioxidant status in tumor tissue were investigated in Dunning 3327 MatLyLu prostatic adenocarcinoma model. Prostate cancer (PCa) was induced by single intradermal injection of 2 x 10(4) MatLyLu cells into the right hind leg of Copenhagen rats. Melatonin (10 mg/kg/daily; IP) or beta-glucan (50 mg/kg/daily; orally) treatments applied alone and together continued for 39 days. Melatonin or beta-glucan treatments alone or together inhibited tumor growth and decreased malondialdehyde (MDA) levels in tumor tissues of Dunning rats. However, there were no significant differences in tumor volumes and MDA levels among treatment groups. Melatonin and melatonin + beta-glucan treatments elevated glutathione (GSH) levels and superoxide dismutase, glutathione peroxidase, and glutathione transferase activities in tumor tissues. However, beta-glucan treatment did not influence GSH levels and antioxidant enzyme activities in tumor tissue of Dunning rats. These results indicate that melatonin and beta-glucan treatments alone or together inhibit tumor progression and oxidative stress in tumor tissues of rats with Dunning PCa.     

Cancer cell motility-inhibitory protein in the Dunning adenocarcinoma model.

Cell motility has been associated with metastatic ability in the Dunning R3327 rat prostatic adenocarcinoma model. Cancer cell motility promoters but not inhibitors have been described by many investigators. Serum-containing and serum-free media conditioned by the nonmotile, nonmetastatic G Dunning subline inhibited the motility of the highly motile, highly metastatic MAT-LyLu subline. Motility inhibition by the G subline-conditioned serum-free media was lost upon heating to 100 degrees C and by treatment with trypsin. The motility inhibitory protein(s) had a molecular weight exceeding 50,000 as determined by diafiltration. G subline-conditioned RPMI 1640 contained several proteins with molecular weights of between approximately 53,000 and 116,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of these bands may represent the first inhibitor of cancer cell motility identified.