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Aim: The aim of this study was to test for the influence of ascorbic acid on tumorigenicity and metastases of implanted PAIII prostate cancer adenocarcinoma cells in syngeneic LW rats., Materials and Methods: Hormone-refractory prostate cancer PAIII cells were implanted subcutaneously into immunologically intact, Lobund-Wistar (LW) rats. Intraperitoneal pharmacological doses of ascorbic acid were administered each day for the ensuing 30 days. On the 40th day, animals were sacrificed. Local tumor weights were measured, and metastases were counted., Results: At the end of the 40 day experimental period, the primary tumors were found to be significantly reduced in weight (p=0.026). In addition, sub-pleural lung metastases were even more profoundly reduced in number and size (p=0.009). Grossly enlarged ipsilateral lymph node metastases declined from 7 of 15 rats to 1 of 15 rats., Conclusion: Pharmacological doses of ascorbic acid suppress tumor growth and metastases in hormone-refractory prostate cancer.

Two common procoagulant activities associated with tumors are tissue factor and cancer procoagulant (CP), an activator of coagulation factor X. We have identified a convenient source of CP in transplanted Lobund-Wistar rat PA3 prostate tumors. CP activity was purified from 5 independent transplanted prostate tumors by column chromatography. The protein activated factor X in the absence of TF and factor VII. An antihuman CP antibody recognized rat CP in an ELISA and inactivated CP activity in a chromogenic assay. Lobund-Wistar prostate tumors may provide a convenient animal model useful in determining the role of CP in cancer development.

Reducing expression of the tissue factor gene in prostate adenocarcinoma cells (PAIII) results in a cell line that, in vivo, mimics the growth of wildtype (wt) PAIII. However, instead of continuing to grow and metastasize as wt PAIII tumors do, tissue factor deficient PAIII (TFD PAIII) masses spontaneously regress after several weeks. Although whole cell vaccines are typically inactivated prior to administration to prevent proliferation within the host, numerous studies have suggested that exposure to live, attenuated, whole tumor cells, and the extracellular microenvironment they recruit, increases immunotherapeutic potential. Here, we provide support for this notion, and a strategy through which to implement it, by demonstrating that subcutaneous vaccinations with the TFD PAIII protect the Lobund-Wistar rat against subsequent wt PAIII cell challenge. TFD PAIII immunized rats suffered significantly less metastasis of wt PAIII challenge tumors compared to unvaccinated naïve controls rats. These results offer the intriguing possibility that the TFD PAIII vaccine is an effective system for the prevention and, possibly, the treatment of prostate cancer.

Lobund-Wistar (LW) rats, which have high testosterone levels, are predisposed to develop hormone-refractory prostate cancer (HRPC) spontaneously and by methylnitrosourea (MNU) induction, and the development of HRPC progresses through 2 stages. This paper reviews several studies in which LW rats were placed on soy-containing diets and were evaluated for development of either spontaneous or MNU-induced prostate cancer. The premalignant, testosterone-dependent stage is inhibited by testosterone deprivation. In the absence of testosterone deprivation, tumorigenesis progresses spontaneously to the testosterone-independent refractory stage. In LW rats: moderate caloric restriction prevented development of spontaneous prostate cancer; dietary 4-hydroxyphenylretinamide prevented MNU-induced prostate cancer; and dietary supplementation with soy protein isolate with high isoflavones prevented spontaneous and induced tumors and led to moderate reduction of serum testosterone. In rats 12 mo of age and younger, changing from the control diet to the soy+isoflavone diet significantly prevented progression of spontaneous tumors to the refractory stage of disease. Tumors that developed spontaneously and after MNU induction showed similar developmental stages and morphology, but MNU-induced tumors had shorter latency periods before development. The accumulated data indicate that soy-based diets are effective in the prevention of prostate cancer.

Research on cancer prevention and therapy must focus on the refractory disease, the fatal end-stage of cancer that develops in patients with organ-related solid tumors. Refractory cancers develop spontaneously in advanced-stage tumors or in relapsed cases after failed therapy. Because neither prevention nor therapy is currently feasible, refractory cancer is a major impediment to survival. There is a great need for an animal model of prostate cancer (PC), one that develops cancer from initial premalignant to the terminal refractory stages. We describe here a model of hormone-refractory prostate cancer (HRPC) that develops spontaneously through two stages by endogenous mechanisms in the Lobund-Wistar (LW) rat. The early premalignant, testosterone (T)-dependent stage is promoted by high levels of endogenous T, and up to age 12 months is reversible by T deprivation; without this intervention, the tumorigenic process progresses to the refractory stage, which is highly aggressive and does not respond to T deprivation or to a wide range of therapies. Initial refractory tumors are palpable at approximately 18 months of age. As they continue to grow, the tumors express characteristics seen in refractory cancers in humans (i.e., hypoxia, expression of hypoxia-inducible factors, and metastasis). Chemically induced HRPCs in LW rats manifest the same two developmental stages, but with shorter latency periods. A transplantable, metastasizing cell line (PAIII) was derived from a germfree LW rat with advanced-stage cancer. Both spontaneous and chemically induced autochthonous HRPC model systems serve as outstanding models for studies on the prevention and therapy of refractory cancer.

Background: Transplantation of the Dunning R-3327 prostate tumor cell line is a common model of prostate cancer, though the rat strain (Copenhagen) from which the cell line was derived is resistant to spontaneous and chemically induced prostate cancer., Methods: To determine if susceptibility to chemically induced prostate carcinogenesis can be transferred from the susceptible Lobund-Wistar (LW) rat strain to the resistant Copenhagen (COP) strain, male COP rats and LW x COP hybrids were administered an intravenous dose (30 mg/kg) of methylnitrosourea (MNU) and implanted three times with silastic capsules containing testosterone propionate (25 mg each) at a 2-month interval. Serum was sampled at 3, 6, 9, and 12 months of age and assayed for testosterone., Results: Serum testosterone was significantly but transiently elevated following implantation of capsules with testosterone propionate, but then decreased by 12 months of age to a level which was significantly less (P < or = 0.001) than in LW rats but not significantly different from COP rats. Prostate cancer did not develop in COP rats, but 36% of LW x COP hybrids developed prostate-seminal vesicle tumors which expanded into the dorsolateral lobes within 10 months of MNU administration; this compares to 90% of LW rats which develop such tumors following this same induction protocol., Conclusions: COP rats lack inherent susceptibility to development of prostate cancer; susceptibility may be transferred from LW rats, or resistance from COP rats, to LW x COP hybrids and is present in the haploid state, consistent with the two-mutation event hypothesis of Knudson which holds that two mutations are required at a genetic locus for development of some cancers., ((c) 2005 Wiley-Liss, Inc.)

Since advanced prostate cancer is difficult to treat, we have chosen a very different approach: the development of vaccines to prevent initial de novo tumor formation. To test the hypothesis that prostate cancer can be prevented by vaccination, Lobund-Wistar (LW) rats were vaccinated subcutaneously with complete Freund's adjuvant (CFA) plus glutaraldehyde-fixed (GFT) whole cell or potassium thiocyanate extract (PTE) preparations derived from in vivo tumors, or with media and CFA (media-vaccinated). Rats were vaccinated each month substituting incomplete Freund's adjuvant for CFA, from age 3 to 12 months, and methylnitrosourea (30 mg/kg) was administered intravenously at 4 months of age. Groups of 30 GFT cell-vaccinated rats showed a 90% reduction, and PTE-vaccinated rats, a 50% reduction in the occurrence of de novo prostate tumors compared with media-vaccinated controls. When splenocytes from vaccinated rats were incubated with tumor cells prior to subcutaneous implantation, PTE-vaccinated rats showed a 80% reduction, and GFT cell-vaccinated rats showed a 40% reduction in the occurrence of tumors, demonstrating a role for the spleen in the protective response. The inflammatory responses in tumors from GFT cell-vaccinated rats and PTE-vaccinated rats were distinguished by an influx of eosinophils compared with the responses in tumors from media-vaccinated rats. These results demonstrate the possibility that prostate cancer can be prevented by immunization with vaccines based on whole tumor-derived vaccines.

Objective: The Fas (CD95) interaction with its receptor Fas ligand (FasL) is one of the main mechanisms of cell apoptosis. High expression of FasL has been consistently observed in a variety of human cancers. In this study, we evaluated FasL and its relationship with apoptosis and proliferation in Lobund-Wistar (L-W) cancers. The L-W rat strain develops spontaneous and induced adenocarcinomas in the anterior prostate and seminal vesicles. Although FasL expression has been observed in a subset of human prostate carcinomas, this multistage model allowed in vivo evaluation of subclones of malignant cells with a single genetic susceptibility., Methods: Apoptosis was evaluated in spontaneous, induced and transplanted tumors as well as metastasis using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique and transmission electron microscopy. Proliferating cell nuclear antigen (PCNA) and FasL expression were detected using immunohistochemistry and analyzed according to the number of positive cells and intensity of staining using a semiquantitive method., Results: Apoptotic indexes were significantly higher in spontaneous tumors compared to induced (p < 0.008), transplanted tumors (p < 0.0112) and metastases (p < 0.009). TUNEL-positive cells were frequently observed in the leukocytic infiltrate of the stroma in transplanted carcinomas and metastases. These findings were confirmed by electron microscopy. FasL expression was not uniformly localized in L-W carcinomas and its highest expression was observed in transplanted tumors and metastasis (p < 0.005). Moreover, PCNA indices were directly correlated with cancers showing high FasL total scores (Hscores)., Conclusions: In this model, high FasL expression was associated with cells displaying low apoptotic indexes and high PCNA index. Therefore, analysis of FasL may have clinical relevance in detecting the malignant potential of prostate cancers.

Background: Gene expression technologies have the ability to generate vast amounts of data, yet there often resides only limited resources for subsequent validation studies. This necessitates the ability to perform sorting and prioritization of the output data. Previously described methodologies have used functional pathways or transcriptional regulatory grouping to sort genes for further study. In this paper we demonstrate a comparative genomics based method to leverage data from animal models to prioritize genes for validation. This approach allows one to develop a disease-based focus for the prioritization of gene data, a process that is essential for systems that lack significant functional pathway data yet have defined animal models. This method is made possible through the use of highly controlled spotted cDNA slide production and the use of comparative bioinformatics databases without the use of cross-species slide hybridizations., Results: Using gene expression profiling we have demonstrated a similar whole transcriptome gene expression patterns in prostate cancer cells from human and rat prostate cancer cell lines both at baseline expression levels and after treatment with physiologic concentrations of the proposed chemopreventive agent Selenium. Using both the human PC3 and rat PAII prostate cancer cell lines have gone on to identify a subset of one hundred and fifty-four genes that demonstrate a similar level of differential expression to Selenium treatment in both species. Further analysis and data mining for two genes, the Insulin like Growth Factor Binding protein 3, and Retinoic X Receptor alpha, demonstrates an association with prostate cancer, functional pathway links, and protein-protein interactions that make these genes prime candidates for explaining the mechanism of Selenium's chemopreventive effect in prostate cancer. These genes are subsequently validated by western blots showing Selenium based induction and using tissue microarrays to demonstrate a significant association between downregulated protein expression and tumorigenesis, a process that is the reverse of what is seen in the presence of Selenium., Conclusions: Thus the outlined process demonstrates similar baseline and selenium induced gene expression profiles between rat and human prostate cancers, and provides a method for identifying testable functional pathways for the action of Selenium's chemopreventive properties in prostate cancer.

Hormone-related cancers in the prostate, breast, endometrium, ovary and testicle account for 30% of malignancies in humans. We have developed a unique model of spontaneous prostate cancer (PC) in Lobund-Wistar [L-W] rats that shares many of its characteristics with the natural history of PC in man, including (a) inherent predisposition, high production of testosterone and aging risk factors, (b) endogenous tumorigenic mechanisms, and (c) early stage testosterone-dependent and late stage testosterone-independent tumors. About 30% of L-W rats on diet L-485 develop spontaneous palpable cancer in the anterior prostate-seminal vesicle (P-SV) complex in average of 20.5 months. At age 12 months early stage spontaneous PC was prevented or reversed by testosterone-deprivation through change of diet from L-485 to soy protein isolate/isoflavone (SPII) diet, thereby preventing the late lethal clinical disease: about 75% of rats at risk of developing testosterone-independent P-SV tumors were free of detectable cancer and about 25% had developed testosterone-independent cancer at age 12 months. The duration of the dependent stage exceeded age 12 months in 75% of the rats at risk. Dietary soymeal, found in most natural ingredient diets, may promote PC tumorigenesis, but only in L-W rats.

Background: Epidemiological surveys recorded that men in the Orient (Japan and China) consuming diets high in soy food were at low risk of developing clinical prostate cancer, compared to a relatively high risk among men in the West who consumed diets low in soy food. Soybeans contain phytoestrogens (isoflavones) with many recorded anticancer mechanisms. The Lobund-Wistar (L-W) rat is a unique model system: approximately 30% develop metastasizing adenocarcinomas spontaneously in the anterior prostate-seminal vesicle complex (P-SV), from which the tumors expand into the dorsolateral lobes. L-W rats are inherently predisposed, possibly by unusually high levels of circulating testosterone (T), to develop P-SV tumors which are T-dependent in the early stages and T-independent in advanced stages of tumorigenesis., Methods: L-W rats were fed two diets from age 2-24 months: 1) natural ingredient diet L-485 (Harlan TekLad Diets, Madison, WI) containing soy meal, or 2) a modified starch-casein diet in which soy protein isolate/isoflavones (SPII) replaced casein as a source of protein., Results: At age 24 months, 3 of 99 (3%) rats on diet SPII and 30 of 100 (30%) rats on diet L-485 developed spontaneous P-SV cancers. Rats on the SPII diet manifested a significant reduction of circulating T, approaching physiological levels. Failure of the rats on diet L-485 to prevent P-SV cancer development suggests that soy meal contained a factor(s) that blocked the antiandrogenic action of the phytoestrogen., Conclusions: The spontaneous development of P-SV cancers was significantly prevented in L-W rats consuming the SPII diet from age 2-24 months, possibly through an agonist effect of the soy-derived phytoestrogens., (Copyright 2000 Wiley-Liss, Inc.)

Based on epidemiological surveys, the low incidence of clinical prostate cancer among aged men in Japan and China were attributed to high consumption of soybean-derived food in which phytoestrogens have numerous anticancer mechanisms. The prostate model in Lobund-Wistar (L-W) rats produce high levels of testosterone (T). They are inherently predisposed to develop induced and spontaneous metastasizing adenocarcinomas, which are (T)-dependent in early stages and T-independent in advanced stages. In the experiment reported here, 2 groups of L-W rats (age 2 months) were fed soy-containing diets: (a) commercial diet L-485 (TekLad) with soy meal; or (b) a soy-free diet (L-474) in which casein was replaced by soy protein isolate/isoflavones (SPII). At age 3 months, all rats were inoculated i.v. with MNU; and 14 months later, 17/58 (29.3%) of rats on diet L-485 developed cancer in avg 12 months, compared to 5/50 (10%) of rats on the SPII diet in avg 12.1 months (P = 0.001). In the latter rats, the serum levels of T, and weights of testes were significantly reduced; but in the former rats, serum levels of T remained elevated, suggesting that soy meal in L-4,85 blocked the estrogenic effects of phytoestrogens.

Susceptibility of Noble strain rats to induced cancers in the accessory sex glands was determined. Following an IV inoculation of methylnitrosourea plus three successive SC implants of testosterone propionate, early stage adenocarcinomas were observed in the seminal vesicles and anterior prostates, but not in the dorsolateral nor in the ventral lobes., (Copyright 2000 Wiley-Liss, Inc.)

Prostate tumors were induced in Lobund-Wistar rats by treatment with N-methyl-N-nitrosourea (MNU) and testosterone propionate (TP). Androgen receptor (AR) expression was confirmed in 16 (100%) of the primary prostate cancers, with strong uniform staining in well-differentiated tumors and more variable AR immunoreactivity in poorly differentiated tumors. Epithelial cell lines were established from nine of the tumors. At early passages, four of the tumor cell lines tested were strongly immunoreactive for AR; however, only two of the cell lines, E2(A) and F2, have remained AR-positive. These cell lines specifically bind 5H-DHT at 40 and 19 fmol/mg protein, respectively, and express a 110 kDa AR immunoreactive protein. Proliferation in in vitro culture of both E2(A) and F2 cells was increased in the presence of 5alpha-dihydrotestosterone (DHT). The antiandrogen, hydroxyflutamide was able to prevent the DHT-induced growth of E2(A) but not F2 cells. Furthermore, hydroxyflutamide alone increased proliferation of F2 cells, suggesting that the androgen signalling pathway in this cell line may be abnormal. Tumorigenicity of the AR-expressing and nonexpressing cell lines was confirmed by xenograft formation following subcutaneous inoculation into intact male nude mice. In summary, carcinogen-induced prostate tumors of Lobund-Wistar rats express AR and two of nine cell lines derived from the tumors express AR. Further evaluation of AR structure in primary prostate tumors forming spontaneously or following MNU and TP induction will determine whether, as in human prostate cancers, disease progression in Lobund-Wistar rats is associated with mutations in the AR gene.

Background: Since prostate cancer (PC) development involves a combination of genetic predisposition and promotional mechanisms, especially the metabolic conversion of testosterone to 5alpha dihydrotestosterone (DHT) by 5alpha reductase, how do mechanisms in man relate to prostate-seminal vesicle (P-SV) tumor development in Lobund-Wistar (L-W) rats? The disease in man and in L-W rats shares developmental mechanisms and characteristics to the extent that prevention of P-SV tumors in L-W rats could be predictive of similar results in man. The epidemiology of PC in man and P-SV tumors in L-W rats indicates that both are hormone-related diseases based on genetic predisposition, high production of androgens (which are activated to DHT by 5alpha reductase), and early development of androgen-dependent and metastasizing late androgen-independent stages of adenocarcinomas, all after long latency periods., Methods: L-W rats at risk of developing spontaneous or induced P-SV tumors were subjected to putative antitumor agents or procedures. These included dietary restriction, testosterone ablation, soybean-derived isoflavones, antiangiogenic linomide, tamoxifen, and a vitamin D analogue., Results: L-W rats subjected to 1) early onset of dietary restriction manifested suppression of spontaneous and induced development of P-SV tumors; 2) testosterone-ablation by nonesterified DHT (NE-DHT) suppressed early onset of induced P-SV tumors and to a lesser extent late onset of spontaneous tumors; 3) diets containing soy protein isolate (high isoflavones) manifested marginal suppressive effects against induced P-SV tumors, but in 12-month-old rats, the development of spontaneous tumors was reduced in incidence; 4) early administrations of antiangiogenic linomide suppressed development of induced P-SV tumors and of transplanted prostate adenocarcinoma III (PA-III) tumors, but linomide had little antitumor effect against large advanced stage tumors; and 5) tamoxifen and vitamin D analogue suppressed development of P-SV tumors. Results in conditions 1-3 were negative when tested against PA-III tumors., Conclusions: Developing stages of P-SV tumors were prevented in L-W rats with autochthonous spontaneous and induced tumors, but most of the agents tested were of no therapeutic benefit against advanced-stage and transplanted PA-III tumors. However, early administrations of antiangiogenic linomide suppressed early growth of induced and transplanted PA-III tumors.

Background: Gene-regulated mechanisms govern tumor development, but the actual development of tumors can be suppressed or promoted by epigenetic factors. Lobund-Wistar (L-W) rats are genetically predisposed to development of spontaneous and induced metastasizing moderately differentiated adenocarcinomas in the prostate-seminal vesicle (P-SV) complex. In L-W rats with one slow-release subcutaneous implant of dihydrotestosterone (DHT) (5alpha-Androstan-17beta-ol-3-one), the development of induced P-SV tumors 14 months later was significantly suppressed, with involution of testes, aspermia, and absence of detectable serum testosterone. The tumor-suppressive effect of DHT was confirmed. Spontaneous P-SV tumors developed in 57 of 220 control L-W rats (26%) at an average age 20 months., Methods: At age 12 months, 70 L-W rats were administered an implant of 40 mg of DHT, and 75 untreated rats served as controls. All rats that developed palpable P-SV tumors were autopsied, and surviving rats were autopsied at age 24 months., Results: At age 24 months, 9 of 70 DHT-treated rats (12.8%) and 20 of 75 DHT-free control rats (26.6%) had developed P-SV tumors spontaneously at average age 20.5 and 20 months, respectively., Conclusions: Slow-release implants of DHT administered to L-W rats at age 12 months reduced by 50% the development of spontaneous P-SV tumors by age 24 months.

Background: Angiogenesis and antiangiogenesis, as applied to oncology, are phenomena in which (1) tumors acquire a new blood vascular system from the host that is needed for their growth progression and metastasis; and (2) factors are produced that interfere with neovascularization, thereby inhibiting growth and metastasis of the tumor. Linomide, a chemical antiangiogenesis agent, inhibited the growth of transplanted tumors in mice and rats and inhibited the early development of metastasizing tumors induced in the prostate-seminal vesicle (P-SV) complex of genetically susceptible Lobund-Wistar (L-W) rats., Methods: L-W rats with small induced P-SV tumors were treated with a recommended dosage of linomide (100 mg/kg BW/day) by the intraperitoneal and oral routes. The rats were monitored for the next 1-2 months, and the primary and metastatic tumors were compared with related data in drug-free tumor-bearing control rats., Results: P-SV tumors in linomide-treated and untreated control rats continued to grow, except that in the former (1) the tumors were marginally smaller, (2) the centers of the primary P-SV tumors had failed to grow, (3) the peripheral areas of the tumors contained actively proliferating tumor cells, and (4) metastatic P-SV tumors in the lungs were disrupted with focal areas of necrosis, but areas of intact tumor cells survived. Spread of tumor cells into the peritoneal cavity was not inhibited. Rats on orally administered linomide lived significantly longer than rats inoculated by the intraperitoneal route and untreated control rats. The dosage of linomide used showed evidence of toxicity., Conclusions: Although primary and metastatic P-SV tumors were damaged in L-W rats treated with linomide, this antiangiogenic drug was of minimal therapeutic benefit to rats in which a palpable P-SV tumor had developed before onset of treatments.

The rate and extent of metastasis by prostate adenocarcinoma-III cells was enhanced in Lobund-Wistar rats by administration of immune-suppressive cyclosporine A. PA-III cells spread from the subcutaneous PA-III-derived tumor, through ipsilateral lymph nodes, to the lungs in which they developed secondary tumors. Swollen lymph nodes, compacted with PA-III cells, indicate that the 'normal' host-engendered level of intravascular non-specific restraints to metastasis were abrogated by CSA.

Lobund-Wistar (L-W) rats are inherently susceptible to spontaneous and induced metastasizing adenocarcinomas in the prostate-seminal vesicle (P-SV) complex. L-W rats were fed soy protein isolates containing high isoflavones (genistein and daidzein) or low isoflavones to determine their effects on development of induced P-SV tumors in two stages of the tumorigenic process. In rats fed the high-isoflavone-supplemented soy diet before initiation by methylnitrosourea (MNU), the incidence of induced prostate-related cancer was reduced and the disease-free period was prolonged by 27% compared with rats fed the same diet but low in isoflavones. Rats fed the same diets, started after MNU, manifested suggestive but less consistent results than those noted above. The incidence rates were of marginal significance, suggesting that the high intensity of the active induced disease may not represent the character of the slower-growing spontaneous (natural) disease. The delay of disease onset is of clinical significance.

Purpose: To study the mechanism of diminished arachidonic acid levels in malignant prostatic tissues., Materials and Methods: Benign and malignant prostate tissues were obtained from human radical prostatectomy specimens and from rats using Pollard's Lobund/Wistar rat prostate cancer model. Fatty acid composition and a variety of enzyme activities involved in maintaining phospholipid fatty acid composition were compared in malignant and benign prostatic tissues., Results: Decreased arachidonic acid levels, previously reported in human prostate cancer, were present in malignant rat as well as in human tissues. There were 21% and 26% decreases of arachidonic acid levels in the rat and human malignant tissues compared with benign tissues. Fatty acid desaturase activity was undetectable. Fatty acyl-CoA hydrolase and synthetase activities were not altered in the malignant tissues. However, there was a 2-fold increase in phospholipase A2 activity and a 4- to 12-fold increase in fatty acyl-CoA lysophosphatidylcholine acyltransferase activity in malignant rat and human prostatic tissues., Conclusions: These data indicate that, in malignant prostate tissues, the fatty acid remodeling mechanism is activated through the deacylation-reacylation cycle. This process may be a result of increased use of arachidonic acid for the formation of prostaglandins that may be crucial for the further development and growth of the malignant tissues.

Two contradictory actions have been ascribed to thalidomide relative to tumor metastasis: immunosuppression and anti-angiogenesis. The latter effect was determined with basic fibroblast growth factor in a rabbit cornea micropocket assay system. The prostate adenocarcinoma (PA-III) transplanted tumor line in Lobund-Wistar (L-W) rats produces a tumor at the subcutaneous implant site from which tumor cells metastasize uniformly only through lymphatic channels through the heart to the lungs in which secondary tumors develop. L-W rats were implanted with PA-III cells and administered, by gavage, thalidomide (50 mg/kg body wt per day) in corn oil. Control rats with PA-III cells were administered corn oil. Autopsy examinations on day 30 revealed that the thalidomide-treated rats developed more metastatic tumor foci in the lungs than in the controls.

Aged Lobund-Wistar (L-W) rats develop: (i) spontaneous and induced metastasizing adenocarcinomas in the prostate and seminal vesicle (P-SV) complex; and (ii) spontaneous hepatomas and hepatocarcinomas. Within the time-frame of 14 months, similar adenocarcinomas were induced in the P-SV complex in 70-90% of younger L-W rats by a single i.v. inoculation of methylnitrosourea (MNU) which was followed by slow release s.c. implants of testosterone propionate (TP). Within the same time-frame, neither MNU nor TP alone induced significant incidences of P-SV tumors; and untreated control L-W rats were disease-free. Methylnitrosourea or TP and combinations thereof did not induce liver tumors. However, when MNU-inoculated L-W rats were fed phenobarbital (PB), they developed (i) metastasizing adenocarcinomas in the P-SV complex and (ii) altered cellular foci and nodules in the livers. Methylnitrosourea induced a high incidence of benign lung adenomas which progressed to lung cancers in numbers which were of marginal significance. Thus, dormant MNU-initiated cells in the P-SV complex were activated by phenobarbital, to produce adenocarcinomas in that complex.

The prostate adenocarcinoma-III (PA-III) cell line manifests a high rate of metastasis from the SC tumor site through lymphatic channels to the lungs in which they produce visible focal tumors. i.v. inoculation of BCG interfered with the passage of PA-III cells to the lungs; but not if the BCG was inoculated IP. IP inoculation of BCG suppressed propagation of PA-III cells in the peritoneal cavity, but there was no effect on metastasis of PA-III cells from a SC PA-III tumor to the lungs. Inoculation of BCG did not induce a systemic anticancer effect unless PA-III cells and BCG occupied the same anatomical compartment. Two compartments are described: intravascular and intraperitoneal.

Adenocarcinomas were induced in the prostate-seminal vesicle complex of Lobund-Wister (L-W) rats by a single IV inoculation of N-methyl-N-nitrosourea. This was followed by three slow-release S.C. implants of testosterone propionate, each at intervals of 2 months. Small (0.5 cm diameter) palpable tumors developed which enlarged during the following month to 3-4 cm diameter. At the latter stage, tumor cells spread via lymphatics to the lungs and/or by direct extension into the peritoneal cavity. Rats with small palpable tumors were inoculated IV with viable Bacillus Calmette-Guerin (BCG). One month later, the rats were killed and examined. Untreated rats with large tumors served as controls. Comparison of the two groups revealed that body weights and tumor sizes were similar and most of them had developed metastatic tumors in the peritoneal cavity. However, lung metastases were rare in the BCG-inoculated rats compared to controls. Spleen and liver weights were significantly heavier in the BCG-treated rats. It is speculated that an intra-vascular mechanism(s), engendered by BCG, immobilized the circulating tumor cells, but not those tumor cells that spread by direct extension from the primary tumor into the peritoneal cavity.

Using a one-stage kinetic chromogenic assay, we studied the procoagulant activity (PCA) of prostatic tissue in an experimental model of prostate cancer in the rat. PCA was present in homogenates of rat prostate glands containing either benign or malignant tumours. The procoagulant activated factor X directly and was provisionally characterised as a tissue factor-factor VIIa complex. There was no significant differences in PCA between control rats and rats exposed to carcinogens that did not develop tumour. Levels in rats that developed tumours were significantly higher (P < 0.01) than all other groups and there was a positive correlation between tumour weight and PCA (r = 0.85, P < 0.001). Furthermore, prostatic PCA levels were higher in the metastasis (P < 0.02). We conclude that PCA reflects the malignant phenotype in this animals, the PCA of the primary tumour was compared with that of the corresponding secondary deposit and levels were higher in the metastasis (P < 0.02). We conclude that PCA reflects the malignant phenotype in this model of experimental prostate cancer and suggest that this parameter is worth evaluating as a potential tumour marker in the human disease.

The complication of metastasis from the primary tumor site to the distant organ is difficult to control. Tumor cells usually spread through the vascular system: lymphatics and/or blood; or by direct extension into adjacent sites. Transplantable prostate adenocarcinoma (PA-III) cells in L-W rats produces a tumor in the implant site and then spreads via the ipsilateral lymphatic route to the lungs in which new visible tumors develop. Viable bacillus Calmette-Guerin (BCG) was inoculated SC or IV into rats which were then inoculated either SC or IV with PA-III cells. They were examined thereafter for primary and for lung tumors. IV-inoculated BCG generated a strong intravascular intervention mechanism on metastatic PA-III cells in L-W rats. This immobilization mechanism was not demonstrable in rats that had been inoculated SC with BCG.

Lobund-Wistar (L-W) rats are susceptible to spontaneous and induced metastasizing prostate cancer. In the search for the initial site of tumor development in the induced disease, rats at risk were examined periodically to acquire this information. In the course of 12 months after the onset of the induction of prostate cancer in L-W rats, 14 of 40 rats (35%) developed visible tumors in the anterior and in the dorso-lateral lobes of the gland. The prostate tumors appeared at 7 months and thereafter. Two of the tumor-bearing rats had developed, in addition, a visible neoplasm in the seminal vesicle. None of the rats with small early tumors had developed visible metastatic tumors, which were manifested in rats with large tumors.

Two models of preventable metastasizing autochthonous prostate adenocarcinoma (PA) have been described in Lobund-Wistar (L-W) rats: spontaneous PAs that develop at a mean age of 26 months; and induced PAs that develop at a mean time of 10.5 months. Both are similar in many respects to the counterpart disease in man. PAs develop spontaneously, and by induction through a combination of N-methyl-N-nitrosourea (MNU)/testosterone treatments. Our investigations with L-W rats show that PA is manifested spontaneously in 26% of aged L-W rats, and by induction in approximately 90% of younger rats. It is characterized by metastatic adenocarcinoma initiated in, and expanding from, the dorso-lateral and anterior lobes, and occasionally in the seminal vesicles. It is regulated by genetic, hormonal, and age-related mechanisms. Spontaneous PAs are prevented by life-long moderate (25%) diet restriction and, in rats at risk of developing induced PA, by early treatments with estradiol, with dihydrotestosterone, with a retinoid, and by castration. While the "premalignant" stages of induced tumorigenesis are susceptible to intervention, the overtly malignant stage resists therapeutic trials with the same agents and procedures. The transition from dependency to autonomy has not yet been defined.

We report for the first time that a synthetic retinoid, N-(4-hydroxyphenyl)retinamide, can prevent the development of both primary and metastatic tumors in an animal model of metastasizing primary prostate cancer. Prostatic adenocarcinomas were induced in high incidence in Lobund-Wistar rats by initiation with methylnitrosourea i.v. and promotion with testosterone. Feeding of N-(4-hydroxyphenyl)retinamide to these rats during the latency period markedly diminished the final incidence of both primary and metastatic prostate carcinomas.

Therapeutic effects of cyclophosphamide (CPA) were compared in L-W rats with (a) transplanted prostate adenocarcinomas (PAs), and (b) with induced autochthonous PAs. All rats, except controls, were treated by IP-administered CPA in slow-release silastic membranes (a) at time 0 after inoculation of transplanted PA cells; or (b) after development of palpable transplanted PAs thereof; or (c) after detection of palpable autochthonous PAs. The treated rats did not manifest evidence of CPA-related toxicity. The transplanted PAs and lung metastases thereof were suppressed by CPA; but, the same treatment schedule with CPA failed to modify continued growth of the autochthonous PAs. This report serves to emphasize the need for test systems of autochthonous model tumors of the counterpart disease in humans.

A model system of prostate adenocarcinoma (PA) in Lobund-Wistar rats is described in which the transplanted tumor cells (PA-III) were inoculated sc over the calvaria. A local tumor developed that, when attached to the bone, caused erosion and fragmentation of the bone, with adjacent osteoplastic changes and metastases to the lungs. The attachment of the tumor to the bone was enhanced if the periosteum was scratched during the inoculation of tumor cells. The destructive effect on bone structure was also induced by in vivo transplanted PA-I and PA-IV cells, but not by PA-II cells. In rats that were pretreated with dichloromethylene diphosphonate, the local PA-III tumors developed with metastases to the lungs, but the calvariae were intact.

Depots of testosterone implanted in Lobund-Wistar (L-W) rats, resulted in increased incidence of prostate adenocarcinomas (PAs) in the dorsal lateral lobe area. In testosterone-treated rats which had been fed the same diet plus 15% corn oil, the prostate tumors were more numerous and they appeared earlier. Rats consuming the same feed containing 15% corn oil (no testosterone) did not produce prostate tumors. Positive correlations were demonstrated between the development of PAs in L-W rats that were treated with testosterone and high fat diet.

Four model systems for prostate adenocarcinomas have been developed in rats: they are designated Dunning, Noble, ACI, and Pollard tumors. They have attributes that make them valuable sources of information, of relevance to the disease in man. The development and use of the Pollard tumors as in vitro and in vivo model systems are reviewed, especially on the phenomenon of metastasis. Specific agents either accelerate or retard the rate and extent of metastatic spread of the prostate tumor cells from extravascular sites to specific target organs. Specific enzymes may be involved in the phenomenon of metastasis.

Aging conventional (CV) and germfree Lobund-Wistar (L-W) rats developed spontaneous tumors, predominantly in the prostate, liver, and adrenal glands. In CV L-W rats a 30% reduction of daily intake (natural ingredient diet L-485) had the following effects: (1) reduction of the incidence of metastatic prostate adenocarcinomas (PA) from 25.7% to 6.3% and extension of the average latent periods from 26.6 to 36.7 months; and (2) reduction of the incidence of hepatomas from 59% to 26% and extension of the average latent periods from 31.3 to 34 months. Adrenal medullary tumors developed in approximately 60% of rats older than 19 months, regardless of dietary intake and microbial status. Stromal hyperplasia developed among 36 of 78 (46%) rats older than age 30 months. Rats with PA were free of stromal hyperplasia, and the reverse was also true. Germfree L-W rats developed all of the above tumors, but the diet-related differences were not as significant as those among the conventional counterpart rats. The incidence of prostatis was reduced from 22% to 6% among the diet-restricted rats, but this lesion did not develop among the germfree rats.

Model systems for prostate cancer in rats have been developed and used for investigations on tumor biology and therapy. The "Pollard tumors" provide a combination of in vitro and in vivo attributes by which investigations can be directed at local tumor development and spontaneous metastasis. The evolution and early applications of this model system are reviewed, and the therapeutic benefits of delayed release of cyclophosphamide are presented.

Three transplantable rat prostate adenocarcinoma cell lines were assessed for patterns of metastasis, ie, routes of dissemination and larger organ(s) selected for implantation. Two lines (I and III) were disseminated only through ipsilateral lymphatic channels to the lungs. The third cell line (II) was disseminated through lymphatic and blood channels to lungs, liver, and kidneys. The pattern of spontaneous metastasis is a characteristic of the tumor cell; but there is evidence that the rate and extent of metastasis can be modified experimentally.

Two rat adenocarcinomas were physically dispersed and propagated in vitro. Epithelial and fibroblast cell lines were cloned from them and the monolayer cell lines derived therof were further characterized. The cells produced acid phosphatase in early in vitro cell passages, and later they turned negative. Fibroblast-like cells produced no tumors when implanted in syngeneic Lobund Wistar rats, but as few as 10 epithelial cells produced metastasizing adenocarcinomas in them. A third prostate tumor has yielded a line of epithelial cells which reproduced the original tumor type in inoculated rats, but the cells have not yet been characterized. Rat prostate adenocarcinomas provided a useful model system for in vitro and in vivo studies on prostate cancer and on metatasis.

Metastatic prostate adenocarcinomas, derived from aging germfree Wistar rats, have been propagated in rats and in tissue culture. A protocol has been developed and demonstrated for assay of treatments which retard or which accelerate the rate and extent of tumor growth and of metastasis in tumor-bearing rats. The pattern of spread has been retarded by cyclophosphamide, aspirin, indomethacin, and Corynebacterium parvum. The spread pattern has been accelerated by oral administrations of sodium barbiturate.

The Lobund-Wistar (L-W) rat is unique in its susceptibility to spontaneous and induced metastasizing prostate adenocarcinomas (PAs). A single IV inoculation of methylnitrosourea (MNU) produced PAs in 20% of L-W rats in 12 months. The combination of MNU plus two to seven slow-release implants of testosterone propionate (TP) induced PAs in 50-90% of rats respectively in an average of 11.5 months. The induction of PAs was prevented by early treatments of rats at risk with estradiol and less so with dihydrotestosterone (DHT). However, on a technical basis, the results were not significant. Treatments of MNU-inoculated rats with estradiol, with DHT, or by castration, at intermediate points in the projected latency time of tumor development, reduced significantly the incidences of PA development. Rats in which overt PAs had already developed in response to 12 months of exposure to implants of TP did not respond to treatment by estradiol, DHT, or castration. Thus there are early stage(s) in induced prostate tumorigenesis in L-W rats that are sensitive to modulating agents.

Germfree (GF) Lobund strain Wistar (LW) rats, fed vegetable diet L-485, have developed prostate adenocarcinomas spontaneously (10% incidence) at average age 34 months. Conventional LW rats, implanted with testosterone at age 4 months, developed a higher incidence of prostate cancer after an average interval of 14 months: 24% had developed gross tumors, and 40% when it included microscopic tumors. Preliminary results indicate that testosterone-treated LW rats that were fed the same diet, which was supplemented with corn oil up to 20% fat, developed prostate cancer after intervals of 6-12 months. Aged GF Sprague-Dawley (SD) rats have not developed prostate cancer spontaneously. Conventional SD rats fed diet L-485 and treated with testosterone developed only prostatitis. Experimental designs should consider genetic susceptibility as a basic prerequisite for studies on experimental prostate cancer.

Natural killer (NK) cells have been proposed to play a significant role in the inhibition of metastasis. The prostate adenocarcinoma (PA-11) in the Lobund-Wistar (L-W) rat provides a unique model of spontaneous metastasis in which to study NK response. Cultured PA-11 tumor cells were shown to be resistant to NK lysis in vitro, and enhancement or inhibition of NK reactivity in vivo using drugs or antiserum did not change the rate or extent of metastasis evident at autopsy. Exposure to PA-11 tumor cells, supernatants from cultured tumor cells, or sera from rats with advanced PA-11 in vitro did not result in inhibition of NK activity. Exposure to PA-11 tumor cells in vivo also did not cause suppression of NK activity. These data indicate that, in the PA-11/L-W system, metastasis is independent of NK activity.

The levels of UDP-galactose: N-acetylglucosamine(beta 1-4)galactosyltransferase activity (GalT-4) were determined in the sera, in solid tumors, and in corresponding cell cultures of rats bearing two lines of prostate adenocarcinomas (PA-2 and PA-3), and in the sera of rats bearing other transplanted and autochthonous adenocarcinomas. Sera and tissues from normal (tumor-free) rats were used as controls. Prostate adenocarcinoma cell cultures had five times greater levels of enzyme activity than did tumor cell infiltrated lymph nodes from animals bearing the prostate adenocarcinomas. The levels of activity in the cells of both prostate tumor lines were equivalent, even though they metastasize through different routes. Serum levels of galactosyltransferase activity were detected in the blood, and in rats bearing a mammary adenocarcinoma with extensive necrosis of the tumor mass (tumor mass greater than 22.0 g/200 g body weight). The increase was three times the control value. The sera of L-W rats bearing prostate tumors (PA-2 and PA-3) were inactive with the GalNAc-containing acceptor, Gm2 (GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4Glc-Cer) but active with either free GlcNAc (Km = 0.25 mM) or LcOse3Cer (GlcNAc beta 1-3 Gal beta 1-4Glc--Cer; GlcNAc--R).

Zymographic analysis of the supernates from confluent cultures of a rat prostate adenocarcinoma cell line, PA-III, revealed the existence of two molecular forms of specific plasminogen activators, one of molecular weight of approximately 80 000 and another of approximate molecular weight of 45 000, in sodium dodecyl sulfate. The low molecular weight form has been purified 364-fold in 66% yield from the culture medium by a combination of gel filtration on Sephacryl S-200 and affinity chromatography on Sepharose 4B-benzamidine. The purified material possessed a specific activity of 192 000 urokinase CTA units mg-1. This enzyme displayed activity toward human Glu1-plasminogen, characterized by a Km of 1.7 +/- 0.2 microM and a Vmax of 0.53 +/- 0.1 pmol of plasmin min-1 unit-1. A synthetic chromogenic substrate, H-D-Ile-Pro-Arg-p-nitroanilide (S-2288), was found for the activator. The enzyme possessed a Km of 0.33 mM and a kcat of 55 s-1 for S-2288. The activator was found to be a serine protease, inhibited by diisopropyl fluorophosphate (iPr2PF). At a concentration of 1 mM iPr2PF, and 30 nM enzyme, the half-time of this inhibition was 3.8 min. The 45 000 molecular weight enzyme was found to be inhibited by rabbit antibodies to human urokinase, thus characterizing the activator as a member of the urokinase class. The 80 000 molecular weight enzyme was not neutralized by anti-human urokinase but was neutralized by rabbit anti-human melanoma activator, likely allowing it to be classified as the tissue activator type.

More than 50% of Lobund-Wistar (L-W) strain rats developed large, palpable prostate adenocarcinomas (PAs) following treatments with N-nitroso-N-methylurea (CAS: 684-93-5) and testosterone propionate [(TP) CAS: 57-85-2], and most of the tumor-bearing rats manifested metastatic lesions. The incubation periods averaged 10.6 months. Within the same timeframe, no L-W rat developed a similar palpable PA when treated only with TP. In L-W rats, TP acted as a tumor enhancement agent, with primary emphasis on the development of prostate cancer.