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Background: The second leading cause of cancer-related deaths in American men is metastatic hormone-refractory adenocarcinoma of the prostate, for which there is currently no effective treatment. Transferrin is abundant in bone stroma and has been found to stimulate models of hormone-refractory metastatic prostate cancer. Suramin, a compound that has been used to treat metastatic prostate cancer, has been demonstrated to antagonize the binding of transferrin to the transferrin receptor and to suppress uptake of iron by hematopoietic cells., Purpose: The purpose of our study was to determine whether transferrin may reverse the inhibitory action of suramin on metastatic prostate-derived cell lines., Methods: Five human prostate cell lines (PC-3, PC-3M, DU-145, TSU-Pr1, and LNCaP) derived from metastatic deposits were examined for response to growth stimulation by apotransferrin, for the presence of transferrin receptors by binding of 125I-labeled transferrin, and for relative transferrin receptor messenger RNA (mRNA) content by ribonuclease protection assays. We measured the amount of growth inhibition by suramin in low serum assays to demonstrate maximal inhibition over the apotransferrin to reverse the inhibition of suramin in these tumors., Results: The results clearly demonstrate that the androgen-insensitive metastatic cell lines (PC-3, PC-3M, DU-145, and TSU-Pr1) demonstrate increased cell numbers when exposed to holotransferrin or apotransferrin, while the androgen-sensitive cell line (LNCaP) did not show any increase. All cell lines demonstrated a similar number of transferrin receptors and transferrin receptor mRNA. We used these maximally inhibitory, but clinically relevant, concentrations of suramin to determine whether transferrin could reverse the inhibition, and it did, but only in the androgen-insensitive metastatic lines. Indeed, in the PC-3 cells, inhibition turned to stimulation with the addition of transferrin, and even at the highest concentration of suramin tested, 400 microM, a concentration that would be toxic to patients, the amount of inhibition by suramin was still reduced by more than 50% by transferrin in TSU-Pr1 cells. In the androgen-sensitive LNCaP cells, however, transferrin had limited ability to block the inhibitory activity of suramin., Conclusions: Concentrations of tumor-stimulating factors, such as transferrin, in the metastatic microenvironment need to be taken into consideration in the use of suramin and suramin-like derivatives. Novel strategies need to be identified that will negate the action of transferrin on androgen-insensitive cells.

This study explored the use of cytokine gene-modified tumor cells as cellular vaccines for the treatment of bladder cancer. The mouse MBT-2 tumor is an excellent model for human bladder cancer. This carcinogen-induced tumor of bladder origin resembles human bladder cancer in its etiology and histology and responds to treatment in a manner similar to that of its human counterpart. In a previous study we have shown that interleukin 2 (IL-2)-secreting, irradiated, MBT-2 cell preparations were capable of curing animals from orthotopically established tumors and engendered protective immunological memory in the cured animals. In this study we have compared the effectiveness of several cytokines and found that while IL-1 alpha, IL-1 beta, and gamma-interferon were only weakly effective in the therapeutic vaccination protocol, granulocyte-macrophage colony-stimulating factor was almost as effective as but not superior to IL-2, as reported previously for another tumor model system. Induction of cytotoxic T-lymphocyte correlated only poorly with the therapeutic benefit of the cytokine gene-modified tumor cell preparations, questioning its prognostic value for the development of improved genetically modified tumor vaccines.

In order to develop an animal model that more closely simulates the organ environment and metastatic routes of human prostatic cancer, R3327-MatLyLu tumors were induced by orthotopic implantation in the ventral prostatic lobe of Copenhagen rats. This procedure reproducibly resulted in metastatic spread of the intraprostatic tumor to the pelvic and retroperitoneal lymph nodes, and invariably to the lungs. Further, a tumor recurrence model was established using an approach that combined orthotopic tumor implantation and subsequent surgical resection of the primary tumor. When prostatectomy was carried out 4 days or more after induction, tumors recurred locally in all animals. The surgical procedures described may provide an animal model to test the in vivo response to experimental adjuvant treatment protocols for advanced prostate cancer. Immunological studies are now in progress using cytokine gene-modified prostatic tumor cells as cellular antitumor vaccines in orthotopically established R3327-MatLyLu tumors.

We have recently cloned a 2.65-kilobase complementary DNA (cDNA) encoding the prostate-specific membrane antigen (PSM) recognized by the 7E11-C5.3 anti-prostate monoclonal antibody. Immunohistochemical analysis of the LNCaP, DU-145, and PC-3 prostate cancer cell lines for PSM expression using the 7E11-C5.3 antibody reveals intense staining in the LNCaP cells with no detectable expression in both the DU-145 and PC-3 cells. Coupled in vitro transcription/translation of the 2.65-kilobase full-length PSM cDNA yields an M(r) 84,000 protein corresponding to the predicted polypeptide molecular weight of PSM. Posttranslational modification of this protein with pancreatic canine microsomes yields the expected M(r) 100,000 PSM antigen. Following transfection of PC-3 cells with the full-length PSM cDNA in a eukaryotic expression vector, we detect expression of the PSM glycoprotein by Western analysis using the 7E11-C5.3 monoclonal antibody. Ribonuclease protection analysis demonstrates that the expression of PSM mRNA is almost entirely prostate specific in human tissues. PSM expression appears to be highest in hormone-deprived states and is hormonally modulated by steroids, with 5-alpha-dihydrotestosterone down-regulating PSM expression in the human prostate cancer cell line LNCaP by 8-10-fold, testosterone down-regulating PSM by 3-4-fold, and corticosteroids showing no significant effect. Normal and malignant prostatic tissues consistently show high PSM expression, whereas we have noted heterogeneous, and at times absent, expression of PSM in benign prostatic hyperplasia. LNCaP tumors implanted and grown both orthotopically and s.c. in nude mice abundantly express PSM, providing an excellent in vivo model system to study the regulation and modulation of PSM expression.

Adenocarcinoma of the prostate is the most common cancer in men. The majority of cancers are discovered once they have already metastasized, and there is no effective therapy for prostatic cancer at this stage. The use of cytokine-secreting tumor cell preparations as therapeutic vaccines for the treatment of advanced prostate cancer was investigated in the Dunning rat R3327-MatLyLu prostatic tumor model. IL-2 secreting, irradiated, tumor cell preparations were capable of curing animals with s.c. established tumors, and induced immunological memory that protected animals from subsequent tumor challenge. Immunotherapy was less effective when tumors were induced orthotopically, but nevertheless led to improved outcome, significantly delaying, and occasionally preventing, recurrence of tumors after resection of the cancerous prostate. Granulocyte-macrophage colony stimulating factor secreting tumor cell preparations were less effective, and interferon-gamma secreting cells had only a marginal effect. Induction of a potent immune response in tumor bearing animals against the nonimmunogenic MatLyLu tumor supports the view that active immunotherapy warrants further investigation as a potential therapeutic approach to prostate cancer.

Adenocarcinoma of the prostate is the second leading cause of cancer deaths in men. The protein kinase C (PKC) family of signal transducing kinases has been implicated in neoplastic transformation and progression in other tissues, and some evidence suggests roles for PKC in prostate growth and neoplasia. We have detected expression of eight specific PKC isozyme mRNAs (alpha, beta, gamma, delta, epsilon, eta, theta, and zeta) in normal rat whole prostate and found some of these to be differentially expressed in certain Dunning R-3327 rat prostatic adenocarcinoma sublines. PKC zeta mRNA was detected in normal prostate and Dunning H tumor, whereas an alternatively spliced form of PKC zeta RNA was found in Dunning G tumor and normal brain. Both forms of PKC zeta RNA were markedly reduced in the androgen insensitive, highly metastatic Dunning AT-3, MAT-Lu, and MAT-LyLu tumors. We have cloned and report the sequence of the novel portion of the alternatively spliced form of PKC zeta RNA, which is polyadenylated and present in cytoplasm.

Recently, a novel M(r) 100,000 prostate-specific membrane glycoprotein (PSM) has been detected by the prostate-specific monoclonal antibody 7E11-C5, raised against the human prostatic carcinoma cell line LNCaP. The PSM antigen is expressed exclusively by normal and neoplastic prostate cells and metastases. We now report the molecular cloning of a full-length 2.65-kilobase complementary DNA encoding the PSM antigen from a human LNCaP complementary DNA library by polymerase chain reaction using degenerate oligonucleotide primers. Analysis of the complementary DNA sequence has revealed that a portion of the coding region, from nucleotide 1250 to 1700, has 54% homology to the human transferrin receptor mRNA. The deduced polypeptide has a putative transmembrane domain enabling the delineation of intra- and extracellular portions of this antigen. In contrast to prostate-specific antigen and prostatic acid phosphatase which are secreted proteins, PSM as an integral membrane protein may prove to be effective as a target for imaging and cytotoxic targeting modalities.

In a pilot study at the authors' institution, 17 patients with stage C carcinoma of the prostate had preoperative therapy with diethylstilbestrol. After 8 to 12 weeks of DES at 3 mg per day, the surgical margins, seminal vesicles, and pelvic lymph nodes were free of tumor in seven patients. In one patient, no residual tumor could be found despite an extensive search, and in two other cases, only small foci of atypical cells could be found. The authors suggest a trial of the proper role of neoadjuvant hormonal therapy in stages B2 and C prostatic carcinoma.

Prostate cancer is the most common type of cancer in men. Despite increased public awareness and new screening methods, a significant proportion of men continue to present with metastatic disease. Most men will respond initially to hormonal intervention; however, given time, the majority will have recurrences of hormonally unresponsive tumor on which present therapies have little impact. Research continues to identify new cellular and molecular aspects of prostate cancer with implications as possible sites of therapeutic intervention.

The prostate is a rich factory of polyamine production. In spite of this, the prostate is also able to accumulate polyamines from the circulation. Intracellular polyamine depletion has been observed to enhance the accumulation of diamines such as putrescine. This needs to be taken advantage of either as a means for imaging prostatic tumour spread to the lymph nodes or as a means of targeting toxins to the prostatic tumour. Putrescine is a small molecule that may prove difficult for use for synthesizing stable toxin derivatives that can take advantage of the targeting potential of the polyamine transporter. The cloning and sequencing characterization of the transporter is under way. When the prostatic transporter is identified, it will be possible to understand the role of the transporter in normal and cancerous prostatic cell growth and functioning and should serve to aid our understanding of how we can better optimize agents to use the transporter for delivery of antitumour agents.

High energy shock waves (HESW) are cytotoxic to tumor cells as determined by vital staining and impaired ability of viable cells to form colonies in a clonogenic assay. In addition, direct exposure of tumor nodules to HESW results in suppression of tumor growth rate. In order to identify histopathologic and ultrastructural correlates of these observations, R3327AT-3 prostatic tumor cells were exposed to HESW in vitro and in vivo. Damage to cells in suspension was manifested by fragmentation of cells to form debris. At the ultrastructural level, mitochondria were swollen and contained distorted cristae following exposure of tumor cells to HESW. In vivo exposure of tumor nodules to HESW did not cause a distinct histopathologic or ultrastructural effect that could be qualitatively distinguished from spontaneously occurring cell death. Hemorrhage and necrosis were observed in muscle and fibroadipose tissue adjacent to tumor. The mechanism of HESW-induced cytotoxicity is not clear from our studies. Evidence of damage of normal tissues exposed in vivo and tumor cells in vitro is reflected in histomorphological changes.

Radiation is increasingly important in the treatment of prostatic carcinoma. The effect and mechanism of action of radiation on prostatic growth was studied using the ventral prostate of the testosterone-treated rat. Prostatic radiation blocked testosterone-stimulated prostatic growth. Electron microscopy showed this inhibition to be associated with specific morphologic alterations. While radiation caused profound inhibition of prostatic DNA synthesis, only modest effects on nuclear uptake of dihydrotestosterone and on prostatic 5alpha-reductase were observed. Based on these animal studies, it is concluded that a major mechanism whereby radiation blocks testosterone-stimulated prostatic growth operates through inhibition of DNA synthesis.

We studied the effect of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, on putrescine uptake by the rat prostate. Using C-14 putrescine, we found a several-fold increase in uptake by both the dorsal and ventral prostates of DFMO-pretreated intact animals, compared with untreated controls. When previously castrated animals were treated with a combination of testosterone and DFMO, the prostatic uptake of exogenously administered C-14 putrescine increased more than tenfold. Under these conditions, DFMO enhanced the uptake by the prostate to a greater extent than by any other tissue studies.

C3H-A-vy and C3H-A-VY-FB mice transferred from our colony to the laboratory of Dr. J. R. Sabine in Australia had a markedly reduced incidence of both hepatomas and mammary tumors. Sabine and co-workers (J Natl Cancer Inst 50: 1237-1242, 1973) attributed the low incidence of tumors in their laboratory compared to the high incidence in this laboratory to the fact that we routinely use red cedar shavings in the bedding, whereas they use sawdust, predominantly Doublas fir. Results of tests in this laboratory showed that the difference in occurrence of tumors could not be attributed directly to either a difference in diet or bedding. There was no evidence that the cedar shavings were carcinogenic. The animals in Australia on bedding without cedar shavings were not as healthy as our animals, in that they did not grow as well and were infested with ectoparasites; apparently these factors caused the reduced occurrence of tumors.

Outcrosses between high tumor mouse strain DD and low-incidence strains BALB/c and C57BL to produce reciprocal F1 susceptible offspring of both types resulted in high incidences of mammary tumors in (DD female X BALB/c male)F1 and (DD female X C57BL male)F1 females but not in the reciprocal hybrids with DD as the male parent. The finding that transmission of murine mammary tumor virus (MuMTV) in DD was through the milk, as most often observed in mouse strains, further supported the uniqueness of strain GR in which MuMTV is transmitted by either parent.

It was previously shown that the rate of disappearance of blood ethanol was identical for two lines of mice selectively bred for differences in sleep-time after ethanol administration. The ED50 values for the loss of righting response with ethanol were significantly different at 3.64 g per kg for the SS line and 1.65 g per kg for the LS line. In the present study the mean sleep time is 367 sec for SS mice and 9342 sec for LS mice. The ED50 values remain essentially the same as previously reported. Unchanged LD50 values for ethanol, however, are not different at 4.8 g per kg for the SS and 4.5 g per kg for the LS line of mice. The ED50 value for loss for righting response following administration of methanol, butanol and t-butanol is approximately 2 fold greater for the SS line of mice than for the LS line. The ED50 values for sodium pentobarbital or ether in the 2 lines of mice for loss of righting response are virtually identical. In addition, the sleep-time values obtained after the administration of pentobarbital, chloral hydrate, trichloroethanol and paraldehyde are not significantly different. These data indicate that while the SS and LS lines of mice differ in central nervous system sensitivity to ethanol, methanol, butanol and t-butanol it is implied that they do no differ in central nervous system sensitivity to other hypnotic agents tested. Proof of this latter suggestion awaits determination of metabolic rates, and brain levels of these other depressants.

Exposure of the Dunning R3327AT-3 rat prostatic carcinoma and SK-Mel-28 human melanoma, in vitro, to high energy shock waves resulted in a reduction in cell viability as determined by trypan blue exclusion and a decrease in the number of colonies formed in a clonogenic assay. Flow cytometric determination of DNA content in R3327AT-3 cells treated in vitro indicated a selective diminution of cells in the G2 and M phases of the cell cycle. When R3327AT-3 cells exposed to high energy shock waves were subsequently injected into rats, or tumor bearing animals were treated by high energy shock waves targeted at the tumor, a delay in tumor growth was observed. These observations indicate that high energy shock waves are cytotoxic to tumor cells in vitro and in vivo. Additional research into the possible use of high energy shock waves in the non-invasive destruction of animal and human tumors is warranted.

The uptake of exogenously administered radiolabeled polyamines by a rat prostate-derived tumor line, the Dunning R3327 MAT-Lu, and various normal tissues was studied. Pretreatment of tumor cells in vitro with alpha-difluoromethylornithine (DFMO), a polyamine synthesis inhibitor, resulted in a markedly enhanced uptake of both [14C]putrescine and [14 C]spermidine. The in vitro uptake of [14C]putrescine by these cells was effectively inhibited by unlabeled spermine, spermidine, 1,8-diaminooctane, 1,7-diaminoheptane, 1,6-diaminohexane, 1,5-diaminopentane, 1,4-diaminopentane, and 1,4-diaminobutane, but less effectively by 1,4-diamino-2,3-butene and 1,4-diamino-2,3-butyne. The diamines, 1,3-diaminopropane and 1,2-diaminoethane, were ineffective in inhibiting [14C]putrescine uptake in vitro into the R3327 MAT-Lu cell line. When tumor-bearing animals were pretreated with DFMO or with DFMO and 5-alpha-dihydrotestosterone propionate, the tumor and prostate uptake of [14C]putrescine and [14C]-cadaverine was enhanced but not substantially increased in other tissues. In contrast to the in vitro results, spermidine and spermine were not enhanced substantially by DFMO pretreatment into any tissue, and their uptake into the tumor actually decreased. Ethylenediamine, which does not utilize the polyamine transport system, did not have its uptake increased into any tissue following DFMO pretreatment. The chemotherapeutic agent, methylglyoxal bis(guanylhydrazone), which utilizes the polyamine transport system for uptake into cells, exhibited uptake behavior different from that of the polyamines. Thus, methylglyoxal bis(guanylhydrazone) uptake into the tumor was not significantly increased or decreased by DFMO or by DFMO + 5-alpha-dihydrotestosterone propionate pretreatment, and only the ventral, but not the dorsal-lateral, lobe of the prostate showed increased uptake of methylglyoxal bis(guanylhydrazone) following DFMO + 5-alpha-dihydrotestosterone propionate pretreatment.

The R3327 MAT-LyLu, a lymphotrophic, metastatic prostate-derived rat tumor was treated with high dose diethylstilbestrol or the chemotherapeutic agent ICRF-159. Both drugs inhibited the growth of the primary tumor and the development of metastases.

The Dunning R-3327-H is a well-differentiated transplantable rat prostatic adenocarcinoma that contains both hormone-sensitive and -insensitive cells. The component composed of hormone-insensitive cells has been permitted to grow in a castrated male, and a new slow-growing, well-differentiated hormone-insensitive subline of the tumor has been established and designated R-3327-HI. In addition, a rapidly growing hormone-insensitive anaplastic tumor has been developed, R-3327-AT. These three tumor lines have been characterized, and their histological and biochemical profiles are compared.

To examine the effectiveness of prostatic acid phosphatase and creatine kinase-BB determinations in detecting prostatic cancer serum from 594 men more than 40 years was assayed for prostatic acid phosphatase with the thymolphthalein monophosphate substrate and a radioimmunoassay kit. Creatine kinase-BB levels also were measured with a radioimmunoassay kit. Patients with benign prostatic hyperplasia had higher prostatic acid phosphatase levels than normal controls. Accordingly, to avoid a high incidence of false positives in patients with benign prostatic hyperplasia the 92.5 percentile level of the patients with benign prostatic hyperplasia (3.9 ng./ml.) was chosen as the upper limit of normal. With this critical value elevated prostatic acid phosphatase levels were observed in 6 per cent of the patients with clinical stage A disease, 8 per cent with stage B, 35 pr cent with stage C and 68 per cent with stage D. The radioimmunoassay was no more effective than the enzymatic assay in detecting prostatic cancer. A correlation between prostatic acid phosphatase levels and patient race was observed, with 80 per cent of the black patients with extracapsular prostatic cancer having elevated prostatic acid phosphatase levels compared to 34 per cent of the white patients with similar stage disease. Creatine kinase-BB was elevated only in patients with advanced disease and was of little value in the diagnosis of prostatic cancer.

We have previously described the inhibitory effects of diethylstilbestrol on the growth of the androgen-independent, anaplastic Copenhagen rat prostatic tumor, R3327AT, which has a low metastatic potential. We have now selected a variant of the R3327AT tumor that metastasizes exclusively to the lungs, and we have designated it the R3327MAT-Lu. In a dose-response protocol, diethylstilbestrol was inhibitory to the primary R3327MAT-Lu tumor, thereby also inhibiting the formation of lung metastases. Inhibition was also observed in this model tumor by the chemotherapeutic agent 1,2-bis(3,5-dioxopiperazin-1-yl)propane.

A primary human renal clear cell carcinoma has been developed as a xenograft (JDF-1). Passage 7 of the JDF-1 tumor retained the microscopic morphology of the primary tumor, electron micrographs have confirmed its epithelial characteristics and karyotyping of a subsequent passage has proved it is not a murine hybrid. Immunoperoxidase studies using a panel of murine monoclonal antibodies demonstrate an antigenic phenotype specific for human renal cancer. In vitro chemosensitivities of the JDF-1 tumor were determined by the double-layer soft-agar clonogenic assay method. JDF-1 showed no significant sensitivities to several standard chemotherapeutic agents, but alpha-2-interferon and difluoromethylornithine in combination synergistically inhibited its growth by 74 per cent.

Coumarin, the parent compound of warfarin, has been observed to stimulate macrophages, increase phagocytosis, and induce changes in lymphocyte-mitogen responsiveness in cancer patients. Coumarin has been reported to have antitumor activity in human melanomas and renal cancer when used in conjunction with the H-2 antagonist, cimetidine. We have observed that coumarin has antiprostatic activity in rats. When coumarin was given to mature rats at a dose of 40 mg/kg, a significant decrease in the size of the prostate, seminal vesicles, and testes was observed. Testosterone levels were unchanged or slightly elevated, consistent with an antiandrogenic-like activity. Similarly, coumarin significantly inhibited the androgen-induced increase in prostatic size when administered to castrated rats receiving testosterone. Coumarin given to rats bearing the R-3327H androgen-sensitive, prostate-derived tumor decreased the size of the primary tumor. The effect was greater than that produced by castration. Coumarin is worthy of further consideration as an agent for use in controlling the normal and abnormal growth of the prostate.

The R3327/MAT-Lu tumor is a metastasizing variant of the Dunning R3327 Copenhagen rat prostatic carcinoma which, when implanted subcutaneously in the flank or intramuscularly in the hind leg, invariably metastasizes to the lungs. We examined treatment with either single dose chemotherapy, surgical excision of the primary tumor, or a combination of tumor excision and postoperative single dose chemotherapy. All 3 regimens significantly reduced the number of lung metastases; however, only the combination of tumor excision, followed by postoperative chemotherapy substantially prolonged survival and produced cures.

Fricke chemical dosimetry is used as an indirect measure of the free radical production of ionizing irradiation. We adapted the Fricke ferrous sulfate radiation dosimeter to examine the chemical effects of high energy shock waves. Significant free radical production was documented. The reaction was dose dependent, predictably increased by acoustic impedance, but curvilinear. A thousand shocks at 18 kilovolts induced the same free radical oxidation as 1100 rad cobalt-60 gamma ionizing irradiation, increasing to 2900 rad in the presence of an air-fluid zone of acoustic impedance. The biological effect of these free radicals was compared to that of cobalt-60 ionizing irradiation by measuring the affect on Chinese hamster cells by clonogenic assay. While cobalt-60 irradiation produced a marked decrease in clonogenic survivors, little effect was noted with high energy shock waves. This suggested that the chemical effects produced by shock waves were either absent or attenuated in the cells, or were inherently less toxic than those of ionizing irradiation.

In in vitro studies with the Copenhagen rat prostate-derived tumor cell lines, the activity of ornithine decarboxylase (ODC) of these lines were as sensitive to inhibition by alpha-difluoromethylornithine (alpha-DFMO) as normal prostatic tissue. The in vitro growth of the tumor in calf serum was inhibited by ODC inhibitors alpha-methylornithine and alpha-difluoromethylornithine. This inhibition was partially reversed by the diamine oxidase inhibitor aminoguanidine. Further, alpha-DFMO was not inhibitory to the growth of these cells in sera which lacked diamine oxidase activity, but was inhibitory when diamine oxidase was added.

We studied the effect of interferon-gamma (IFN-gamma) and mouse L-cell interferon (IFN-beta) on the proliferation of a mouse bladder tumor, MBT-2. A liquid culture clonogenic assay was used, and a linear relationship was obtained between the number of cells plated and the number of colonies formed. When the cells were assayed in the presence of various doses of murine IFN-gamma or IFN-beta, colony formation was inhibited in a dose-dependent manner. Partially purified IFN-gamma was more effective than IFN-beta in inhibiting MBT-2 colony formation in that IFN-beta exhibited a 50% inhibition dose of approximately 1000 units/ml, while the 50% inhibition dose for the partially purified IFN-gamma was approximately 70 units/ml. The 50% inhibition dose for recombinant IFN-gamma was 700 units/ml, suggesting that multiple lymphokines were active in the partially purified preparation. Further studies with partially purified IFN-gamma showed that the inhibitory effect was time dependent with the maximal effect observed after 48 hr of exposure in a 5-day assay. Treatment of partially purified IFN-gamma for 24 hr at pH 2.0 resulted in the abrogation of the antiproliferative effect. Studies in which partially purified IFN-gamma preparations were treated with a monoclonal antibody against IFN-gamma also resulted in abrogation of antiproliferative activity, confirming the nature of the antiproliferative agent to be IFN-gamma. Further studies showed that murine recombinant IFN-gamma also inhibited MBT-2 proliferation in a dose-dependent manner, confirming that IFN-gamma alone mediates antiproliferative activity. Combinations of IFN-beta and recombinant IFN-gamma acted synergistically in the inhibition of MBT-2 proliferation.

The effects of the ornithine decarboxylase suicide substrate, difluoromethylornithine (DFMO), and of cyclophosphamide, individually and in combination, on the growth of the R3327MAT-Lu prostate derived tumor were determined. DFMO decreased the growth rate and resulted in a 75% reduction in DNA content compared to the control group. Cyclophosphamide produced a greater inhibition of growth and resulted in a 96% reduction in DNA content relative to the control. DFMO in combination with cyclophosphamide provided no greater inhibition of tumor growth than that of cyclophosphamide as a single agent. Further, in the schedule used in this experimental protocol the toxicity to the host of the drug combination was additive.

Clinical trials have utilized intermittent diethylstilbestrol diphosphate (DES) therapy in advanced symptomatic prostatic carcinoma to diminish the morbidity of standard endocrine therapy. To determine the effect of intermittent DES administration on the Dunning R3327 rat prostatic adenocarcinoma 60 days following tumor implant, 6 groups were randomly assigned: control (N = 8), castrate (N = 10), high dose DES (N = 8, 1.6 micrograms/ml DES continuously in drinking water), low dose DES (N = 10, 0.4 microgram/ml continuously in drinking water), intermittent high dose DES (N = 10, 1.6 micrograms/ml DES in drinking water for 1 week, then off for 3 weeks), and intermittent low dose DES (N = 10, 0.4 microgram/ml DES for 1 week, then off for 3 weeks). Results indicate that low or high dose DES, and intermittent low or intermittent high dose DES during the week of administration were able to reduce serum testosterone to castrate levels (0.1 ng/ml). After withdrawal of intermittent DES, serum testosterone returned toward control levels (1.0 ng/ml). Initial mean tumor burden between control and treatment groups was not significantly different. All DES exposed rats had a tumor volume at death (range, 15.6-18.3 cm3) smaller than control (mean, 25.4 cm3) or castrate (mean, 40.8 cm3) rats. Despite this significant survival advantage from the time of randomization was achieved only in castrate (median survival, 331 days) or high dose DES (median survival, 359 days) groups compared to control (median survival, 225 days). Similarly, significant prolongation in tumor doubling time was achieved only by rats receiving castration or high dose DES. Intermittent DES administration controls tumor volume but does not provide a survival advantage. In this respect, intermittent DES is inferior to castration.

The transplantable R-3327 prostate adenocarcinoma (Dunning tumor) has been studied in the Copenhagen rat. We present information about androgen-dependent and hormone-independent tumors in regard to metastasis, growth rate, extrapolated cell kinetics, and response to treatment.

The role of antibody-dependent cell-mediated cytotoxicity (ADCC) in cytotoxicity mediated by antibody-armed effector cells was examined. Cytotoxicity mediated by unsensitized human peripheral blood mononuclear cells (MNC) directed against the human bladder cancer cell line, 253J, was significantly enhanced when MNC were preincubated with a rabbit anti-253J serum and then washed free of unbound antibody. Nonadherent, Fc receptor positive MNC were the effector cells and the arming antibody was IgG. Supernatants of 24-hour cultures and armed MNC exhibited antibody activity in ADCC assays, suggesting the ADCC was in part responsible for the arming phenomenon.

Using Northern blot analysis, we have demonstrated that mRNA for transforming growth factor alpha (TGF-alpha) was expressed in five malignant kidney tissue specimens but was not detected in their autologous nonneoplastic homologues. In addition, the expression of epidermal growth factor (EGF) receptor mRNA in these malignant tissues was 2- to 3-fold greater than in nontransformed tissues. In two cases examined using immunohistochemistry, we were able to correlate the increased expression of the mRNA with an increase in protein expression. Since TGF-alpha is known to bind to the EGF receptor, the finding of an increased expression of both TGF-alpha and EGF receptor mRNA in kidney tumor tissue suggests that interaction between TGF-alpha and the EGF receptor may play a role in promoting transformation and/or proliferation of kidney neoplasms, perhaps by an autocrine mechanism.

The clonogenic assay is an in vitro chemosensitivity test performed on tumor specimens and has had limited success in predicting or confirming patient response to chemotherapy. We investigated this assay system, using a 1-hour method, in 37 renal cell carcinoma specimens to determine its clinical usefulness. These specimens also were graded independently by a pathologist with respect to cell type, mitotic figures and degree of differentiation. Only 22 specimens formed at least 50 colonies per 500,000 cells plated and few specimens displayed any chemosensitivities. Marked variations of colony counts among duplicate controls or drug-treated samples demonstrated further the unreliability of the assay. Of several factors responsible for the poor performance of the assay the 2 outstanding problems were losses of clear cells in variable amounts (unique to renal cell carcinoma), and the inability to generate and maintain single cell suspensions. Pathologic correlations confirmed that predominantly clear cell carcinomas did not form colonies as well as granular cell carcinomas. Owing to these problems in its present form the clonogenic assay is not useful clinically and is unsuited particularly for renal cell carcinoma.

The mitochondria of carcinoma cells retain the permeant cationic compound rhodamine 123 longer than the mitochondria of normal epithelial cells. The possibility of exploiting this difference in the chemotherapy of a murine renal adenocarcinoma was investigated. Rhodamine 123 exhibited anticarcinoma activity in mice and this activity was potentiated by 2-deoxyglucose and methylglyoxal bis(guanylhydrazone), a chemotherapeutic agent that is toxic to mitochondria. Prolonged retention of rhodamine 123 by renal tumor cells compared with normal renal epithelial cells was demonstrated by flow cytometry, perhaps explaining its antitumor activity. A combination of both mitochondrial toxins, rhodamine 123 and methylglyoxal bis(guanylhydrazone) produced the longest survival and had the greatest antitumor effect.

We have previously demonstrated that prostate and prostate-derived rodent tumors can be manipulated into increasing their accumulation of radiolabeled putrescine by alpha-difluoromethylornithine (DFMO)-induced depletion of intracellular putrescine and spermidine. As methods which increase intracellular accumulation of cytotoxic agents often increase the chemotherapeutic effectiveness of the agent, we examined whether an alkylating derivative of putrescine would be cytotoxic to tumor cells. We present here our findings on the cytotoxicity of the aziridinyl derivative of putrescine (AZP) against prostatic cancer cells. The apparent Km for putrescine was 2.5 microM with or without DFMO pretreatment and the apparent Ki for AZP was 1 microM with or without DFMO pretreatment. Intracellular polyamine depletion by DFMO pretreatment resulted in a 3.7-fold greater accumulation of AZP compared to non-DFMO-treated cells. The growth inhibitory activity of AZP was increased with prior polyamine depletion by DFMO with the 50% effective dose decreasing from 18 microM to 2.1 microM. Putrescine was able to block the cytotoxic effect of AZP. Putrescine was also able to rescue the AZP-treated PC-3 cells for up to 6 h following a 1-h exposure to AZP. It appears that aziridinylputrescine behaves like putrescine in that it competes with putrescine for uptake into the cell and, like putrescine, has its uptake into the cell increased by prior polyamine depletion. It differs from putrescine in that it expresses cytotoxic activity and inhibits the growth of the human prostate-derived PC-3 cell line. This cytotoxic activity is also increased by prior polyamine depletion. The cytotoxic behavior of AZP is dependent both on the concentration and duration of exposure. Putrescine can rescue the cells from the effect of AZP. AZP is a potentially useful cytotoxic analogue that utilizes the polyamine transport system for its uptake into the cell.

A polypeptide isolated from tissue extracts of renal adenocarcinoma was mitogenic for BALB/c 3T3 cells and human umbilical vein (HUV) cells in culture. It also demonstrated angiogenic ability using the chorioallantoic membrane bioassay. Using heparin-sepharose affinity chromatography the purified protein eluted with a NaCl concentration between 1.4 and 1.8 M and demonstrated a molecular weight of approximately 17,000 daltons based on SDS polyacrylamide gel electrophoresis. Half maximal stimulation of tritiated thymidine incorporation into BALB/c 3T3 cells was achieved by 1.6 ng./ml. of the heparin binding material. Western blot analysis using antibodies specific to basic fibroblast growth factor (bFGF) only or acidic FGF (aFGF) only demonstrated that the purified protein binds to the former and not the latter. The characteristics of this material, in effect the elution profile off heparin-Sepharose, the molecular weight, angiogenic activity and the results of western blot analysis, suggest that this growth factor is similar to the family of basic fibroblast growth factors.

Expressed prostatic secretions and extracts of benign prostatic hyperplasia tissue contain a polypeptide growth factor(s) that stimulates the uptake of tritium-labeled thymidine by cultured 3T3 fibroblasts. Mitogenic activity was present in expressed prostatic secretions and extracts of benign prostatic hyperplasia tissue. The apparent molecular weights of the mitogenic fractions were estimated to be 300,000, 150,000 and 60,000 daltons for prostatic tissue extracts, and 30,000 daltons for expressed prostatic secretions. Bioassays yielded a mean of 27 units of mitogenic activity per mg. protein in expressed prostatic secretions obtained from men with normal and enlarged prostate glands. There was no difference in bioassayable mitogenic activity in the expressed prostatic secretions from normal and benign prostatic hyperplasia samples but gel filtration studies revealed a high molecular weight component present only in samples from men with prostatic enlargement. A dialyzable low molecular weight inhibitor of fibroblast growth was found in the prostatic tissues and expressed prostatic secretions. We report the characterization studies and discuss the possible roles of growth factors in the pathogenesis of benign prostatic hyperplasia.

Mammary tumorigenesis in genetic crosses between the high mammary tumor incidence GR and the low incidence C57BL mouse strains is highly correlated with murine mammary tumor virus expression in milk. Although the F1 and first backcross females had a mammary tumor incidence which was consistent with a single dominant gene segregation, the tumor incidence in the critical second backcross segregants disproved the single gene hypothesis. Genetic factors were clearly involved in regulation of virus expression which in turn correlated with both tumor incidence and tumor latency; these complex phenotypes are however best explained as threshold or quasicontinuous characters. As predicted from this model, the age specific incidence of mammary tumors showed a broad peak at 14-19 mo of age with no evidence of an early or late phase. Hematopoietic tumors showed no correlation with virus expression or mammary tumorigenesis suggesting different etiologies for these tumors.