Detection of metastatic tumors in nude mice: brief communication.

Homozygous nude (nu/nu) mice were inoculated ip with either highly malignant human bladder transitional cell carcinoma or human prostate adenocarcinoma. These animals were subsequently given injections of normal human T-lymphocytes to restore the known T-lymphocyte deficiency present in homozygous nude mice. Metastatic spread of the prostate and bladder carcinomas was evident in mice given human T-lymphocytes. Although tumor growth was observed at the sites of tumor inoculation, no tumor spread was observed in mice not receiving T-lymphocytes.

Time- and concentration-dependent inhibition of the clonogenic growth of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide-induced murine bladder tumor cell lines by cis-diamminedichloroplatinum(II).

The influence of the concentration and time of exposure to cis-diamminedichloroplatinum on the inhibition of the clonogenic growth of three N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide mouse bladder tumor cell lines was evaluated in a tumor colony assay. Drug testing was performed in the murine model, and tumor cells were removed from the animals for in vitro testing. Murine drug testing revealed marked cis-diamminedichloroplatinum sensitivity of all three mouse bladder tumor lines. One-hr incubation in cis-diamminedichloroplatinum was an adequate time of drug exposure to produce in vitro colony survival curves predictive of in vivo sensitivity to the drug. Furthermore, it was found that 6- to greater than 24-hr exposure to the drug was required to produce colony survival curves in the tumor colony assay predictive of tumor sensitivity. High drug concentrations using 1-hr drug incubation or continuous incubation in drug both produced colony survival curves predictive of tumor sensitivity. Both methods, however, would require higher products of the drug concentration multiplied by time curves than could theoretically be clinically achievable in the murine model. Until pharmacokinetic data on cis-diamminedichloroplatinum are available in this murine model, higher drug sensitivity boundaries than are presently being used for other chemotherapeutic agents will have to be utilized when testing these mouse bladder tumor cell lines for their sensitivity to cis-diamminedichloroplatinum in a tumor colony assay.

Oncodevelopmental enzymes of the Dunning rat prostatic adenocarcinoma.

In this paper, data are presented which demonstrate that adenylate kinase and creatine kinase are oncodevelopmental enzymes in the rat prostate. The Dunning tumor (dorsal rat prostate) was used as a model system; four sublines of the tumor (R3327-H, R3327-AT, MAT Lu, and MAT LyLu) were studied. The tumor lines were maintained as solid tumors in syngeneic rats (Copenhagen) and as monolayers in tissue culture. The appearance of adenylate kinase with malignant transformation of the dorsal prostate was demonstrated. The disappearance of the CK-M subunit of creatine kinase and decreasing levels of creatine kinase were demonstrated with increasing anaplasia. The lactate dehydrogenase (LDH) concentration increased with increasing anaplasia, and the LDH isoenzyme pattern shifted to a more glycolytic pattern (LDH-4, LDH-5). The malignant isoenzyme pattern was reversible with the use of a differentiating agent (dimethyl sulfoxide). Prostates from neonatal rats and castrated adult male rats exhibited patterns of creatine kinase and adenylate kinase similar to those of the undifferentiated tumor. The oncofetal isoenzyme pattern of the castrated rat prostate was reversible with physiological levels of exogenous testosterone.

Development and application of basic research techniques in bladder cancer research.

The growth of transitional epithelial cells with different growth media and growth supports was examined. Sephadex G-10, Bio-Gel P-20, Bio-Glas-1000, DEAE-Sephadex A-50, DEAE-cellulose, CM-Sephadex C-50, acid-soluble collagen, and immobilized collagen fibers were used to enhance plating efficiency. Acid-soluble collagen layers optimally increased the plating efficiency of primary cultures of bladder carcinoma. Media alterations with serial combinations of fetal calf, newborn calf, calf, bovine, and bull serum with minimum essential medium, Roswell Park Memorial Institute Tissue Culture Medium 1640, Connaught Medical Research Laboratories Medium 1066, Medium 199, Grand Island Biological, National Cancer Tissue Culture 135, 1415, McCoy's 5A, and National Cancer Institute medium were established. No promotion of cell division was noted with any one of these basic medium formulations.

Heterotransplantation of a human prostatic adenocarcinoma cell line in nude mice.

Nude mice of NIH/Swiss background were utilized for the heterotransplantation of a tissue culture cell line derived from a human prostate adenocarcinoma metastatic to the brain. These cells, which had been grown in vitro for 13 passages, formed solid tumors when injected s.c. into nude mice. The cell line DU 145 has been passaged 60 times in vitro over a period of 18 months. Tumors removed from the mice were serially transplanted to additional mice and reestablished in vitro. Light-microscopic analysis of the tumor grown in nude mice revealed a strong similarity to the patient's metastatic tumor. The ultrastructure of the tumor cells propagated in nude mice was compared to that of the original human tumor cells and to the tissue culture cells, both before and after passage in nude mouse. No major differences were detected. Karyotypic analysis of the tumor cells grown in vitro before mouse passage, grown in nude mouse, and grown in vitro after mouse passage indicated chromosomal identity and consistent marker chromosomes: three large acrocentric chromosomes and metacentric minute chromosomes.

Correlation of apparent molecular weight and antigenicity of viral proteins: an SDS-page separation followed by acrylamide-agarose electrophoresis and immunoprecipitation.

A simple method is described which combines a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SOS-PAGE) in the first demension with a second electrophoresis, at right angles to the first, into an agarose matrix. The proteins, separated by SDS-PAGE, are exposed to appropriate antisera after the second stage electrophoresis and immunoprecipitates form in the agarose corresponding to the relative electrophoretic mobilities of proteins in the first stage SDS-PAGE separation. The method thus provides a simple, reproducible means for correlating antigenicity with apparent molecular weight of proteins. The technique is qualtitative, but requires smaller quantities of antisera than more conventional immunoelectrophoretic methods such as rocket electrophoresis.

Serial carcinoembryonic antigen assays in patients with metastatic carcinoma of prostate being treated with chemotherapy.

Serial carcinoembryonic antigen (CEA) assays were conducted in patients with endocrine-unresponsive prostatic adenocarcinoma who were being treated with multidrug chemotherapy. Changes in CEA correlated with the clinical status of the patient in 70 per cent of the determinations and were more accurate than acid phosphatase in monitoring the response to treatment.

Combined therapeutic effects of an immunomodulator, PSK, and chemotherapy with carboquone on rat bladder carcinoma.

Responses of bladder cancer in ACI rats to combination therapy with an immunomodulator, PSK, and an alkylating agent, carboquone, are reported. PSK is a protein-bound polysaccharide isolated from Basidiomycetes, and carboquone is the alkylating agent 2,5-bis(1-aziridinyl)-3-(2-hydroxy-1-methanoxyethyl)-6-methyl-p- benzoquinone carbonate, molecular weight 3,214. The immunomodulator, PSK, was shown to enhance the effectiveness of the chemotherapeutic agent, carboquone. The therapeutic effect of combination treatment was monitored by measuring growth rates of tumors transplanted SC and by measuring decreases in metastatic spread to lungs in tumor-bearing animals. Effects of PSK on host immunity were monitored by measuring serum levels of immunosuppressive substance.

Autoradiographic localization of tritium-labeled dihydrotestosterone in human vas deferens.

The human vas deferens was examined autoradiographically for the presence and distribution of androgen receptors. Samples of vas deferens from the region proximal to the testis and the region at the internal inguinal ring were incubated in vitro with tritium-labeled dihydrotestosterone ([3H]-DHT). Frozen sections of tissue were mounted on autoradiographic emulsion-coated slides and exposed for up to three weeks to demonstrate cells with nuclear accumulations of radioactive hormone. Quantitation of autoradiograms was performed with a Zeiss Videoplan morphometric analysis system. Cells in all five tissue layers of the vas deferens were able to bind androgen receptors in the nucleus, as evidenced by superimposition of silver grains over the nuclei of cells in external, middle, and internal smooth muscle layers, as well as in epithelial and subepithelial stromal cells.

A preliminary study of urinary transferrin as a marker for prostatic cancer.

Traditional serum markers used in the diagnosis of prostate cancer lack sensitivity and specificity. Prostatic fluid is in direct contact with the prostate epithelium and, thus, has been investigated as a better source for potentially useful markers. Since prostatic fluid contents can enter the urine directly through the urethra, without prerequisite entry into blood, proteins present in significant quantities in prostatic fluid represent candidate markers for entry into the urine, particularly in diseases affecting the prostate epithelium, such as adenocarcinoma. High concentrations of transferrin in prostatic fluid led us to examine urine transferrin levels, using an immunoturbidimetric technique. Urine transferrin was significantly increased in 18 out of 22 patients with prostate cancer in comparison to age-matched controls. Since there was no evidence of increased transferrin excretion, we suggest that prostatic fluid is the source of transferrinuria.

Renal carcinosarcoma. Ultrastructure and transplantation into athymic mice.

An adult renal carcinosarcoma was studied by light and transmission electron microscopy. A portion of the tumor was transplanted and serially passed in athymic mice. The neoplasm consisted in part of typical renal cell carcinoma. The sarcomatous portion showed definite differentiation into bone, cartilage, and skeletal and smooth muscle. Hilar lymph nodes were involved by carcinoma, whereas the renal vein and recurrent tumor in the flank incision consisted of sarcoma. Only the sarcomatous tissue was transplanted into mice, and no carcinoma appeared during serial passages.

Prostate and transitional cell carcinoma: radioimmunoassay of viral tumor-associated antigens.

Partially purified extracts from human urothelial tumors were utilized as competing antigens in competition radioimmunoassays in conjunction with purified RNA viral interspecies proteins and the respective antibodies to these viral proteins in efforts to detect the presence of one of the structural components of type C RNA viruses, the p30 core protein. Some antigen present in extracts of 25% of the bladder and 22% of the prostate tissues assayed demonstrated cross-reactivity with the viral p30 protein used in the radioimmunoassay system. These findings suggest that some human urothelial tissues contain a protein similar to the p30 core protein of the C-type RNA viruses and that this protein might prove useful in clinical surveys of patients with urogenital tumors.

In vitro growth inhibition of Dunning rat prostate tumor by bone marrow factor.

Normal Copenhagen rat bone marrow was assayed for growth inhibition of cultured MAT LyLu rat prostate tumor cells. A marrow-derived factor was identified that had significant growth inhibitory activity in vitro against MAT LyLu as well as against DU-145 human prostate tumor and MBT-2 mouse bladder tumor cells but that was noninhibitory to normal rat fibroblasts. The factor was stable to degradation by acid, heat, freezing, trypsin, and carboxypeptidase B. The factor was nonreactive with Coomassie blue, and the molecular weight was estimated as less than 620 daltons. A similar factor was identified in normal human and normal rat sera. The presence of this factor in bone marrow may explain the absence of osseous metastases in the Dunning rat prostate tumor model.

Conventional chemotherapeutic agents combined with DMSO or DFMO in treatment of rat prostate carcinoma.

Chemotherapy for prostatic carcinoma is usually reserved for those patients who have failed conventional therapy. These patients generally are in poor health and tolerate chemotherapy poorly. If doses of conventional agents could be decreased without altering cytotoxic activity, then conventional chemotherapy could become an attractive treatment modality. Dimethylsulfoxide and difluoromethylornithine have been shown to induce differentiation in some tumor systems. Growth alteration effects of these two agents were investigated individually and in association with conventional chemotherapeutic agents cyclophosphamide, cisplatin, and fluorouracil in an experimental prostatic cancer model. Copenhagen rats had subcutaneous tumors induced by injections of cells cultured in vitro from a subline of the Dunning rat prostatic tumor, MAT LyLu. Treatment with chemotherapeutic agents individually and associated with differentiation agents was initiated when tumors were palpable. Tumor growth rates and rat body weights were monitored in all groups. The differentiation agents used singly were not able to retard significantly tumor growth rates. In higher doses, each conventional agent used singly significantly retarded tumor growth. Used in combination, the differentiation agents induced cytodestructive properties of lower doses of conventional agents, but some combinations also increased host toxicity. These data suggest that differentiation agents may provide additional antineoplastic benefits when administered in combination with selected chemotherapeutic agents in the management of prostatic cancer.

Effect of DMSO and DFMO on rat prostate tumor growth.

An anaplastic, metastatic subline of the Dunning rat tumor was exposed to non-cytodestructive doses of the cellular differentiation agents dimethylsulfoxide and difluoromethylornithine. Copenhagen rats hosting prostate tumors were evaluated by comparing solid tumor growth resulting from injection of treated cells with solid tumor growth of untreated control cells. Results showed significantly slower solid tumor growth after a 15 day in vitro exposure of cells to either agent, after oral treatment of host animals for 20 days with either agent before injection of untreated tumor cells, and after oral treatment of host animals with either agent initiated on the day of untreated tumor cell injection. Treatment of animals with established tumors with either agent also had an inhibitory effect on tumor growth, and the effect was related to the length of treatment. Thus, exposure of these highly malignant rat prostate carcinoma cells to non-cytotoxic doses of either agent induced slower tumor growth rates. Treatment with either agent could have selected for a slower growing population of tumor cells. Since a slowing of cell cycle transit times is an early indicator of cellular differentiation, these results could reflect an increase in the capacity of the malignant cells to differentiate.

Effects of the immunomodulator PSK on growth of human prostate adenocarcinoma in immunodeficient mice.

Tumor growth alterations were studied using an immunomodulator, PSK. Four human prostate tumor lines were grown in two types of immunodeficient mice. Two of the lines were selected because they are able to metastasize to lungs in host animals. Outbred NIH Swiss athymic mice having normal natural killer cells and athymic Beige mice deficient in natural killer cells were used as animal hosts. PSK treatment was given to tumor-bearing hosts to some animals soon after solid tumors were injected and to others after solid tumors were well-established. Low dose cyclophosphamide was given to some animals to decrease host natural killer cells and polyinosinic-polycytidylic acid (poly I:C) was given to other animals to increase natural killer cell activity. Measurement of tumor doubling times, host survival and metastatic capabilities showed that either poly I:C or PSK treatment in NIH Swiss animals soon after tumor cells were injected significantly increased tumor doubling times and host survival and decreased the incidence and number of metastatic lung lesions. Two of the tumor lines incapable of metastasizing in NIH Swiss mice were metastatic in the Beige athymic, natural killer-cell-deficient animals.

Combined therapeutic effects of conventional agents and an immunomodulator, PSK, on rat prostatic adenocarcinoma.

Growth alteration effects of an immunomodulator, PSK, were investigated individually and in association with conventional chemotherapeutic agents cyclophosphamide, cisplatin and fluorouracil in an experimental prostatic cancer model. Copenhagen rats had subcutaneous tumors induced by injections of cells cultured in vitro from a highly metastatic hormonally unresponsive subline of the Dunning rat prostatic tumor, MAT-LyLu. Treatment with conventional agents and the immunomodulator agent individually and in combination began three days after tumor cell inoculation. PSK used alone was not able to significantly influence tumor growth. In appropriate doses, each conventional agent significantly retarded tumor growth. Used in combination, PSK and conventional agents retarded tumor growth locally and decreased metastatic spread of the tumor. Animals receiving combination therapy had increased life spans over those animals receiving single standard chemotherapeutic agents. Immunomodulation with PSK may enhance the antineoplastic effects of chemotherapeutic agents and offer a treatment option for hormone resistant prostatic cancer.

C-type virus particles in human urogenital tumours after heterotransplantation into nude mice.

C-type viruses were formed in heterotransplants of 5/14 human urogenital tumours which had been serially transferred in nude mice of NIH(S) background. Except for one case in which C-type particles were present in the epithelial cells as well as the connective tissue, the viruses were only found within the stroma of the heterotransplanted tumours. Peroxidase labelling with anti-mouse serum demonstrated that the connective tissue supporting the transplanted human tumours was of mouse origin. Competition radioimmunoassays demonstrated that MuLV interspecies viral protein was present in high titre in the transplanted tumour extracts and also in extracts of 2 spontaneous mouse-tumour extracts. These data suggest that endogenous viruses of the nude mice are activated by the graft, and only subsequently infect the human tumour cells and form particles.

Oncornavirus-like protein expression in human prostatic tissue.

A sensitive competition radioimmunoassay using 125 I-labelled p 30 interspecies antigen, antiserum specific to the interspecies antigen of the feline leukaemia virus, and aqueous tissue extracts from prostate was used to examine benign hyperplastic prostates for the presence of protein components able to complete with the interspecies viral antigens. Six of 20 prostatic nodular hyperplastic tissues were competitive in radioimmunoassay with the 125 I-labelled viral antigen for binding sites on the antiviral antibodies. These findings suggest the presence of oncornavirus-like proteins in prostatic nodular hyperplasia. No correlation could be made between the presence of competing protein and histological features of acute or chronic prostatitis and squamous metaplasia.

Detection of oncornavirus antigenic activity in human urothelial tissues.

Our studies indicate that certain urologic tissues demonstrate evidence of proteins which compete with the interspecies antigens of C-type ribonucleic acid viruses for binding sites of the feline and murine oncornavirus antiglobulins. This fact indicates either the association of viral antigens within these tissues or the presence of a protein so similar to the interspecies antigen that it cross reacts. Studies are now underway to localize reacting antigens within these urologic cells, to correlate the immunologic properties with enzymatic properties known to be specific for oncornaviruses and to assay the activity of patient sera with isolated interspecies antigens. Sera from patients with urologic malignancies will be tested to determine whether the sera can effectively absorb reactivity from tissue extracts or compete with reagent rabbit antisera raised against the interspecies component of the viruses. Should either activity be detected in patient sera, clinical screening will be undertaken to determine if this approach is applicable to early detection of urologic malignancies.