Growth modulation effects of synthetic steroids on rat prostatic adenocarcinoma and human breast adenocarcinoma cells in vitro.

Treatment of carcinoma of the reproductive organs using both an antiproliferative agent to inhibit cell growth and a cytotoxic agent to kill the neoplastic cells represents an attractive approach to therapy. By using steroids to achieve both effects, it may be possible to avoid some of the dose-limiting side effects of conventional antiproliferative or cytotoxic agents. We screened in vitro 23 selected steroids against four rat prostatic carcinoma cell lines and one human breast carcinoma cell line. Effects of the steroids were measured using a tritiated thymidine uptake assay to assess antiproliferative activity and a colorimetric enzyme-based assay to assess cytotoxicity. Several of the steroids had marked cytostatic and cytotoxic properties rendering them worthy of further evaluation, both separately and in combination.

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.

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.

Oncogene expression in prostate cancer: Dunning R3327 rat dorsal prostatic adenocarcinoma system.

Steady-state levels of myc, fos, p53, sis, and neu mRNAs were measured in eight variants derived from the Dunning R3327 rat prostate adenocarcinoma and compared to levels in normal dorsal prostate. Expression of the myb and erbB oncogenes in the Dunning tumors was below the limits of detection. Myc, p53, and sis mRNA levels in all tumors were at or above control levels. Fos mRNA levels were below control levels in four of five anaplastic tumors and were above control levels in the remaining tumors. A comparison of mRNA levels along the two Dunning lineages revealed that increased expression of these oncogenes did not correlate with tumor progression.

Expression of ras proto-oncogenes in the Dunning R3327 rat prostatic adenocarcinoma system.

Steady-state levels of c-Ha-ras mRNA were measured in eight sublines of the Dunning R3327 rat prostatic adenocarcinoma. As a control, normal dorsal prostate tissue was studied. Increased expression of c-Ha-ras is associated with tumor progression in one lineage of the Dunning R3327 system (H to AT1 to MAT-Lu and MAT-Ly-Lu). Here ras mRNA increases as the tumor advances from androgen dependence and a high degree of differentiation to an anaplastic aneuploid phenotype with high metastatic potential. However, in the other Dunning lineage (H to HI to HI-F to AT3), expression of c-Ha-ras is variable and does not correlate with tumor progression. Immunocytochemistry showed that levels of the c-Ha-ras p21 protein paralleled steady-state mRNA levels in variants. Transfection assays, using NIH/3T3 cells, suggested that the ras loci were not activated in the R3327 tumors. Levels of c-Ki-ras mRNA were also measured in the Dunning tumors; these did not correlate with tumor progression in either lineage. Expression of N-ras mRNA was not detected in the Dunning 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.

Serum hormone levels among patients with prostatic carcinoma or benign prostatic hyperplasia and clinic controls.

This study sought to identify differences in serum hormone levels between prostatic cancer (CaP) patients, benign prostatic hyperplasia (BPH) patients, and clinic controls (CC). Serum testosterone, estradiol, and prolactin values were obtained from 35 CaP, 42 BPH, and 161 CC patients attending a single medical center between January 1984 and April 1985. Relative risk estimates adjusted for age and race were calculated to compare hormone values between each case group and the CC. The distributions of hormone values and the testosterone to estradiol (T/E) ratios were grouped into thirds with the lowest third forming the reference category. The relative risk estimates for BPH in the middle and high thirds of testosterone were greater than unity (1.26 and 2.10, respectively), whereas the relative risk estimates in the middle and high thirds of estradiol were less than unity (0.63 and 0.35, respectively). For the middle and high thirds of the T/E ratio, the relative risk estimates for BPH showed statistically significant three- to fourfold increases. Modest depression of serum testosterone and estradiol was noted for CaP patients compared to CC, although the differences were not statistically significant. This depression was interpreted to be a likely result of the malignant process rather than a cause of it, whereas the development of clinically evident BPH was felt to be a biologically plausible response to an elevated T/E ratio.

Medical, life-style, and occupational risk factors for prostate cancer.

Information on suspected risk factors for prostate cancer was obtained from in-person interviews as part of a case-control study of tissue sex hormone receptors and serum hormone levels. The risk factors examined were medical history (including venereal disease), sexual history, smoking, alcohol consumption, and occupational exposures. Study subjects were 40 prostate cancer patients and 64 benign prostatic hyperplasia controls who were newly diagnosed during 1984-1985 at North Carolina Memorial Hospital in Chapel Hill. Subjects were white and black men aged 50 years and older. Comparisons of cases' and controls' past medical histories did not support a venereal disease hypothesis of prostate cancer etiology. The most prominent finding is an association with farming employment: 75% of cases compared to 38% of controls reported farmwork occupations. Exposures to pesticides and herbicides, while more common among the patients, did not account for the association detected for farming. No relationship was observed with cadmium exposure, the most frequently cited occupational risk factor for prostate cancer.

Androgen receptors detected by autoradiography in prostatic carcinoma and benign prostatic hyperplastic tissue.

Androgen receptor (AR) content in prostatic tissues from patients with either cancer or benign prostatic hyperplasia (BPH) is of interest from at least two standpoints: receptors may be a feature of the pathogenesis of these conditions, and they may be important to the management and prognosis of prostatic cancer patients. For these reasons, a quantitative autoradiographic assay for AR content in prostatic tissues has been developed. Application of autoradiography to rodent tissues yielded results that were highly correlated with those from biochemical assays. Thus, the autoradiographic analyses with human tissues reported in this paper were undertaken. Average AR content in 22 prostatic carcinomas was lower than that in tissues from 14 patients with BPH; the median values of the affinity index, the quantitative estimate of receptor content, were 7.0 and 12.0, respectively. For the cancer tissues, a trend of declining receptor content with advancing stage of disease appeared but was not statistically significant. No association between receptor content and degree of tumor aggressiveness as measured by Gleason score and MD Anderson score was evident. Patient age and race were not related to receptor content in either type of tissue.

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.

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.

Adenylate kinase: an oncodevelopmental marker in an animal model for human prostatic cancer.

Data are presented demonstrating that adenylate kinase (AK; EC 2.7.4.3) is an oncodevelopmental enzyme in the prostate of Copenhagen rats. We selected the Dunning tumor (dorsal rat prostate) as a model system because it most nearly approximates the human pathology. Four sublines of the tumor (R3327-H, R3327-AT, MAT Lu, and MAT LyLu) were studied. The tumor sublines were maintained as solid tumors in syngeneic rats and as monolayers in tissue culture. AK activity appeared in conjunction with malignant transformation of the dorsal prostate. We also determined the normal developmental enzyme pattern: AK was present in prostates of newborns, but was undetectable in prostates of adults. However, AK increased after castration. Therefore, we propose AK as a potential oncofetal tumor marker in prostatic cancer.

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.

Autoradiographic localization of estrogen and androgen target cells in human and rat prostate carcinoma.

The distribution of estrogen target cells within the Dunning R3327-H rat prostate tumor following intravenous injection of tritiated estradiol into rat hosts was compared to the distribution obtained following incubation of a 2 mm. sample of the tumor with tritiated estradiol in organ culture. No difference was observed, indicating that the in vitro method was an effective approach for autoradiographic analysis of tumor biopsy samples. Subsequently, tumor samples were excised from solid tumors of R3327-H and R3327-MAT LyLu tumors growing in Copenhagen rats. These tumor models were chosen as representatives of hormone sensitive (R3327-H) and hormone insensitive (R3327-MAT LyLu) tumors. Normal rat dorsal prostate and human tumor biopsy samples were also studied. Autoradiographic studies were performed in vitro utilizing tritiated estradiol and tritiated dihydrotestosterone to compare the distribution of estrogen and androgen target cells. The present research demonstrated that 1) similar patterns of nuclear uptake of steroids are obtained with in vivo and in vitro autoradiographic techniques, 2) estradiol receptors occur primarily in extra-acinar epitheloid cells in both rat and human prostate carcinomas, 3) these epithelioid cells are not characteristic of the normal rat dorsal prostate, 4) androgen receptors occur in both acinar and stromal epithelioid cells in rat and primarily in acinar epithelial cells in human tumors and 5) in vitro autoradiographic methods can provide insight into differences in sensitivity to steroids which may be of diagnostic importance in the treatment of cancer.

Dimethylsulfoxide-induced changes in a rat prostate adenocarcinoma.

A variety of agents can induce mammalian tumor cell lines to acquire characteristics of the normal cell counterpart. Dimethylsulfoxide (DMSO) has been an effective differentiating agent in many tumor cell lines. In the present study a Dunning rat prostate tumor subline, MAT LyLu, available as an in vitro continuous cell culture was treated with 2.25% DMSO (vol/vol). Treated MAT LyLu cells had a decreased growth rate, saturation density, and clonogenicity, an increased doubling time, and alterations in enzyme activity and tumorigenicity when compared to untreated MAT LyLu cells. The cell viability of treated cells at the saturation density was greater than 90%. MAT LyLu cells treated with DMSO and then removed from DMSO (posttreated) when compared to untreated cells had similar growth rates, doubling times, clonogenicities, enzyme activities, and tumorigenicities. Posttreated MAT LyLu cells had a different growth pattern than untreated MAT LyLu cells. Posttreated cell viability at saturation density was greater than 90%. This investigation demonstrated that a rat prostate adenocarcinoma grown in medium containing 2.25% DMSO acquired characteristics consistent with differentiated prostate cells. Posttreated MAT LyLu cells were similar in many characteristics to untreated cells but were not identical. The alterations noted were not cytotoxic and were not completely reversible. The results of this study correlated with the observations of other investigators who have studied mammalian tumor cell lines exposed to DMSO.

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.

In vitro characterization of MAT LyLu: a Dunning rat prostate adenocarcinoma tumor subline.

Prostate carcinoma has been a therapeutic challenge. The Dunning tumor, a rat prostate adenocarcinoma tumor model, has been used to evaluate prostate carcinoma treatment protocols. The Dunning tumor subline, MAT LyLu , as described in this report, has been established and characterized as an in vitro continuous cell culture. The cell culture has been stable for greater than 60 passages. The in vitro characteristics of the MAT LyLu cell culture, such as growth rate, loss of contact inhibition, clonogenicity, morphology and tumorigenicity, are consistent with the malignant characteristics of the Dunning tumor subline. The MAT LyLu cell culture has enzyme activities which can be used to characterize the cell line. The establishment of MAT LyLu as a continuous cell culture should provide a controlled approach to evaluate the etiology and treatment of prostate carcinoma.

Effect of media and growth promoters on in vitro cultivation of normal murine urinary bladder epithelium.

Growth potential of normal murine epithelium was investigated by means of 3 culture media: RPMI 1640 with fetal calf serum, Dulbecco's with fetal calf serum and McCoy's with horse serum. The effect of 3 growth promoters was tested separately with each of the 3 media. A total of 886 explants were cultured from 29 murine bladders and epithelial outgrowth was obtained in 35.2 per cent. The growth medium 1640 with 20 per cent fetal calf serum and epithelial growth factor produced the most abundant outgrowth of explants. McCoy's medium containing 15 per cent horse serum produced significantly lower outgrowth compared to the medium containing fetal calf serum (p less than 0.001). Epidermal growth factor has a stimulating effect and horse serum has an inhibitory effect on growth of normal murine epithelial cells.