Intermediate filament expression and the progression of prostatic cancer as studied in the Dunning R-3327 rat prostatic carcinoma system

To evaluate if there is any consistent relationship between the expression of intermediate filament proteins (IFP), particularly keratins, and the degree of malignancy of prostatic cancer cells, a series of nine Dunning rat prostatic cancer sublines that span the entire spectrum of progression of prostatic cancer were studied immunocytochemically by the use of a variety of antibodies specific for keratins, vimentin, or desmin. For the keratin studies, monoclonal antibodies with either a general reactivity to several keratins or highly specific for either luminal or basal epithelial cells of the normal rat prostate were used. By use of an antibody specific for luminal cell keratin 18, the luminal tumor cells of the well-differentiated, slow-growing H and HI-S sublines were positively stained. In most of the sublines with a more advanced state of progression (i.e., the moderately differentiated, moderately fast growing HI-M; the poorly differentiated, faster growing HI-F; and the anaplastic, very fast growing AT-1, AT-2, and MAT-Lu tumors), however, no expression of keratin specific for luminal cells was detected. In addition, several of the most advanced sublines (i.e., AT-1, AT-2, and MAT-Lu) were negative using any of the keratin antibodies. In contrast, several of the other sublines with the most advanced degree of progression (i.e., the anaplastic, very fast growing MAT-LyLu tumor derived from the AT-1 subline; and the anaplastic, very fast growing AT-3 tumor, derived from the HI-F subline), however, were positively stained with the keratin antibody specific for the luminal cells. By use of the keratin antibody specific for the basal cells of the normal rat prostate, the basal tumor cells of the well-differentiated slow-growing H and HI-S tumor were positively stained. This positive staining for basal cell keratin was also found in the HI-M and HI-F tumors, while the AT-1, AT-2, MAT-Lu, MAT-LyLu, and AT-3 were negative with this antibody. Thus, a loss in staining for basal cell keratin was consistently associated with the most advanced state of tumor progression. Vimentin-positive staining was demonstrated either alone or with keratin-positive staining in part of the epithelial cancer cells of all the sublines. An increase in the positive staining for vimentin was consistently associated with a more advanced state of tumor progression. Desmin-positive staining was found only in smooth cells present within the various tumor sublines.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of adrenalectomy,flutamide, and leuprolide on the growth of the dunning rat R-3327 prostatic carcinoma

The R-3327 prostatic tumor implanted in the male Copenhagen x Fischer F₁ rat continues to grow because androgen is being supplied from an endogenous source. It follows that regimens which decrease the availability of androgen will retard the growth rate of the tumor. These experiments showed that castration, the antiandrogen flutamide, and the luteinizing hormone-releasing hormone (LHRH) agonist leuprolide inhibited tumor growth. Adrenalectomy alone had no significant effect on tumor size and did not further retard the growth of the tumor in castrated rats. Further, under the conditions of this study, there was no significant difference in tumor growth rates between the groups of rats treated with either flutamide alone or flutamide combined with leuprolide. Total ablation of androgen may not be needed for maximal inhibition of tumor growth.     

Androgen stimulated chemotherapy in the dunning R-3327 prostatic adenocarcinoma

Summary This study was undertaken to determine whether hormonal stimulation followed by chemotherapy with a cell-cycle specific agent would improve the effectiveness of the chemotherapy in a prostatic adenocarcinoma model.One hundred Copenhagen rats were randomised into 5 equal groups and injected subcutaneously with 2×10⁷ cells of Dunning G strain prostatic adenocarcinoma. The groups were treated in the following fashion: 1. sham operated controls, 2. castration, 3. castration and methotrexate, 4. castration, testosterone and methotrexate and 5. castration and testosterone. When the tumours became palpable, all animals received the surgery to which they were randomised. Subsequent hormonal and chemotherapy was started 1 week thereafter. Therapy was given for 5 consecutive days followed by a 16-day recovery period and then continued in a cyclical fashion. Serial measurements of animal weights and tumour size were obtained. Analysis of tumour growth was restricted to the first 29 days of therapy because of a rapid decline in animal survival beyond that point.The group treated with castration, testosterone, and methotrexate inhibited tumour growth more than any other group and was the only group that was significantly different from control (P<0.05).Dr. Grossman is a Ferdinand C. Valentine Fellow of the Section of Urology, New York Academy of Medicine     

Comparison of the R-3327H rat prostatic adenocarcinoma to human benign prostatic hyperplasia and metastatic carcinoma of the prostate with regard to steroid hormone receptors

Quantitative analyses of cytosolic steroid hormone receptors were performed on nine tumors from the transplantable rat prostatic adenocarcinoma R-3327H. Androgen receptors and estrogen receptors were found in eight of nine and five of six tumors, respectively. None of the tumours analyzed contained detectable progestin or glucocorticoid receptors (four and seven tumors, respectively). The apparent equilibrium dissociation constants for the androgen and estrogen receptors were 0.7-4.3 nM and 0.6-1.8 nM, respectively. The apparent equilibrium Bmax values (maximum number of binding sites) were 1,500-25,000 fmoles/gm tissue for the androgen receptor and 640 to 5,800 fmoles/gm tissue for the estrogen receptor. A comparison between the receptor contents of the R-3327H rat tumor and human benign prostatic hyperplasia and metastatic carcinoma of the prostate showed that the rat tumor was different from the human tissues in several respects. Hence, the search for an optimal animal model for prostatic carcinoma in man must be continued.     

Effect of early exposure of flutamide on subsequent growth of transplantable rat prostatic tumor (dunning R-3327)

Rat transplantable prostatic tumors (Dunning R-3327) were treated with flutamide before tumors grew palpable, in order to examine the effect of short term treatment of antiandrogen for prostatic cancer in latent period on the growth after appearance of tumor. Flutamide delayed an appearance of the tumor nodule and retarded the growth rate in proportion as treatment began earlier. Flutamide also reduced final tumor volume. Flutamide-treated tumors histologically consisted of small or dilated glandular structure with an increase in stromal area, but androgen receptors were preserved. Flutamide-treated tumor showed slow growth with androgen sensitivity when transplanted to intact rats, showing prolonged influences of antiandrogen on tumor growth. There was no significant difference between flutamide-treated and control groups in weight of accessory sex organs and serum androgen or estrogen levels. In conclusion, flutamide treatment may retard an appearance of prostatic cancer concomitant in benign prostatic hyperplasia. © 1994 Wiley-Liss, Inc.     

Proliferation of Dunning R-3327 rat prostatic adenocarcinoma and of its sublines (CUA and CUB)

Proliferating cells in tumors of the androgen-dependent R-3327 and its partially dependent and autonomous sublines, CUA and CUB, were examined. Two types of cells, myoepithelial cells and large stromal cells, in the R-3327 have a high proliferating ability, as estimated by thymidine uptake. These cells were also observed in the normal dorsolateral prostate of rats, the lobes from which R-3327 originated. CUA is a squamous carcinoma, which proliferates at the border of the epithelial layer and large stromal cells. The tumor cells of CUB appear as clusters of large stromal cells which have a strong potential for dividing. Therefore, R-3327 contains two types of dividing cells, and their uncoordinated proliferation may be responsible for causing the different features seen in the sublines.     

Differential effects of prolactin on rat dorsolateral prostate and R3327 prostatic tumor sublines

Many investigators have reported effects of the pituitary hormone, prolactin, on the physiology and biochemistry of the rat prostate gland, particularly the lateral or dorsolateral lobe. The Dunning R3327H is a transplantable rat prostatic adenocarcinoma derived from a spontaneous tumor of the Copenhagen rat dorsolateral prostate. This study describes and compares morphological and physiological effects of prolactin on rat dorsolateral prostate and two sublines of the Dunning tumor. Ectopic pituitary grafts were used to induce chronic hyperprolactinemia in castrated rats receiving androgen supplement to provide a relatively controlled hormonal environment in which the effects of prolactin were maximally and consistently observed. Gravimetric and biochemical analyses, as well as ultrastructural study, provided evidence of prolactin's stimulatory effect on dorsolateral prostate growth and secretory activity. Hyperprolactinemia stimulated the growth of the well-differentiated, androgen-dependent R3327/3219 tumor subline with an increase in weight, volume and the total content of DNA, protein and zinc. There were no changes in tumor morphology. In contrast, the anaplastic androgen-independent R3327/150 tumor subline did not respond to graft-induced hyperprolactinemia. This differential response of the two R3327 tumor sublines attests to the complexity of prolactin's effects on prostatic tissue and to the extent of the deterioration of endocrine control that often accompanies tumor progression. Prolactin binding in the R3327 sublines was studied using immunohistochemical staining and radioligand assay, but produced complex results which raise questions about the discrepancy between hormone binding and biological action of prolactin in prostatic tissues.     

Expression of endogenous receptors for neoglycoproteins in Dunning R3327 rat prostatic carcinoma

An important animal model for human prostatic adenocarcinoma is the Dunning R3327 rat carcinoma. In the present study this tumor was further characterized by analyzing the expression of endogenous sugar-binding proteins using glycohistochemistry and immunocytochemistry as well as affinity chromatography and gel electrophoresis. Our glycohistochemical and glycobiochemical results provide evidence for the presence of specific receptors for various carbohydrate moieties. Remarkably, basal cells of the Dunning tumor contain an endogenous lectin with specificity for beta-galactosides that is not found in basal cells of the normal rat prostate. This finding was corroborated using polyclonal antibodies against an immunologically related beta-galactoside-specific lectin from bovine heart. Basal cells of prostatic carcinoma may therefore behave different from normal basal cells. This difference could have a significant impact on the development of prostatic cancer.     

Direct effects of oestradiol on growth and morphology of the dunning R3327H prostatic carcinoma

Summary Male Copenhagen x Fischer F₁ rats were implanted with Dunning R3327H prostatic carcinoma, castrated when the tumours became palpable and were then treated with testosterone, testosterone in combination with oestradiol or oestradiol alone for four weeks. Treatment with oestradiol produced the smallest tumours. The testosteronestimulated growth of tumours was inhibited by oestradiol. The adenocarcinoma was moderately to well-differentiated. Morphometric analysis of the composition of the tumours showed that oestradiol stimulated tumour stroma and inhibited glandular epithelium. These effects were produced concomitantly with decreased overall tumour growth. Testosterone stimulated all cell types of the tumour.     

Inhibitory effects of the nucleoside analogue gemcitabine on prostatic carcinoma cells

Gemcitabine (2≺,2≺difluoro-2≺deoxycytidine, dFdC) is a synthetic antimetabolite of the cellular pyrimidine nucleotide metabolism. In a first series of in vitro experiments, the drug showed a strong effect on the proliferation and colony formation of the human androgen-sensitive tumor cell line LNCaP and the androgen-insensitive cell lines PC-3 and DU-145. Maximal inhibition occurred at a dFdC concentration as low as 30 nM. In contrast to the cell lines which were derived from metastatic lesions of prostate cancer patients, no inhibitory effects were found in normal primary prostatic epithelial cells at concentrations up to 100 nM. The effect of gemcitabine was reversed by co-administration of 10–100 μM of its natural analogue deoxycytidine. In view of a future clinical application of this anti-tumor drug in advanced prostatic carcinoma, we have compared the effect of gemcitabine on prostatic tumor cells with that on bone marrow granulopoietic-macrophagic progenitor cells, because neutropenia is a common side effect of gemcitabine treatment. The time course of action on the two kinds of cells was markedly different. Colony formation of tumor cells was inhibited by two thirds at a gemcitabine concentration of about 3.5 nM. The same effect on granulopoietic-macrophagic progenitor cells required a concentration of 9 nM. Co-administration of deoxycytidine to gemcitabine-treated tumor cell cultures completely antagonized the effect of gemcitabine whereas addition of deoxycytidine after 48 hr of gemcitabine treatment could not prevent gemcitabine action on the tumor cells. In contrast, more than half of the granulopoietic-macrophagic progenitor cells could still be rescued by deoxycytidine administration after 48 hr. These findings and the marked difference in the susceptibility of neoplastic and normal prostatic cells suggest that gemcitabine is a promising substance which should be further evaluated as to its efficacy in the treatment of advanced prostatic carcinoma. © 1996 Wiley-Liss, Inc.     

Estramustine potentiates the effects of irradiation on the dunning (R3327) rat prostatic adenocarcinoma

The present study was designed to determine if estramustine phosphate (EMP) could potentiate the effects of irradiation on the Dunning (R3327) prostatic adenocarcinoma in rats. Two groups of male Copenhagen × Fisher F1 rats carrying bilateral tumors in the flank were used. Irradiation was given with a linear accelerator 6 MV, in a dose of 6 Gy/day for 4 days to the tumor on one side while the tumor on the other side served as control. EMP (360 μg/24 hours) was administered with osmotic pumps to one group of rats for 2 weeks, starting 1 week before irradiation. Tumor growth was calculated by measuring tumor volume, and tumor blood flow was measured 8 weeks after treatment. Irradiation alone effectively delayed tumor growth and EMP enhanced these effects. Tumor blood flow was stimulated by EMP treatment irrespective of radiotherapy. Volume density of tumor epithelium was effectively decreased by irradiation but no significant effects could be seen after EMP. In conclusion, the present study shows that EMP potentiates irradiation on rat prostatic adenocarcinoma, and further evaluation of this therapeutic approach in the clinical treatment of prostatic carcinoma is thus justified. © 1994 Wiley-Liss, Inc.     

Androgen receptor binding characteristics in the cytosol of the rat dorsolateral prostate gland and the dunning R-3327 prostatic adenocarcinoma

The incidence of prostatic cancer is highly correlated with advanced age, and it has been suggested that changes in androgen binding may be important in age-associated alterations in growth regulatory mechanisms of prostatic epithelial cells. In this study the effects of age on androgen binding characteristics in the dorsolateral prostate glands of young and aged Copenhagen rats were determined and the binding properties in the Dunning R3327/130 subline of rat prostatic adenocarcinoma were characterized. Tritium-labeled and nonlabeled methyltrienolone analogs (R1881) were used to study the binding properties of 5α-dihydrotestosterone receptor in the cytosol of tumors and prostate glands. Binding of R1881 was low but specific for the androgen receptor as shown by competition studies in which nonlabeled R1881, 5α-dihydrostestosterone, and testosterone competed successfully with ³H-R1881 for binding sites, but 17β-estradiol and low levels of progesterone did not. In Copenhagen dorsolateral prostate, Scatchard analysis suggested a single class of binding sites. In young animals (three to five months) the average binding capacity was 10.36 fmol/mg cytosol protein with a dissociation constant (K_d) of 2.28 nmol/L. The dorsolateral prostate of aged rats (11–16 months) showed no significant difference in specific binding characteristics as compared to the younger age group. Specific binding of ³H-R1881 in R3327/130 tumor was saturable with a single class of high-affinity binding sites having an average binding capacity of 64.77 fmol/mg cytosol protein and a K_d of 2.76 nmol/L. These data show that the tumor had approximately 6.5 times the number of binding sites as did the normal Copenhagen rat dorsolateral prostate gland. However, no age-related changes were detected through 11–16 months of age in the androgen binding characteristics of normal rat dorsolateral prostate gland that could be correlated with the higher concentration of androgen binding sites in the R3327/130 tumor subline.     

Growth-stimulating effect of adrenal androgens on the R3327 Dunning prostatic carcinoma

Summary Adrenal androgens are discussed as a reason for tumor progression after androgen ablation therapy. Because of the difference in the secretion of androgens by the adrenals of humans and rats, there is no reliable tumor model to study the role of adrenal androgens in tumor progression. Therefore, the main adrenal androgens were administered to rats in order to mimic human endocrine conditions. Application of dehydroepiandrosteron-sulfate (DHEA-S) alone or a mixture of androstendione (A), 11-hydroxyandrostendione (OHA), dehydroepiandrosterone (DHEA), and its sulfate (DHEA-S) to castrated rats caused only a slight increase of prostate and seminal vesicle weights. Contrary to these findings, growth of the R3327 prostatic carcinoma in castrated rats was greatly stimulated by these adrenal androgens up to the level of the intact control. Thus, in spite of androgen ablation, tumor progression could be induced by exogenous adrenal androgens.     

Growth hormone receptor expression in the Dunning R 3327 prostatic carcinoma of the rat.

The Dunning R3327 rat carcinoma is an important model for human prostate adenocarcinoma. In the present study this tumor was further characterized by immunohistochemical demonstration of receptors for growth hormone (GH-R). Weak GH-R immunoreactivity was present in the secretory epithelial cells of the tumor acini. Large epithelial cells which were localized at the periphery of the acini and large cells in the stroma, which are probably derived from the epithelium ("Large neoplastic epithelial cells"), displayed a strong staining with one of the monoclonal antibodies (Mab 263) to GH-R. The presence of GH-R receptors in proliferating prostatic tumor cells supports the concept that GH reacts directly on prostate target tissue to facilitate tumor cell growth.     

Investigation of different combinations of estrogen therapy and radiation therapy on prostatic adenocarcinoma (R-3327)

The relative effectiveness of different combinations of estrogen therapy and radiation therapy against the R-3327 prostatic adenocarcinoma of the Copenhagen rat was studied. Because of similar actions of estrogens and radiation in the cell cycle, and possibly antagonistic effects reported in the clinical literature, we looked for an antagonism between these two therapeutic modalities. Radiation therapy consistently showed a greater tumor inhibitory effect than estrogen therapy alone at the dose tested. Combinations of radiation therapy with hormonal manipulation did not appear to show a greater inhibition of tumor growth than radiation therapy alone. There also did not appear to be an antagonistic effect between these two modalities in this system.     

Effects of cyclophosphamide alone and scheduled methotrexate-5-fluorouracil combination chemotherapy on transplantable R-3327 prostatic adenocarcinoma in F1 hybrid male rat

Male F1 hybrid rats bearing the R-3327 transplantable adenocarcinoma demonstrating similar growth patterns within the original sample of animals were carefully separated into control and treatment groups for each growth pattern. This assured treatment of tumors with similar cell kinetics within each group. In one group, cyclophosphamide (100 mg/kg) was injected intraperitoneally once every four weeks for eight weeks (total of two doses). In the second group, methotrexate (7.5 mg/kg) followed in ninety minutes by 5-fluorouracil (50 mg/kg) were injected intraperitoneally once each week for eight weeks (total of eight doses). These drug dosages did not depress the white cell count of the animals, and they tolerated the medicines well. Excellent suppression of tumor growth was obtained in each group with the cyclophosphamide treatment group being significant at the 0.01 level and the methotrexate-5-fluorouracil treatment group being significant at the 0.05 level.     

Prediction of metastatic potential of aspirated cells from the Dunning R-3327 prostatic adenocarcinoma model.

Pathological grading systems have failed to assess accurately the prognosis of patients with prostatic carcinoma. A visual grading system of cancer cell motility has described successfully the metastatic potential of sublines of the Dunning R-3327 rat prostatic adenocarcinoma model maintained in vitro and in vivo. Clinical application of this technique would be facilitated if it could be performed upon aspirated cells. We compared the motility of cells obtained by biopsy and fine needle aspiration from in vivo Dunning tumors and determined if the motility of aspirated cells could predict metastatic potential. Specimens from the same tumor were obtained by fine needle aspiration and incisional biopsy from three sublines of low (less than 10%) and three sublines of high (greater than 90%) metastatic potential. Membrane ruffling, pseudopodal extension and cellular translation of 100 cells were graded using time-lapse videomicroscopy. Cells obtained by biopsy and aspiration had similar average (analysis of variance, p greater than 0.7) and variation (coefficient of variation, aspirated = 18.6%, biopsy = 17.7%) of motility. Aspirated cells from three low metastatic sublines (membrane ruffling 5.20 +/- SEM 0.41, pseudopodal extension 4.10 +/- 0.57 and cellular translation 3.07 +/- 0.45) were distinguished from cells of three high metastatic sublines (membrane ruffling 7.10 +/- 0.15, pseudopodal extension 6.93 +/- 0.22 and cellular translation 5.47 +/- 0.25). Cellular translation, the best discriminator, correctly classified the metastatic potential of the subline of origin in 82% of 60 individual cells. A grading system based upon the motility of aspirated cancer cells should be studied in human prostatic carcinoma.     

Effect of castration, DES, flutamide, and the 5 alpha-reductase inhibitor, MK-906, on the growth of the Dunning rat prostatic carcinoma, R-3327.

Male rats bearing implants of the Dunning rat prostatic carcinoma, R-3327, were used in a 42-day study to determine the effect of castration or orally administered flutamide (FL), DES (diethylstilbestrol) or the 5 alpha-reductase inhibitor, MK-906, on the growth of this androgen-responsive cancer. The rate of growth and final weights of the tumor and the ventral prostate (VP) were all reduced (P less than 0.05) by castration. Flutamide (25 mg/kg/day) significantly decreased tumor and VP weights in intact rats and castrates given 100 micrograms/day (SC) of testosterone propionate (TP) or dihydrotestosterone propionate (DHTP). It also significantly retarded tumor growth rate in TP- or DHTP-treated castrates and was marginally effective in intact animals. DES (100 micrograms/kg/day) reduced (P less than 0.05) tumor and VP weights of intact rats but did not significantly affect tumor growth rate or weight in castrates given TP or DHTP. These results indicated that the effect of DES on tumor growth is caused by its inhibition of the secretion or release of the gonadotropins necessary for testicular androgen production. MK-906 (25 mg/kg/day) affected neither the gross nor the histomorphology of the tumor in intact rats or castrates given TP or DHTP. Further, it caused no histological changes in the testes of intact rats. It did, however, significantly reduce VP weight in intact animals and TP-treated castrates but not in those given DHTP. This illustrates that the anti-androgenicity of MK-906 stems from its inhibition of DHT formation. The failure of MK-906 to influence tumor growth in the TP-treated castrates strongly suggests that the R-3327 tumor can respond to testosterone directly. If that is true, then its growth is unlikely to be affected by a pure 5 alpha-reductase inhibitor such as MK-906. In ancillary experiments, tumors from MK-906-treated animals were found to have reduced levels of DHT and, when assayed in vitro, to have a reduced capacity to convert [3H]-T to [3H]-DHT.