An immunohistochemical technique for localization and semiquantitation of estramustine-binding protein (EMBP) in rat and human prostate.

An immunohistochemical technique for determination of "estramustine-binding protein" (EMBP) in rat prostate is described. The localization and staining intensity of this protein were correlated to prostatic morphological structures in intact animals and at different time intervals after androgen deprivation by castration. EMBP was found almost exclusively in epithelial cells, while the fibromuscular stroma seemed to be negative. Intracellularly, immunostaining was confined to the cytoplasm, but was absent in nuclei. In intact rats, acinar lumina demonstrated heavy immunostaining, indicating secretion of EMBP. Orchiectomy caused a diminution of EMBP expression as well as secretion, suggesting that EMBP synthesis is under androgenic regulation. Human benign hyperplastic and cancerous prostatic specimens were also examined. All human specimens examined so far exhibited positive epithelial staining although of varying intensity. Therefore, this immunohistochemical technique may be used for studying the correlation of EMBP with tumor malignancy grade and for clinical investigations of how various treatments affect EMBP expression in prostatic carcinomas.

Effect of treatment on the expression of estramustine-binding protein (EMBP) in prostatic cancer patients: an immunohistochemical study.

Estramustine-binding protein (EMBP) was determined immunohistochemically in prostatic cancer (PC) specimens taken from patients before and after therapy. The EMBP staining intensity was correlated with the tumor malignancy grade in untreated PCs. The effect of various treatments (i.e., androgen-withdrawal therapy, treatment with estramustine phosphate or radiation) on the expression of EMBP was also investigated. Although a rabbit polyclonal antiserum raised against rat EMBP was used all through the study, all untreated PCs (n = 53) examined so far displayed a positive immunoreaction. The staining intensity was higher in moderately and poorly differentiated than in well-differentiated tumors. Furthermore, two types of staining patterns were observed, that is, a diffuse type in about 70% and a focal in the remaining cases, which might reflect the multifocal appearance of PCs. The prognostic significance of these staining patterns is discussed. Irrespective of the treatment used, EMBP staining was reduced to lower or undetectable levels in PCs where cytological as well as clinical regression were indicated after 6-30 months of therapy. In nonresponders or patients with refractory disease, however, EMBP expression reappeared and returned to pretreatment levels. In a short-term follow-up, the diminuation of EMBP was evident as early as 10 days after androgen-withdrawal therapy and persisted as long as the patient responded to therapy. When estramustine phosphate was given as secondary treatment to hormone refractory PCs, EMBP decreased to undetectable levels in 3/4 of the specimens, suggesting response to therapy. In conclusion, a decreased EMBP staining was well correlated with favorable cytological regression as well as with clinical regression, whereas unchanged staining intensity was indicative of clinical progression and a poor cytological regression grade. The high levels of EMBP in moderately and poorly differentiated tumors as well as in relapsing PCs, despite continued androgen withdrawal, strongly support a regulation of EMBP that is not under androgenic control. Based on the present findings, we suggest the use of EMBP as a therapy marker. Provided that immunohistochemical measurements can be performed on fine-needle aspirates, EMBP analysis may be a direct and early means to distinguish between responding patients and nonresponders.

Regulation of prostatic growth. Growth-limiting mechanisms in the rat ventral prostate.

The possibility that a prostatic chalone is a factor regulating growth of the gland was investigated in rats submitted to hemiprostatectomy to reduce the level of a hypothetical circulating chalone. No evidence of regeneration was found 4 weeks postoperatively. Orchiectomized rats with intrasplenic, intramuscular or intraprostatic implants of ventral prostatic tissue from other orchiectomized rats, were given androgen. The cell divisions in the prostatic tissue did not end until the total cell number was 150-170% of that in control rats. These results indicate that other factors than a prostatic chalone have a dominant role in the mechanism limiting prostatic growth. As introduction of an artificial stroma led to increased prostatic growth, it appears likely that the fibromuscular stroma is one of the factors controlling growth of the prostatic epithelial cells.

Ultrastructure and immunohistochemistry of the lateral prostate in aged rats.

Ultrastructural, histological, and immunohistochemical studies were performed on lateral prostates of 1) aged rats from different strains, 2) rats permitted different levels of sexual activity, and 3) castrated rats. Antibodies against the following proteins were used as immunohistochemical markers: SVS II from seminal vesicle, LP 28 from lateral prostate, acid phosphatase isoenzymes from ventral prostate, transglutaminase from coagulating gland, and a commercial monoclonal antibody against cytokeratin. SVS II is a marker of lateral prostatic secretion, while immunoreactions to LP 28 and acid phosphatase (pI 7.1) were cytoplasmic. In aged animals the amount of intracellular secretion is decreased, and focally metaplastic transformation can be visualized by using immunohistochemical markers. Epithelial ultrastructure varied considerably with experimental conditions. Intensive sexual activity resulted in increased polymorphism and increased number of secretory granules within the glandular cells, while castration was followed by a rapid loss of secretory material. Also, in rats older than 10 months, a reduction in the number of secretion granules was common. The epithelium developed a positive immunoreaction to transglutaminase antibodies that were not observed in juvenile glands. Cells, presumably macrophages, which had an intense immunoreactivity for transglutaminase, were increased in number both within and outside the prostatic acini of aged rats. The possible interaction between secretory SVS II, a substrate of transglutaminase, the release of this enzyme from macrophages or its reflux from coagulating glands, the spontaneous cellular exfoliation that is due to decreased androgen levels, and dietary noxae may be of importance in the development of lateral prostatic nonbacterial inflammation in aged rats.

Treatment of prostatitis in the rat.

A spontaneous, nonacute, age-dependent prostatitis was found in a high incidence in the lateral prostatic lobes of Lewis rats. Such rats were treated with methylprednisolone, indometacin , testosterone, hexyloxyphenylproprionate , polyestradiol phosphate, various antibiotics, or were caged together with female rats. The effect of the different treatment modalities was evaluated microscopically by blind observation of the degree of inflammatory reaction in the lateral prostate. Methylprednisolone and the testosterone ester caused a reduction of the inflammatory reactions and so did caging with female rats. The similarity of the rat prostatitis to the human condition may suggest the possibility of using corticosteroids or androgens for the treatment of patients with nonacute prostatitis.

Chemotherapy principles in the treatment of prostatic cancer.

The efficacy of chemotherapy for prostatic cancer is difficult to evaluate owing to the low incidence of measurable indicator lesions and the resulting need for indirect response criteria. Although complete regressions remain exceptional, a number of agents, eg, doxorubicin and cisplatin have been shown to be effective in the treatment of this disease. So far, combinations of effective agents with or without concomitant hormone therapy have not proven to be more effective than single agents. Androgen priming has considerable theoretical appeal and deserves further consideration. A higher effectiveness of chemotherapeutic agents might be obtained by linkage to various carriers. Estramustine phosphate is an example of such a complex that has a cytotoxic effect in test systems in which estrogen has no effect and in patients with hormone-refractory prostatic cancer. The use of hormonal and other carriers that could increase the specificity of chemotherapeutic agents deserves extensive exploration.

Uptake in vivo of 45Ca in male accessory sex organs of the rat: effect of estramustine phosphate and diethylstilbestrol diphosphate.

The radioactivity following a single i.v. injection of 45Ca into male rats was found to be significantly higher in seminal vesicles, dorsolateral prostate, coagulating glands and ventral prostate than in muscle. The acute effect of a single dose given intraperitoneally of estramustine phosphate (Estracyt) and diethylstilbestrol diphosphate (Honvan) were examined on 45Ca uptake. Generally, the 45Ca concentration in the accessory sex organs with the exception of the dorsolateral prostate increased after administration of estramustine phosphate. Diethylstilbestrol phosphate treatment also significantly increased 45Ca uptake in the ventral prostate when measured on the basis of tissue wet weight but not on the basis of protein. The implications of these findings are discussed in relation to the therapeutic action of Estracyt in prostatic carcinoma.

Antitumour activity, toxicity and disposition of LS 1727, a nitroso-chloroethyl carbamate of 19-nortestosterone, in rats and mice.

LS 1727, a nitroso-chloroethyl carbamate of 19-nortestosterone, given intraperitoneally had a high cytostatic activity against some experimental tumours. In vitro studies showed that the tested tumours differed in their ability to hydrolyze LS 1727. The hydrolytic capacity was related to the sensitivity to treatment with LS 1727. Distribution studies with double-labelled LS 1727 demonstrated that the chloroethyl-part of the molecule was retained in dimethylbenz(a)anthracene-induced mammary tumours in the rat. Our findings suggest that the antitumour activity of LS 1727 is exerted by alkylating metabolites released at hydrolysis of the compound. LS 1727 had no oral antitumour activity probably due to pre-systemic hydrolysis. When given intravenously, hydrolysis of LS 1727 in lungs caused severe pulmonary toxicity already at low doses.

Effect of LS 1727, a nitrosocarbamate of 19-nortestosterone, on the R-3327 rat prostatic adenocarcinoma.

LS 1727, a nitrosocarbamate of 19-nortestosterone did not affect the growth of the androgen-dependent R-3327 rat prostate adenocarcinoma. Such treatment markedly increased the weight of the ventral prostate and reduced body weight. The androgenic character of LS 1727 was demonstrated in an experiment in which LS 1727 was found to reduce the uptake of tritiated dihydrotestosterone in both the ventral prostate and the tumors. The androgenic growth-stimulating action of LS 1727 on the tumors may have counteracted the cytostatic, growth-inhibitory action of the compound.

Androgen and collagen as growth regulators of the rat ventral prostate.

With the aim of studying a possible role of the fibromuscular stroma as growth regulator for the rat ventral prostate the weight, DNA content, and hydroxyproline content were analyzed in the ventral prostate of rats of varying age. Exogenous androgen increased the age-dependent weight and contents only in young and old rats. Orchiectomy reduced prostatic weight, DNA content, and hydroxyproline content although to different extent, and exogenous androgen restored the weight and contents. Exogenous estrogen to intact animals had effects similar to orchiectomy. Estrogen combined with androgen increased DNA content over that found in animals given androgen only, while weight and hydroxyproline content were not changed. It is concluded that the endogenous androgen determines the size of the prostate in young and old animals. Androgen also determines prostatic collagen content. The good correlation between prostatic DNA and hydroxyproline indicates a more or less fixed number of epithelial cells per amount of collagen. A hypothesis suggesting a crucial role for prostatic collagen in the growth-limiting mechanism in the prostate is presented.

Direct and indirect effects of docosanol (IK.2), the active principle in Tadenan, on the rat prostate.

To better understand the mode of action of Tadenan, a drug used in the treatment of benign prostatic hyperplasia, the effect of its active principle docosanol, IK.2, was investigated in rats. IK.2 had no effects on the weight and histologic appearance of the prostate in intact rats but increased the RNA/DNA quotient in the ventral prostate. The plasma concentrations of luteinizing hormone and testosterone were reduced. In orchiectomized animals IK.2 increased the weight of the prostate and the adrenals. In adrenalectomized, orchiectomized animals IK.2 did not increase prostatic weight but on the contrary caused a further weight reduction. IK.2 had a thymolytic effect in intact rats but not in adrenalectomized rats in which the thymus weight was increased. The results indicate that IK.2 increases adrenal steroid secretion. The supposedly higher concentration of adrenal androgens causes a stimulation of the prostate most easily discernible in orchiectomized animals. The further weight reduction of the ventral prostate in orchiectomized, adrenalectomized animals, and the increased thymus weight in adrenalectomized animals after IK.2 administration may suggest that IK.2 has effects other than the stimulatory effect on the adrenals.

A rat prostatic adenocarcinoma as a model for the human disease.

A transplantable, metastasizing prostatic adenocarcinoma (Tumor I) in Lobund Wistar rats was examined for activity and distribution of five hydrolytic enzymes and for ability to accumulate radioactive zinc. The results suggest that the tumor had arisen in the ventral lobe of the prostate and that its growth was not affected by orchiectomy, adrenalectomy, or replacement treatment with exogenous androgen or corticosteroids. The androgen independency of the tumor was further shown by the low uptake of 3H-testosterone, in contrast to the high uptake in the ventral prostate. Tumor growth was retarded by Cytoxan but not by 5-fluorouracil, Estracyt, or streptozotocin, three agents clinically effective in the treatment of some patients with prostatic cancer resistant to endocrine therapy. It is concluded that this tumor in Lobund Wistar rats may be an adequate model for human prostatic cancers resistant to the agents mentioned above.

Chalones and stroma as possible growth-limiting factors in the rat ventral prostate.

In an attempt to determine the mechanism that limits the growth of the rat ventral prostate, atrophied ventral prostates from orchiectomized inbred rats were implanted into the ventral prostate of other inbred rats. Under the influence of endogenous or exogenous androgen the DNA content of the ventral prostates was increased above normal. This indicates that there is no soluble growth inhibitor diffused in the normal prostate in a concentration sufficient to block cell divisions of the epithelial cells. In other experiments pieces of plastic sponge were implanted into the rat ventral prostate. Orchiectomy and subsequent androgen treatment led to an increased cell number in the prostate. Histologic examination revealed an ingrowth of loose connective tissue and also of normal-looking prostatic tissue in the sponge. The results suggest that implantation of pieces of sponge induced increased growth of the fibromuscular stroma and a secondary proliferation of the epithelial cells. A growth control of the prostatic epithelial cells by the fibromuscular stroma is suggested.

Pilot study on the growth inhibition by estramustine phosphate (Estracyt) of rat mammary tumours sensitive and insensitive of oestrogen.

Rat mammary tumours induced by 7,12-dimethylbenz(a)anthracene had a higher concentration of 3H and 14C than muscle after injection of estramustine phosphate labelled with 3H in the oestrogen moiety and 14C in the alkylating moiety. Thin-layer chromatography showed that dephosphorylated estramustine phosphate was present in the tumours but no free oestradiol-17beta. The uptake of the drug in the tumours was parallelled by a dose dependant retardation of tumour growth and a prevention of tumour number increase. Estramustine phosphate also retarded growth of mammary tumours resistant to treatment with oestradiol-17 beta. It is concluded that estramustine phosphate has a greater effect on tumour growth than oestrogen.

Prostatitis in the rat.

A high incidence of spontaneous, non-acute, age-dependent prostatitis was observed in the lateral prostate of Copenhagen rats and Wistar rats. The lumen of infected acini was filled with polymorphonuclear leucocytes, shed epithelial cells and cell residues. Epithelial cells lining such acini showed degenerative changes. Lymphocytes and macrophages were seen in the stroma. A histochemically observed increase in acid phosphatase and beta-glucuronidase activity in affected epithelial cells may indicate an increased lysosomal activity. Some bacteriological cultures of infected lateral prostates were positive for Proteus vulgaris and diphtheroids. It is suggested that this spontaneous rat prostatitis may be a useful model for the study of the pathogenesis and treatment of human non-acute prostatitis.