Prostate intraepithelial neoplasia in Noble rats, a potential intermediate endpoint for chemoprevention studies.

In most prostate chemoprevention studies conducted with animal models, the incidence and multiplicity of tumours have been used as endpoints for efficacy. However, the latency of tumours is usually over 1 year, making these studies costly and time consuming. The main purpose of this study was to assess the utility of prostate intraepithelial neoplasia (PIN), induced in Noble rats by continuous testosterone + oestradiol (T + E) administration, as a potential intermediate endpoint biomarker of efficacy in chemoprevention studies. Noble rats at the age of 12 weeks were treated for 36 weeks with T + E given subcutaneously via Silastic capsules. The incidence and multiplicity of PIN were assessed in various prostate glands by serial sections generated at three separate tissue levels. The efficacy of dehydroepiandrosterone (DHEA) and DHEA 8354 (1000 and 2000 mg/kg diet), difluoromethylornithine (DFMO) (1000 and 2000 mg/kg diet) and oltipraz (125 and 250 mg/kg diet) to inhibit PIN was assessed in two independent sets of experiments. T + E induced multiple PIN in the dorsolateral prostate (DLP) of 80-100% of the animals. DHEA and DHEA 8354 did not affect the incidence but decreased the multiplicity of PIN in the DLP, from 3.2 +/- 1.0 in control group to 1.5 +/- 1.0 in the low-dose and to 1.6 +/- 0.6 in the high-dose group for DHEA (P<0.05 and P<0.02, respectively), and to 1.9 +/- 0.8 in the high-dose (P<0.05) DHEA 8354. Both agents did not affect PIN in anterior prostate, seminal vesicles or ventral prostate. In a second experiment, DFMO and oltipraz were found not effective in inhibiting PIN. In this study, we provide new evidence that PIN in Noble rats, induced by continuous T + E treatment, is a useful intermediate endpoint for determining the efficacy of DHEA and other potential chemopreventive agents. The hormonal pathogenesis, high multiplicity, short latency, preferential location in the DLP, similarity in morphology and biology to PIN of human prostate, and the sensitivity to agents that suppress prostate carcinogenesis, makes PIN in Noble rats a promising intermediate endpoint for chemoprevention studies.

Chemopreventive agents induce a senescence-like phenotype in rat mammary tumours.

Terminal replicative senescence (TRS) is a physiological process associated with terminal differentiation, shortening of the telomere, and lack of proliferative activity. Immortalised and tumour cells have lost their differentiation potential and the ability to develop a senescence phenotype. Recently, others and we [11] have observed that some antitumour agents and radiation induce a senescence-like phenotype (SLP) in human immortalized and tumour cell lines. The main purpose of this study was to identify senescence-like cells (SLC) in mammary tumours of rats and assess whether chemopreventive agents that have been used for the prevention and/or treatment of breast cancer can induce a SLP in tumour cells. Sprague-Dawley rats with N-methyl-N-nitrosourea (MNU)-induced mammary tumours were randomised and treated with tamoxifen, vorozole, 4-(hydroxyphenyl)retinamide (4-HPR), or 9-cis-retinoic acid (9cRA). The SLC in mammary tumours were identified and characterised by: (a) SA-beta-Gal staining method, which has been considered specific for human cells in TRS (b) staining for lipofuscin, which, although not specific, accumulates in the cytoplasm of cells in senescence; (c) lack of 5-Bromodeoxyuridine (BrdU) labelling after continuous (7 days) infusion of BrdU via osmotic pumps; (d) 90 degrees side light scatter (9OLS) as evaluated by flow cytometry; and (e) decreased telomerase activity. We found that in control tumours, SA-beta-Gal-positive cells were rare (below 1.0%) among the tumour cells, stroma fibroblast, myoepithelial and endothelial cells. SA-beta-Gal-positive cells increased significantly in the tumours treated with chemopreventive agents and this was associated with a lack of proliferative activity, increased cell granularity, lipofuscin accumulation, and decreased telomerase activity. Thus, in this study we provide for the first time evidence that cells in replicative senescence are present in mammary tumours of rats and that chemopreventive agents can suppress tumor growth by a novel cellular mechanism, inducing a SLP in the tumor cells.

p27Kip1-deficient mice exhibit accelerated growth hormone-releasing hormone (GHRH)-induced somatotrope proliferation and adenoma formation.

p27Kip1 (p27) controls cell cycle progression by binding to and inhibiting the activity of cyclin dependent kinases. Disruption of the p27 gene in mice (p27-/-) results in increased body growth with a disproportionate enlargement of the spleen, thymus, testis, ovary and pituitary. The increase in pituitary size is due to selective hyperplasia of the intermediate lobe (IL) while the anterior lobe (AL) is not overtly affected. p27 heterozygous mice (p27+/-), as well as p27-/- mice, are hypersensitive to radiation- and chemical-induced tumors compared to wildtype (p27+/+) littermates. Therefore, unlike classical tumor suppressors, only a reduction in p27 levels is necessary to predispose tissues to secondary tumor promoters. Consistent with these studies is the fact that the p27 gene sequence and mRNA levels appear normal in human pituitary adenomas while p27 protein levels are decreased. Therefore, a reduction in p27 levels could be sufficient to sensitize pituitary cells to tumorigenic factors. To test this hypothesis, metallothionein promoter-driven, human growth hormone-releasing hormone (MT-hGHRH) transgenic mice, that exhibit somatotrope hyperplasia before 9 months of age and subsequent adenoma formation with 30 - 40% penetrance, were crossbred with p27+/- mice for two successive generations to produce p27+/+, p27+/- and p27-/- mice that expressed the hGHRH transgene. At 10 - 12 weeks of age, p27-/- and p27+/+, hGHRH mice were larger than their p27+/+ littermates and displayed characteristic hyperplasia of the IL and AL, respectively. Expression of the hGHRH transgene in both p27+/- and p27-/- mice selectively expanded the population of somatotropes within the AL, where pituitaries of p27+/-, hGHRH and p27-/-, hGHRH mice were two- and fivefold larger than p27+/+, hGHRH pituitaries, respectively. There was also a synergistic effect of hGHRH transgene expression and p27-deficiency on liver, spleen and ovarian growth. At 6 - 8 months of age, 83% of p27+/-, hGHRH mice displayed macroscopic AL adenomas (>100 mg), while all pituitaries from p27+/+, hGHRH mice remained hyperplastic (<20 mg). In contrast to the dramatic effects of p27-deficiency on hGHRH-induced organ growth, elimination of p53, by crossbreeding MT-hGHRH mice to p53-deficient mice, did not augment the hyperplastic/tumorigenic effects of hGHRH transgene expression. Taken together these results demonstrate that a reduction in p27 expression is sufficient to sensitize somatotropes to the proliferative actions of excess GHRH, resulting in the earlier appearance and increased penetrance of hGHRH-induced pituitary tumors.

Cell proliferation and apoptosis during mammary carcinogenesis in pituitary isografted mice.

In the present study, pituitary isografted animals serve as a model for evaluating the changes in differentiation, cell proliferation and programmed cell death (apoptosis) in mammary epithelial cells during carcinogenesis. The percentage of bromodeoxyuridine (BrdU)-labeled ductal and alveolar cells was significantly higher in pituitary isografted animals than in non-isografted control animals. BrdU-labeled cells increased in lobular hyperplastic nodules, keratinized nodules and mammary carcinomas; similar changes were observed with apoptotic cells, which were rare in mammary glands of adult non-isografted animals (one to three apoptotic cells per 2000 mammary epithelial cells), but their number increased in hyperplastic lesions and mammary carcinomas. Among hyperplastic nodular lesions, variants with high, moderate and low proliferative activity and/or apoptotic cell death were identified, which suggests that they may have different growth potentials and different propensities for malignant transformation. After removing pituitary isografts, apoptosis occurs in hyperplastic lesions but not in mammary carcinomas-implying that malignant tumors are hormone-independent. The dynamics of the changes in apoptotic cell death among various hyperplastic lesions after removal of pituitary isografts suggests that these lesions are composed of heterogeneous cell populations, as far as the initiation of apoptosis is concerned. Our data indicate that apoptosis can be used together with cell proliferation as a potential marker in characterizing the growth potential and phenotypic diversity of hyperplastic, premalignant and malignant mammary gland lesions.

Ovarian and endometrial endometrioid carcinoma: diagnostic implications of flow cytometry.

Surgical specimens from two patients with simultaneously occurring ovarian and endometrial endometrioid carcinoma were studied by flow cytometry (FCM) and routine histological examination. Tissue derived from the uterine and the ovarian lesion showed similar FCM curves in each case. The information obtained from FCM was more relevant than the study of frozen histological sections as far as confirming or ruling out a common histogenesis was concerned. Study of paraffin sections provided additional data, suggestive of metastatic spread from ovary to uterus in Case 1, and from uterus to ovary in Case 2.