[CANCER RESEARCH 54, 5766-5770, November 15, 19941 Advances in Brief Prostatic Localization of Spontaneous Early Invasive Carcinoma in Lobund-Wistar Rats1 Thomas D. Pugh, Chawnshang Chang, Hiroji Uemura, and Richard Weindruch2 Departnwn:s of Medicine IT. D. P.. R. W.J and Human Oncology (C. C., H. U.]. University of Wisconsin. Madison. Wisconsin 53706 and Geriatric Research, Education and Clinical Center, Wm. S. Middleton Memorial Veterans @ Hospital, Madison, Wisconsin 53705 [T. D. P., R. W.J Abstract Animal models of human prostate cancer are very limited in number but are of obvious importance to develop. Dr. Morris Pollard (M. Pollard, J. NatI.Cancerlust.,51:1235—1241, 1973)hasreported thatLobund Wistar rats develop spontaneous metastatic prostatic cancer when they become old (—‘25% incidence after 25 months). A chemically induced form of the disease has also been described in Lobund-Wistar rats. However, recent reports suggest that most of the chemically induced adenocarcino mas are not prostatic in origin, with most arising in the seminal vesicle, and thereby raise questions about the origin of the spontaneous cancers. We herein report cancer spontaneously arising in the lateral lobes of the prostates in Lobund-Wistar rats. One of 8 rats killed at 16 months of age showed prostatic carcinoma in situ. Two of 39 rats killed at 20 months displayed early invasive adenocarcinomas with no signs of metastases. Because sectioning of the prostates in this study was lhnited to face sections from a single block for each rat, it is highly probable that the true incidence of dysplasias and carcinomas is underestimated by these data. Dysplastic or neoplastic changes were not seen in either the seminal vesicles or other portions of the prostatic complex. The nuclei of adeno carcinoma cells showed less labeling with antibody to the androgen hor mone receptor than did normal cells. These data strongly support the validity of the Pollard model of spontaneous prostate cancer in Lobund Wistar rats. Introduction A 1991 summary report (1) of the “National Cancer Institute Roundtable on Prostate Cancer: Future Research Directions―empha sized the need to develop appropriate animal models of human pros tate cancer. The Lobund-Wistar (L-W)3 rat, studied by Pollard and his colleagues (2, 3), is one of three rat models of spontaneous prostate cancer described to date (reviewed in Refs. 4 and 5). The L-W rat is unique among the rat models and potentially advantageous because the prostate cancer reportedly occurs spontaneously in —25%of old animals and is metastatic. L-W rats also develop a chemically induced form of the disease. However, a recent report (6) stated that most (73%) of the chemically induced adenocarcinomas originate in the seminal vesicle (and not in the prostate). Other reports (7, 8) also show that the chemically induced tumors can arise in nonprostatic urogenital tissues. Together, these fmdings, coupled with the fact that Pollard's experiments (2) on the spontaneous cancer did not utilize timed autopsies to study the early stages of the disease, raise questions about the site of origin of the spontaneous cancer. In the present study, Received 7/26/94; accepted 10/3/94. The costs of publicationof this articlewere defrayedin partby the paymentof page charges.This articlemustthereforebe herebymarkedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicatethis fact. I This research was supported by National Institute on Aging Grant ROl AG 10536. This is Publication 94-20 from the Geriatric Research Education and Clinical Center at the VeteransAdministrationMedicalCenterin Madison,WI. 2 To whom requests for reprints should be addressed, at Department of Medicine, University of Wisconsin, 2245 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706. 3 The abbreviations used are: L-W, Lobund-Wistar; PBS, phosphate-buffered saline; AR, androgen receptor. L-W rats were killed at 3, 16 and 20 months of age and examined for the histopathological status of the prostatic complex. In this report we describe the occurrence of early invasive carcinomas confined to the lateral lobe of prostates from L-W rats. Materials and Methods Experimental Design. Fifteen-month-old male L-W rats were purchased from Hilltop Lab Animals (Scottdale,PA). Otheryoung (1-month-old)male L-W mice were purchasedfrom the same source and used later as young (3-month-old)controls.WhileatHilltopLabAnimalstheratswerehousedtwo percage andgiven free access to Agway RMH3500 ratchow. Upon arrivalin Madison, the rats were singly caged with filter tops at the American Associ ation for Accreditationof LaboratoryAnimal Care-certifiedSharedAging Rodent Facility at the VA Medical Center. At their arrival and monthly thereafter,sentinel ratswere certified free of pathogenicviruses and Myco plasma. The rats continued to receive the Agway RMH 3500 diet ad libitum until reaching 19 months of age. With the exception of the dietary regimens after 19 months of age, the rats were maintained under conventional condi tions: 24 ±2°C, 50 ±10% relative humidity, 12—16 air cycles of air exchange/h, 12-h light/12-h dark light cycle, and free access to acidified water. After the 19th month of age, the rats were divided into four groups and fed one of fourdiets as partof a largerstudyon the effects of changesin calorieand/or fat intake startedin late-middleage on the developmentof prostatecancer. These diets arenot detailedhereinbecauseonly one monthof feedingoccurred by 20 months of age and intergroup differences in prostate histology or cancer were not apparentat this age. Rats were killed at three ages, 3 months (“young,― n = 8), 16 months(“middle age,― n = 8), and 20 months(“late middle age,― n = 39), with an overdose of pentobarbital and the organswere harvested immediately. Organ weights were recorded for the prostatic com plex, the seminalvesicles (includingthe coagulatinggland),testes, andseveral other organs. The weighed samples were flash frozen in liquid nitrogenfor subsequent biochemical analyses. IlistopathologicalEvaluation. A segmentof the prostaticcomplexfrom each rat,comprisinga representativesampleof the dorsolateralobes andthe urethra,and cross-sectionalcuts from the middle portions of both seminal vesicles with the adheringcoagulatinggland (anteriorlobe of the prostate) were fixed with 4% paraformaldehyde in 0.1 Mphosphatebuffer(pH 7.4) for 2—3h, dehydrated in a graded series of ethanol and embedded in paraffin. Remainingportionsof the prostaticcomplexwere frozenin liquidN2 for later molecular analysis. Ten sections from each prostatewere cut at 4—5 @m, deparaffinizedand stainedwith hematoxylinand eosin or used for immuno histochemistry (see below). Immunohistochemlstry. Sections cut at 4-5 p.m were deparaffinized, then pretreated with Tissue Unmasking Fluid (Signet Laboratories, Dedham, MA), heatedin a waterbathat 85°C for 25 min, andthenremovedfromthe bathand cooled for 10 mm. Sections were rinsed in PBS at room temperature and incubatedwithpolyclonalrabbitanti-androgenreceptorovernightat4°C in a humidified chamber. The polyclonal antibody used in this study was raised againstthe purifiedhumanandrogenreceptorpeptide(40%of the N-terminal domain and 25% of the DNA-bindingdomain)—bacterial trpEfusion protein and its specificity were confirmed by sucrose gradient centrifugation and immunoprecipitation assays (9, 10). Sections were rinsed in PBS and a bioti nylatedsecondantibody(goat anti-rabbitIgG)appliedfor 0.5 h. Afterrinsing with PBS, the localization of the antibodies was visualized with avidin-biotin 5766 Research. on September 3, 2021. © 1994 American Association for Cancer cancerres.aacrjournals.org Downloaded from