[CANCER RESEARCH 5l. 4415-4422, August 15. 1991| Hereditary Renal Cell Carcinoma in the Rat Associated with Nonrandom Loss of Chromosomes 5 and 61 Kenji Funaki,2 Jeffrey Everitt, Mitsuo Oshimura, Jerome J. Freed, Alfred G. Knudson, Jr., and Cheryl Walker1 Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709 ¡K.F.,J. £.,C. W.]; Tottori L'niversity, Yonago, Japan ¡M.O./; and Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 fJ. J. F., A. G. K.] ABSTRACT A large body of work exists documenting the induction of RCC in rats by various chemical carcinogens (1), although the A spontaneous form of renal cell carcinoma occurs in rats that arises use of these animals as models for studying the mechanism of as the result of the inheritance of a mutation in a single autosomal gene. Cytogenetic analysis was performed on seven cell lines and four primary RCC and extrapolating this information to humans have been tumor cell preparations derived from this hereditary form of renal cell limited by the fact that spontaneous RCC in rats is very rare carcinoma. Banded karyotypes prepared from these seven lines exhibited (28). However, a line of Long-Evans rats has been developed loss and/or partial deletion of both chromosomes 5 and 6. Translocations that carries a single gene mutation that predisposes to renal involving chromosome 4, resulting in a net loss of genetic material located cell carcinoma (29). The mutation has an autosomal dominant near the centromere (4qll), were observed in three of the cell lines. pattern of inheritance with 100% penetrance (30). Rats that Monosomy, translocation, and breakage of chromosome 5 involving band inherit the mutation develop tumors bilaterally (30), and tumors 5q31 and monosomy and partial deletion of chromosome 6 involving band have been detected in these animals as early as 4 months of 6q22-q24 were independently observed in primary tumor cells from three age.5 Tumors develop spontaneously in animals that are heter of four tumors examined. Monosomy of chromosome 4 was observed in cells from a single tumor. The smallest region of deletion of chromosome ozygous for the mutation, but the mutation is lethal when 6 common to all the cell lines and tumor cells was 6q24, suggesting the homozygous (30), suggesting that at least one normal copy of presence of a tumor suppressor gene at this locus. These results indicate this gene is required for normal growth and development. that loss of genes located on chromosomes 4, 5, and 6, possibly tumor While it is clear that a single gene mutation is responsible suppressor gene(s), may be important for tumor development and/or for the inherited susceptibility to renal cell carcinoma in this progression in rat renal cell carcinoma and is consistent with the hypoth rodent model (29, 30), the location and nature of this gene esis that a gene locus on one of these chromosomes may be the site of defect are unknown. The pattern of inheritance of this suscep the original predisposing mutation. tibility and histological similarity to the human disease are both consistent with the predisposing mutation having occurred in a tumor suppressor gene. As a first step in determining whether INTRODUCTION loss of a tumor suppressor gene function might play a role in Histologically, RCC4 in rats resembles that in humans (1, 2) tumor development in this animal model, we examined cell and in both species is thought to arise from cells of the proximal lines and tumor cells derived from these hereditary rat renal convoluted tubule (1). In humans, RCC occurs in both a spon cell carcinomas for loss or partial deletion of specific taneous and a hereditary form. Both forms of the disease are chromosomes. associated with loss of genetic material on human chromosome 3 (p arm) (3-13), suggesting the presence of a tumor suppressor MATERIALS AND METHODS gene in this region. Loss or partial deletion of chromosome 3 is the most frequent cytogenetic alteration observed in renal Cell Lines. Renal cell carcinomas from individual rats carrying the Eker mutation (29) were explanted into tissue culture for the establish cell carcinoma, with alterations of 3p reported in some studies ment of cell lines (31).' Seven cell lines from ERCs were used in this in >95% of nonpapillary renal cell carcinomas examined (4). analysis: ERC 15; ERC 17; ERC 18; ERC 19; ERC 21; ERC 24; and In at least one hereditary renal cell carcinoma, inheritance of a ERC 37. Cell lines were maintained in medium consisting of 50% balanced translocation involving chromosome 3 has been re Dulbecco's minimal essential medium (high glucose) and 50% Ham's ported (7), and von Hippel-Lindau disease, which predisposes F-12, supplemented with 10% fetal bovine serum and ferrous sulfate to renal cell carcinoma as well as other tumors, shows consistent (1.6 x IO"6 M), sodium selenite (5 x IO"8 M), vasopressin (1 x IO"5 loss of heterozygosity for alíeleson 3p in tumor DNA (14, 15). units/ml), cholesterol (1 x 10~8 M), hydrocortisone (2 x IO"7 M), transferrin (10 ng/ml), triiodothyronine (I x IO"9M), and insulin (25 In addition to chromosome 3, trisomy of chromosome 7 also /¿g/ml)(hereafter referred to as complete DF8 medium). All cell lines occurs frequently in human renal cell carcinoma (16, 17), and were grown at 37°Cin a humidified atmosphere of 5% CO2 and have the presence of an alteration on both chromosomes 3 and 7 may be associated with a more aggressive tumor phenotype been in continuous culture for over 1 year. Isolation of Primary Tumor Cells. Five female rats (A-E) and 1 male (16). The same region of chromosome 3p which is deleted in rat (EKT-1), carrying the Eker mutation, were sacrificed at approxi renal cell carcinoma is also frequently lost in other human mately 16 months of age, and the tumors were removed for cytogenetic tumors such as those arising in the lung (18-25), cervix (26), analysis. Tumor tissue was minced finely and placed in a spinner flask and mesothelioma (27). with 2.5% trypsin in Ca2*,Mg2+-free phosphate-buffered saline and incubated at 37°C.Aliquots were removed from the flasks at 30-min Received 2/15/91: accepted 6/10/91. The costs of publication of this article were defrayed in part by the payment intervals, and the trypsin was neutralized by the addition of serum. of page charges. This article must therefore be hereby marked advertisement in Cells were then pelleted and resuspended in complete DF8 medium accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and plated into a 50:50 mixture of complete DF8 medium and Swiss ' These studies were supported in part by Department of Health and Human 3T3 fibroblast-conditioned media. Primary cultures were obtained from Services Grants CA-06927 and CA-43211. 2 Present address: Tottori University. Yonago. Japan. four of five tumors, isolated from females B, D, and E and male EKT- 3To whom requests for reprints should be addressed, at Chemical Industry 1 (tumor F). Institute of Toxicology. P. O. Box 12137. Research Triangle Park. NC 27709. ' The abbreviations used are: RCC, renal cell carcinoma: ERC, Eker rat renal *J. Everitt. unpublished observations. carcinoma. 6J. Freed and A. Knudson. manuscript in preparation. 4415 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1991 American Association for Cancer Research. RENAL CELL CARCINOMA AND NONRANDOM LOSS OF CHROMOSOMES 5 AND 6 Cytogenetic Analysis. Cultures were exposed to colcemid (0.02 ng/ The chromosome number and modal karyotype for each cell ml) for 1.5-2 h before reaching confluency. Cultured cells were detached by treatment with 0.05% trypsin:EDTA for 5 min at 37°Cand centri- line are presented in Table 1. Four of the seven cell lines were aneuploid with chromosome number distributions ranging be fuged. Cell pellets were resuspended in a hypotonie solution of 0.075 M KC1 for 15 min at 37°Cand then fixed with methanohacetic acid tween In and 4n, and the remaining lines had chromosome numbers in the In or 4n range. Diploid and tetraploid cells (3:1, v/v). After three changes of fresh fixative, cell suspensions were dropped onto glass slides and air dried. Preparations stained with a 2% from the same cell line consistently contained identical chro Giemsa solution were used for chromosome counts. The chromosome mosome rearrangements, indicating that the tetraploid cells number in 45 to 100 metaphases was counted to construct the chro originated from the diploid ones. Modal chromosome numbers mosome number distribution. For chromosome banding analysis, de- were diploid or hypodiploid in five cell lines and hypotetraploid stained preparations were stained by a Q-banding method (32). Banded in two cell lines, suggesting that specific chromosome losses metaphases (10 to 15 from each cell line and 14 to 23 from each tumor) had occurred in the ERC cell lines. Karyotypes of the cells were used for determining karyotypes of the cells. The karyotype that examined revealed that each cell line had some recurrent and was most frequently observed in each cell line was judged to be modal. common chromosome changes in spite of a wide variety of Chromosome rearrangements were classified into common and recur numerical and structural alterations and that in some cases the rent abnormalities. The former is an abnormality observed in >80% of the cells examined from a cell line, and the latter is one found in >20% same chromosome changes occurred in three or more cell lines of cells examined. Primary dermal fibroblasts carrying the Eker muta (Tables 1 and 2).