Carcinogen-specific gene expression profiles in short-term treated Eker and wild-type rats indicative of pathways involved in renal tumorigenesis

Eker rats heterozygous for a dominant germline mutation in the tuberous sclerosis 2 (Tsc2) tumor suppressor gene were used as a model to study renal carcinogenesis. Eker and corresponding wild-type rats were exposed to genotoxic aristolochic acid (AA) or non-genotoxic ochratoxin A (OTA) to elucidate early carcinogen-specific gene expression changes and to test whether Eker rats are more sensitive to carcinogen-induced changes in gene expression. Male Eker and wild-type rats were gavaged daily with AA (10 mg/kg body weight) or OTA (210 microg/kg body weight). After 1, 3, 7, and 14 days of exposure, renal histopathology, tubular cell proliferation, and Affymetrix gene expression profiles from renal cortex/outer medulla were analyzed. AA-treated Eker and wild-type rats were qualitatively comparable in all variables assessed, suggesting a Tsc2-independent mechanism of action. OTA treatment resulted in slightly increased cortical pathology and significantly elevated cell proliferation in both strains, although Eker rats were more sensitive. Deregulated genes involved in the phosphatidylinositol 3-kinase-AKT-Tsc2-mammalian target of rapamycin signaling, among other important genes prominent in tumorigenesis, in conjunction with the enhanced cell proliferation and presence of preneoplastic lesions suggested involvement of Tsc2 in OTA-mediated toxicity and carcinogenicity, especially as deregulation of genes involved in this pathway was more prominent in the Tsc2 mutant Eker rat.     

Promotion by sodium barbital induces early development but does not increase the multiplicity of hereditary renal tumors in Eker rats

Induced cell proliferation is important in the mode of action of many non-genotoxic renal carcinogens. Since Tsc2 mutant (Eker) rats are genetically predisposed to the development of renal cell tumors, they provide a useful animal model in which to study the action of renal carcinogens. Sodium barbital was used as a model non-genotoxic renal carcinogen to test whether a concentration that increased renal tubular proliferation without severe nephrotoxicity would enhance tumor induction in a hereditary tumor model. First, a subchronic concentration-response study was conducted in wild-type male Long-Evans rats to determine increased cell proliferation without severe nephrotoxicity. Rats were dosed with sodium barbital in the feed at 0, 50, 250, 500, 1000, 2000 or 4000 p.p.m. for 3 or 8 weeks. Cell proliferation within the cortex and nephrotoxicity were quantitated. Enhanced proliferation with minimal nephrotoxicity occurred at 500 p.p.m. A second study was conducted in male Tsc2 mutant rats given sodium barbital in the feed at 0, 100 or 500 p.p.m. from 9 weeks of age to either 6 or 12 months of age. An additional group of rats was treated with sodium barbital for 6 months and then provided control feed until 12 months of age. Rats necropsied at 6 months of age had a concentration-dependent increase in preneoplastic and total renal lesions. Sodium barbital-treated rats necropsied at 12 months of age had numbers of lesions that were not different from controls. Total combined preneoplastic and neoplastic lesions in the 6 month, high dose group was the same as the 12 month control group. These data show that sodium barbital caused progression to the stage of spontaneous renal lesions in Tsc2 mutant rats but did not increase their overall number. These data suggest that enhanced cell proliferation without significant cytotoxicity exerted a promotional influence in this hereditary model.     

Niban gene is commonly expressed in the renal tumors: a new candidate marker for renal carcinogenesis

Functional inactivation of tuberous sclerosis 2 gene (Tsc2) leads to renal carcinogenesis in the hereditary renal carcinoma Eker rat models. Recent studies revealed a role of tuberin, a TSC2 product, in suppressing the p70 S6 kinase (p70S6K) activity via inhibition of mammalian target of rapamycin (mTOR). Phosphorylated S6 protein, a substrate of p70S6K, was expressed in the early lesions in Eker rats, and this expression was suppressed by the treatment of rapamycin, an inhibitor of mTOR. We previously isolated the novel gene Niban expressed in renal carcinogenesis of Eker rats. In this study, we demonstrated that the expression of Niban was detected from early preneoplastic lesions in Eker rats. Interestingly, in contrast to the phosphorylated S6 protein, the expression of Niban was unchanged and early lesions still remained even after treatment with rapamycin. These results might suggest the existence of another pathway independent of mTOR-S6K pathway in Tsc2 mutant renal carcinogenesis. In addition, Niban was also expressed in other renal carcinoma models, including Tsc1 and Tsc2 knockout mice, and various types of human renal cell carcinomas. Thus, Niban was commonly expressed in renal carcinomas and might be a new marker for renal carcinogenesis.     

Allelic Loss at the Tuberous Sclerosis (Tsc2) Gene Locus in Spontaneous Uterine Leiomyosarcomas and Pituitary Adenomas in the Eker Rat Model

Hereditary renal carcinomas (RCs) develop in virtually all Eker rats by the age of one year. Investigation of extra-renal primary tumors co-occurring in Eker rats late in life (at 2 years) additionally revealed enhanced development of hemangiosarcomas of the spleen, uterine leiomyosarcomas and pituitary adenomas, although the demonstrated predilection for these extra-renal tumors was not as complete as with RCs. We identified the germline mutated tuberous sclerosis ( Tsc2 ) gene as the predisposing Eker gene and revealed the tumor suppressor nature of Tsc2 gene function in renal carcinogenesis. In the present study, we examined allelic loss at the Tsc2 gene locus in uterine leiomyosarcomas and pituitary adenomas developing in hybrid F1 rats carrying the Eker mutation as well as in pituitary adenomas from non-carrier rats. We detected loss of heterozygosity in 4 of 11 uterine leiomyosarcomas (36%) and 11 of 31 pituitary adenomas (35%) from Eker rats but in none of 9 pituitary adenomas from non-carrier rats ( P <0.05), suggesting that inactivation of the Tsc2 gene is also a critical event in the pathogenesis of these extra-renal tumors. Our present data indicate that there might be different pathways for tumorigenesis of pituitary adenomas between Eker and non-carrier rats.     

Renal carcinogenesis, hepatic hemangiomatosis, and embryonic lethality caused by a germ-line Tsc2 mutation in mice

Germ-line mutations of the human TSC2 tumor suppressor gene cause tuberous sclerosis (TSC), a disease characterized by the development of hamartomas in various organs. In the Eker rat, however, a germ-line Tsc2 mutation gives rise to renal cell carcinomas with a complete penetrance. The molecular mechanism for this phenotypic difference between man and rat is currently unknown, and the physiological function of the TSC2/Tsc2 product (tuberin) is not fully understood. To investigate these unsolved problems, we have generated a Tsc2 mutant mouse. Tsc2 heterozygous mutant (Tsc2+/-) mice developed renal carcinomas with a complete penetrance, as seen in the Eker rat, but not the angiomyolipomas characteristic of human TSC, confirming the existence of a species-specific mechanism of tumorigenesis caused by tuberin deficiency. Unexpectedly, approximately 80% of Tsc2+/- mice also developed hepatic hemangiomas that are not observed in either TSC or the Eker rat. Tsc2 homozygous (Tsc2-/-) mutants died around embryonic day 10.5, indicating an essential function for tuberin in mouse embryonic development. Some Tsc2-/- embryos exhibited an unclosed neural tube and/or thickened myocardium. The latter is associated with increased cell density that may be a reflection of loss of a growth-suppressive function of tuberin. The mouse strain described here should provide a valuable experimental model to analyze the function of tuberin and its association with tumorigenesis.     

Generation of Metastatic Variants of Eker Renal Carcinoma Cell Lines for Experimental Investigation of Renal Cancer Metastasis

We and others have demonstrated that a mutation in Tsc2 is the rate-limiting step for renal carcinogenesis in the Eker rat model. Although inactivation of Tsc2 results in development of renal tumors, it is not sufficient for metastatic renal cell carcinomas (RCs) in the Eker rat. To investigate the additional genetic event(s) necessary for cancer metastasis, we have established highly metastatic S-Lk9d-SLM cell lines from a non-metastatic RC cell line (Lk9dL) by co-implantation with a foreign body (gelatin sponge). Since these cell lines were remarkably different in metastatic performance (all and none, respectively) despite having the same genetic background, they should be useful experimental tools to investigate metastasis-promoting events in renal carcinogenesis.     

p53 status in spontaneous and dimethylnitrosamine—induced renal cell tumors from rats

Rats carrying the Eker tumor–susceptibility mutation (Eker rats) are predisposed to developing renal cell carcinoma. Rats heterozygous for the Eker mutation develop spontaneous multiple bilateral renal cell tumors by the age of 1 yr. In a previous study, Eker-mutation carrier and noncarrier rats were exposed to the renal carcinogen dimethylnitrosamine (DMN), and male rats carrying the Eker mutation exhibited a 70-fold increase in the induction of renal adenomas and carcinomas when compared with noncarrier rats. In this study, spontaneous and DMN-induced rat renal cell tumors (adenomas and carcinomas) were analyzed for mutations of the p53 gene by direct sequencing of cDNA polymerase chain reaction products. There were no mutations in p53 cDNA derived from renal tumors from six untreated rats. Mutations were found in one of 15 of the DMN-induced tumors: a transition at codon 140, CCT → CTT, in a renal adenoma. Additionally, seven cell lines derived from spontaneous renal cell tumors did not contain mutations in p53. The low frequency of p53 mutations (one of 21 renal cell tumors and none of seven cell lines derived from renal cell tumors) indicates that the development of both spontaneous and carcinogen-induced renal tumors involved a non–p53-dependent pathway. As p53 is infrequently mutated in human renal cell carcinomas and in rat renal mesenchymal tumors, it is likely that a tumor suppressor gene or genes other than p53 are involved in the development of renal cancer. © 1995 Wiley-Liss Inc.     

Interstitial chromosomal deletion of the tuberous sclerosis complex 2 locus is a signature for radiation‐associated renal tumors in Eker rats

Ionizing radiation can damage DNA and, therefore, is a risk factor for cancer. Eker rats, which carry a heterozygous germline mutation in the tumor‐suppressor gene tuberous sclerosis complex 2 (Tsc2), are susceptible to radiation‐induced renal carcinogenesis. However, the molecular mechanisms involved in Tsc2 inactivation are unclear. We subjected Fischer 344 × Eker (Long Evans Tsc2^+/?) F1 hybrid rats to gamma‐irradiation (2 Gy) at gestational day 19 (GD19) or postnatal day 5 (PND5) and investigated the patterns of genomic alterations in the Tsc2 allele of renal tumors that developed at 1 year after irradiation (N = 24 tumors for GD19, N = 10 for PND5), in comparison with spontaneously developed tumors (N = 8 tumors). Gamma‐irradiation significantly increased the multiplicity of renal tumors. The frequency of LOH at the chromosome 10q12 region, including the Tsc2 locus, was 38%, 29% and 60% in renal carcinomas developed from the nonirradiated, GD19 and PND5 groups, respectively. Array comparative genomic hybridization analysis revealed that the LOH patterns on chromosome 10 in renal carcinomas were classified into chromosomal missegregation, mitotic recombination and chromosomal deletion types. LOH of the interstitial chromosomal deletion type was observed only in radiation‐associated carcinomas. Sequence analysis for the wild‐type Tsc2 allele in the LOH‐negative carcinomas identified deletions (nonirradiated: 26%; GD19: 21%) and base‐substitution mutations (GD19: 4%). Reduced expression of Tsc2 was also observed in the majority of the LOH‐negative carcinomas. Our results suggest that interstitial chromosomal deletion is a characteristic mutagenic event caused by ionizing radiation, and it may contribute to the assessment of radiation‐induced cancer risk.     

Presence of a modifier gene(s) affecting early renal carcinogenesis in the Tsc2 mutant (Eker) rat model

The Eker (Tsc2 mutant) rat model of renal carcinoma is an example of Mendelian dominantly inherited predisposition to a specific cancer. Effects of genetic background on renal carcinogenesis in the Eker rat model (Eker/Eker > Eker/BN strain) indicate the presence in the BN rat genome of a modifier gene(s) that suppresses tumorigenesis. The identification of such a modifier gene(s) might help clarify the diversity of tuberous sclerosis in humans. i) We found that preneoplastic lesions in 8-week-old F1 rats [(Eker x LE) and (Eker x BN)] were more numerous in the LE strain than in the BN strain although the difference was not large. ii) We next administered N-ethyl-N-nitrosourea (ENU; single injection, i.p.) at the age of 4 weeks to amplify the strain difference in tumorigenesis, as we had done in an earlier study to identify the predisposing gene. iii) This experiment was also done in BN congenic Eker rats to confirm the strain difference in tumorigenesis. Preneoplastic lesions were fewer in BN congenic rats than in Eker rats by a factor of 100. We used this ENU system to perform a backcross experiment [F1(Eker x BN) x Eker] and finally succeeded in mapping a new modifier locus on rat chromosome 5 (the LOD score of the D5Rat12 was 3.13).     

Aberrant expression of HMGA2 in uterine leiomyoma associated with loss of TSC2 tumor suppressor gene function

Unregulated proliferation of mesenchymal cells in leiomyomas, lipomas, hamartomas,and other diseases has been linked to the high mobility group (HMGA) family of DNA architectural proteins. HMGA genes are primarily expressed during embryonal development and silenced in adult tissues but can become reactivated in neoplasia as a result of chromosomal rearrangements. Although the genetic data suggesting a role for HMGA proteins in tumorigenesis are compelling, the biological role of these proteins in mesenchymal proliferation and differentiation is incompletely defined. Uterine myometria and spontaneous leiomyomas from the Eker rat, which carries a germ-line mutation in the tuberous sclerosis complex-2 (Tsc2) tumor suppressor gene, were analyzed for genetic defects in and expression of the Tsc2 and HMGA proteins. Eker leiomyomas exhibited a 50% incidence of loss of the wild-type Tsc2 allele and an almost uniform loss of protein expression, implicating loss of function of the Tsc2 gene in these tumors. Concomitantly, HMGA2 protein, which was completely absent in normal myometria, was expressed in 16 of 19 Eker leiomyomas. HMGA1 was expressed in both leiomyoma and normal myometria. No structural alterations were observed at the HMGA2 locus in either primary rat leiomyomas or leiomyoma-derived cell lines that expressed HMGA2. These data support a role for HMGA2 in the development of smooth muscle neoplasms and suggest HMGA2 expression is a point of convergence between the human disease and the Eker rat model. Furthermore, these data indicate that aberrant HMGA2 expression can result from dysfunction of the Tsc2 tumor suppressor gene, in the absence of structural alterations involving the HMGA2 locus.     

Age dependence of radiation‐induced renal cell carcinomas in an Eker rat model

(Cancer Sci 2010; 101: 616–623) Exposure to carcinogens early in life may contribute to cancer development later in life. The amount of radiation exposure children experience during medical procedures has been increasing, so it is important to evaluate the radiation risk of cancer in developing organs. Toward this goal, we assessed the risk of developing renal cell carcinoma using Eker rats as a kidney tumor model. F1 hybrids of male Eker (Tsc2 mutant) and female F344 rats were irradiated with 0.5 or 2 Gy gamma radiation on gestation days 15 and 19, and on postnatal days 5, 20, and 49. At 27 weeks of age, kidneys were examined for proliferative lesions. Preneoplastic lesions such as phenotypically altered tubules increased after postnatal irradiation as a function of age‐at‐irradiation, and hyperplasia were greatly increased after perinatal and postnatal irradiation. In contrast, development of adenoma and adenocarcinoma were evident in animals irradiated at perinatal ages, being maximal at gestational day 19. The frequency of LOH at the Tsc2 locus was unexpectedly low – 0% (0 of 4) for the unirradiated control, and 17% (6 of 35) for the irradiated group. Irrespective of LOH, the mTOR (mammalian target of rapamycin) pathway, which is negatively regulated by the Tsc1/2 complex, was activated in both benign and malignant lesions, as evidenced by phosphorylation of S6 ribosomal protein and 4E‐BP1. This suggests that the wild‐type Tsc2 allele may be functionally inactivated. In conclusion, actively growing kidneys in perinatal‐aged (F344 × Eker) F1 rats (Tsc2^+/?) are at risk for radiation‐induced malignant transformation of the renal epithelium associated with mTOR activation.     

A Novel Gene "Niban" Upregulated in Renal Carcinogenesis: Cloning by the cDNA-amplified Fragment Length Polymorphism Approach

A modified AFLP (amplified fragment length polymorphism) method was employed to isolate genes differentially expressed in renal carcinogenesis of Tsc2 gene mutant (Eker) rats. One gene, selected for further investigation, was named "Niban" ("second" in Japanese), because it is the second new gene to be found after Erc (expressed in renal carcinoma) in our laboratory. Importantly, "Niban" is well expressed even in small primary rat Eker renal tumors, more than in progressed cell lines, and is also expressed in human renal carcinoma cells, but not in normal human or rat kidneys. Chromosome assignment was to RNO 13 in the rat, and HSA 1. This "Niban" gene is a candidate as a marker for renal tumor, especially early-stage renal carcinogenesis.     

A novel "Nihon" rat model of a Mendelian dominantly inherited renal cell carcinoma.

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Sprague-Dawley strain in Japan, and named the rising "Nihon" rat. In this strain, RCs develop from early preneoplastic lesions, which begin to appear at 4 weeks of age, forming adenomas by the age of 16 weeks. The RCs are predominantly of clear cell type. Southern blot, northern blot and SSCP analyses revealed no change in the Tsc1, Tsc2, VHL, and c-Met genes. Thus, the Nihon rat should be a valuable experimental model for understanding renal carcinogenesis, especially clear cell type, which is common among human RCs.     

A novel gene "Niban" upregulated in renal carcinogenesis: cloning by the cDNA-amplified fragment length polymorphism approach.

A modified AFLP (amplified fragment length polymorphism) method was employed to isolate genes differentially expressed in renal carcinogenesis of Tsc2 gene mutant (Eker) rats. One gene, selected for further investigation, was named "Niban" "second" in Japanese), because it is the second new gene to be found after Erc (expressed in renal carcinoma) in our laboratory. Importantly, "Niban" is well expressed even in small primary rat Eker renal tumors, more than in progressed cell lines, and is also expressed in human renal carcinoma cells, but not in normal human or rat kidneys. Chromosome assignment was to RNO 13 in the rat, and HSA 1. This "Niban" gene is a candidate as a marker for renal tumor, especially early-stage renal carcinogenesis.     

A Novel "Nihon'Rat Model of a Mendelian Dominantly Inherited Renal Cell Carcinoma

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Sprague-Dawley strain in Japan, and named the "Nihon'rat. In this strain, RCs develop from early preneoplastic lesions, which begin to appear at 4 weeks of age, forming adenomas by the age of 16 weeks. The RCs are predominantly of clear cell type. Southern blot, northern blot and SSCP analyses revealed no change in the Tsc1, Tsc2, VHL , and c-Met genes. Thus, the Nihon rat should be a valuable experimental model for understanding renal carcinogenesis, especially clear cell type, which is common among human RCs.     

Increased susceptibility to in vitro transformation of cells carrying the Eker tumor susceptibility mutation

Rats carrying the Eker tumor susceptibility mutation are geneticallypredisposed to renal cell carcinoma. Rats heterozygous for theEker mutation (Eker carriers) develop multiple bilateral renalcell carcinomas by the age of 1 year. Using an in vitro ratkidney epithelial (RKE) transformation assay developed in ourlaboratory, proximal tubule cells derived from known Eker ratcarriers (+/ek) and non-carriers (+/+) were exposed to the carcinogenN-methy-N '-nitro-N-nitrosoguanidine (MNNG), to determine ifcells derived from Eker carriers were more susceptible to invitro transformation than cells derived from noncarrier animals.The percent transformation frequency following MNNG treatmentwas 7.5-fold higher in cells derived from carrier animals whencompared to cells from non-carrier animals. This increased susceptibilityto transformation due to inheritance of the Eker mutation isconsistent with a predisposition resulting from inactivationof a tumor suppressor gene. The increased susceptibility ofkidney epithelial cells carrying the Eker mutation may proveuseful in the further development of the RKE transformationassay as a sensitive tool to identify potential renal carcinogens.In addition, because transformation frequency in the RKE assaymeasures a very early step in multistage transformation, theseresults also suggest that alterations (by Loss of Heterozygosityor mutation) at the Eker tumor susceptibility locus are an earlyevent in the development of renal tumors in the rat.     

Amelioration of sodium barbital-induced nephropathy and regenerative tubular hyperplasia after a single injection of streptozotocin does not abolish the renal tumor promoting effect of barbital sodium in male F344/NC4 rats

The renal tumor promoting activity of barbital sodium (BBNa) and the role of renal tubular cell hyperplasia in tumor promotion following initiation with streptozotocin (STZ) was investigated. Six week old male F344/NCr rats were given STZ as a single i.p. injection of 50 mg/kg body wt and beginning 2 weeks later were fed diets containing 0 or 4000 p.p.m. of BBNa until they were killed at 33, 52 or 72 experimental weeks for histological evaluation and determination of levels of renal DNA synthesis by bromodeoxyuridine (Brdu) immunohistochemistry. A promoting effect on renal carcinogenesis was found by 72 weeks, but not at 33 or 52 weeks, confirming that prolonged administration of BBNa is necessary to promote renal tubular cell neoplasms. The promoting effect was evident as a higher incidence of large renal tubular tumors after 52 weeks, rather than an increase in number of dysplastic tubules, putative preneoplastic lesions. These findings suggest that the targets for the promoting activity of BBNa may be dysplastic lesions which may progress to tumors. Detailed examination by the step section technique through the entire kidneys revealed that STZ or BBNa administration induced a high incidence of putative preneoplastic renal tubular lesions (dysplasias) which seemed to be derived from the P1 or P2 segment of the nephron, also a site of high Brdu labeling index (LI) associated with BBNa toxicity. STZ administration was also associated with attenuation of BBNa-induced nephropathy and with diabetes from pancreatic islet degeneration and atrophy. The reduction in severity of nephropathy was correlated with a reduction in the LI of the renal cortical and medullary tubules, but not with the renal tumor incidence. These results indicate that decreased DNA synthesis in target cells does not eliminate tumor promoting activity in renal tubular epithelium. In addition, BBNa alone induced papillomas of the transitional epithelium of the renal pelvis and renal tubular cell adenomas.     

Inhibitory Effect of Potassium Citrate on Rat Renal Tumors Induced by N-Ethyl-N-hydroxyethylnitrosamine Followed by Potassium Dibasic Phosphate

Potassium dibasic phosphate (PDP) was administered at a concentration of 10% by weight in basal diet to unilaterally nephrectomized Wistar rats previously given 1000 ppm N-ethyl-N-hydroxyethyl-nitrosamine (EHEN) in the diet for 2 weeks. To study the effect of alkalinization on renal mineralization, some animals concomitantly received 5% potassium citrate (PC). Feeding PDP alone promoted adenomatous hyperplasias, which were regarded as preneoplastic lesions, as well as renal cell tumors in EHEN-initiated rats, whereas the addition of PC to PDP diets reduced the promoting effect. Histopathology, serum biochemistry and urinalysis indicated retardation of renal calcium crystallization by PC. Two other phosphate salts, sodium phosphate (SP) and calcium phosphate (CP), were also administered. SP showed a slight promoting effect on adenomatous hyperplasias and a 2-fold increase in the yield of renal cell tumors, while CP induced a clear reduction of both lesions, over EHEN alone. The promoting effects of both PDP and SP and the inhibitory effect of PC were somewhat correlated to 5-bromo-2'-deoxyuridine labeling indices, the degree of nephropathy, and mineralization in the kidney. Immunohistochemically, the nephropathy induced by phosphate salts was not linked to α_zu-globulin. A pathogenesis for renal carcinogenesis is suggested in which nephropathy associated with mineralization enhances the development of renal cell tumors.     

Altered expression of transforming growth factor-α: An early event in renal cell carcinoma development

Transforming growth factor-α (TGF-α) is a multifunctional cell regulatory protein with a wide range of effects on cell growth and differentiation and has been implicated in the neoplastic transformation of a variety of cell types. Altered expression of TGF-α and its cognate receptor (epidermal growth factor receptor) is enhanced in human and rat renal cell carcinomas. The objective of the study reported here was to determine whether altered TGF-α expression is an early or late event in renal tubular oncogenesis. The immunohistochemical expression of TGF-α was studied in preneoplastic renal tubular lesions in a rat model of hereditary renal cell carcinoma. Strong TGF-α immunoreactivity was present at all stages of renal cell tumor development, including the earliest detectable dysplasias. In contrast, the non-neoplastic regenerating tubular epithelium of rat degenerative nephropathy did not stain for TGF-α, although this tissue exhibited a proliferative capacity similar to that observed in the dysplastic and neoplastic lesions. This study indicated that altered TGF-α expression was detectable early in the development of renal cell tumors and may be an important feature of the transformed phenotype. Mol. Carcinog. 19:213–219, 1997. © 1997 Wiley-Liss, Inc.