[CANCER RESEARCH 59, 4651– 4657, September 15, 1999] Identification of Genetic Loci Controlling Hepatocarcinogenesis on Rat Chromosomes 7 and 10 1 Maria R. De Miglio, Federico Canzian, Rosa M. Pascale, Maria M. Simile, Maria R. Muroni, Diego Calvisi, Giovanni Romeo, and Francesco Feo 2 Department of Biomedical Science, Division of Experimental Pathology and Oncology, University of Sassari, I-07100 Sassari, Italy [M. R. D.M., R. M. P., M. M. S., M. R. M., D. C., F. F.], and Unit of Genetic Cancer Susceptibility, International Agency for Research on Cancer, Lyon F-69372 cedex 08, France [F. C., G. R.] ABSTRACT Neoplastic liver nodules and hepatocellular carcinomas (HCCs) were induced, by “resistant hepatocyte” model, 32 and 70 weeks after initiation with diethylnitrosamine, respectively, in F344 Brown Norway (BN), and (BNxF344)Fl rats. Nodule number/liver (N) did not significantly differ among rat strains, whereas nodule mean volume (V) and nodule volume fraction (VF) were higher in susceptible F344 than in resistant BN and BFF1 strains and were predictive of subsequent development of HCCs. Genomic scanning of 157 backcross BFFlxF344 rats with 190 polymorphic microsatellites, and linkage analysis, revealed two quantitative trait loci (QTL) on chromosomes 7 and 10, which showed significant linkage with VF, and two QTL on chromosomes 4 and 8, which showed suggestive linkage with V and VF. On the basis of phenotypic patterns of homozy- gous and heterozygous backcross progeny and of allelic distribution pat- tern, QTL on chromosomes 10, 8, and 4 were tentatively identified as resistance loci, and QTL on chromosome 7 was identified as susceptibility locus for rat hepatocarcinogenesis. An analysis of interactions allowed us to identify additional putative QTL on chromosomes 5 and 8 and sug- gested an additive effect of loci on chromosomes 10, 8, and 4 for VF and V. These data are the first to identify chromosomal regions containing putative susceptibility/resistance loci for rat hepatocarcinogenesis, which seems to be highly complex in terms of the number of genetic factors involved. INTRODUCTION HCC 3 is the sixth most common cancer in the world (1). Its incidence is high in specific areas (Western Africa and East Asia), where it is associated with environmental exposure to hepatitis B virus, hepatitis C virus, and Aflatoxin B1 (2), and it is rising in low-incidence Western countries, after an increase in the incidence of hepatitis B and C (3). HCCs do not develop in all individuals at risk, which suggests a role for various etiological factors, including the genetic substrate. An association between a L-myc intronic polymor- phism and the risk of HCC has been reported in humans (4). Never- theless, familial clusters of HCCs have been observed very rarely in humans, which suggests a minor role for genetic predisposition or a complex genetic predisposition to HCC. In this latter case, we may hypothesize the existence, in the human population, of several sus- ceptibility and resistance alleles, each contributing to risk (5, 6). The study of murine models of hepatocarcinogenesis has evidenced a very complex interplay of genetic factors. Thus far, seven suscep- tibility loci (Hcs1 to Hcs7) and two resistance loci (Hcr1 and Hcr2) have been identified in crosses between susceptible and resistant mice strains (7–12). Two more susceptibility genes (Hcf1 and Hcf2) abro- gate hepatocarcinogenesis inhibition by ovarian hormones in the mouse (13). Very little is known about inherited predisposition to HCC in rat. A putative suppressor gene (rcc + ), critical for determin- ing the sensitivity of rats to DENA-induced liver carcinogenesis, has been identified in MHC-recombinant rat strains congenic for the MHC and its linked region grc (14). Partial deletion of WD gene, encoding a copper-transporting protein expressed almost exclusively in liver (15), results in hepatitis in LEC rats, 60% of which survive and undergo malignant transformation (16). On the other hand, rat liver preneoplastic and neoplastic lesions, induced in different models, exhibit various commonalties with analogous human liver lesions, with respect to cytological and biochemical changes (17), molecular alterations [including p53 mutation (18) or deletion (19, 20)], c-myc rearrangement and amplification (21, 22), and chromosomal aberra- tions (19, 20, 23, 24). Thus, the evaluation of the mechanisms of genetic susceptibility to hepatocarcinogenesis in rat and a comparative evaluation of these mechanisms in different rodent species could suggest some possible mechanisms for understanding the genetics of human hepatocarcinogenesis. We focused our efforts on the genetic control of rat liver tumors in a hybrid BFF1 rat strain, generated in our laboratory by crossing the phylogenetically distant (25) BN (B), resistant, with the F344 (F), susceptible, rat strain (26). Resistance of BN rats to hepatocarcino- genesis is genetically transmitted as a dominant character to BFF1 hybrids (26). As previously observed in mice (27), this genetic trait influences growth ability and progression of initiated cells to HCC, more than the initiation stage (26). Herein, we performed a linkage analysis in a backcross progeny generated by crossing BFF1 with F344 rat strains. We found significant and suggestive linkage of four unlinked loci, mapped on chromosomes 4, 7, 8, and 10, with the development of neoplastic lesions. MATERIALS AND METHODS Rats. Fisher 344 and BN rats (140 –160 g, at the beginning of the experi- ment) were obtained from Harlan-Nossan (Correzzana, Milan, Italy). BN females were crossed with male F344 rats to obtain BFFl (26), and BFFlxF344 backcross rats. Animals were bred and maintained in our laboratory. They were fed, throughout the study, a standard G.L.P 4RF21 diet (Mucedola s.r.l. Settimo Milanese, Milan, Italy), and tap water ad libitum, and were housed individually in suspended wire-bottomed cages in a room with constant tem- perature (22°C) and humidity (55%) and with a 12-h light (6 a.m.– 6 p.m.) and dark cycle. Phenotyping of Parental Strains and Backcross Rats. Premalignant and malignant lesions were induced in 25 F344, 25 BN, 25 BFF1 rats, and 157 backcross rats by the RH protocol (28), which included initiation with a necrogenic dose of DENA (150 mg/kg) and feeding, after repair, of a 0.02% 2-acetylaminofluorene diet, with a partial hepatectomy at the midpoint of this feeding. Seven to twelve rats of each parental strain were killed 32 weeks after initiation, and all of the other surviving rats were killed at 70 weeks. All of the backcross rats were killed at 32 weeks. The livers were resected and rapidly cut into 2–3-mm slices to identify gross neoplastic nodules and HCCs. Small Received 12/7/98; accepted 7/20/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by grants from Associazione Italiana Ricerca sul Cancro, Ministero dell’Universita ` e Ricerca Scientifica e Tecnologica, and Assessorato Igiene e Sanita ` RAS. M. R. D. M. is a recipient of a fellowship “Liliana Momigliano Sacerdote-Martha Galle” of the Fondazione Italiana per la Ricerca sul Cancro (FIRC). 2 To whom requests for reprints should be addressed, at Department of Biomedical Science, Division of Experimental Pathology and Oncology, University of Sassari, Via P. Manzella, 4, I-07100 Sassari, Italy. Phone: 39 – 079-228307; Fax: 39 – 079-228305; E- mail: feo@ssmain.uniss.it. 3 The abbreviations used are: HCC, hepatocellular carcinoma; BN, Brown Norway; DENA, diethylnitrosamine; GST-P, glutathione S-transferase (placental); LOD, logarithm of the odds; QTL, quantitative trait locus/loci; N, nodule number/liver; RH, resistant hepatocyte, V, nodule mean volume, VF nodule volume fraction. 4651 Research. on February 18, 2016. © 1999 American Association for Cancer cancerres.aacrjournals.org Downloaded from