Genome-wide loss of heterozygosity analysis of chemically induced rat hepatocellular carcinomas reveals elevated frequency of allelic imbalances on chromosomes 1, 6, 8, 11, 15, 17, and 20

Abstract

Neoplastic development is a multistep process that involves the stochastic accumulation of heritable genetic alterations in proto-oncogenes, DNA repair genes, and tumor suppressor genes. Loss of heterozygosity (LOH) analysis has been used successfully to identify the genetic determinants of neoplastic development, including tumor suppressor genes, in several species and organs but not in the rat liver. We report the results of a sensitive genome-wide LOH analysis of rat hepatocellular carcinomas (HCCs). Heterozygous rats (Wistar-Furth x Fisher 344) were subjected to an Initiation-Promotion-Progression (IPP) protocol of hepatocarcinogenesis. Two weeks after initiation (by partial hepatectomy, 10 mg/kg diethylnitrosamine), the rats were placed on a diet containing 0.05% phenobarbital (PB). After 24 wk of PB promotion, the rats received either 100 or 1 50 mg/kg ethylnitrosourea. Hepatocellular tumors were resected after a total of 76wk of PB promotion. LOH analysis was completed on 26 HCCs by using 60 microsatellite markers covering all 20 rat autosomes and chromosome X. While 85% of the HCCs had one or more allelic imbalances, the average HCC had 3.3 allelic imbalances (range 0-9). A conditional hypothesis-testing method called the Hot-Cold model was used to determine the location of statistically significant elevations in the frequency of allelic imbalances. Elevated allelic imbalances were observed on chromosomes 1q, 6, 8, 11, 15, 17, and 20p. Together, these allelic imbalances suggest that the retinoblastoma and insulin-like growth factor genes as well as the resistance to chemical carcinogenesis (rcc) locus may be involved in HCC development in the rat but that LOH of the p53 gene is not. The elevated rate of allelic imbalances on chromosomes 8,11, and 17 may indicate the location of undiscovered tumor suppressor genes important to neoplastic development in rat liver. Microdissection-based LOH analysis of HCC revealed that contamination of non-neoplastic and nonhepatocellular tissue was not masking LOH in the whole-tumor analysis. There were no statistically significant differences in the frequency of allelic imbalances between HCC of any differentiation state (histological grade). To the degree that it does not reflect differences in etiological factors, the absence of allelic imbalances in chromosomal regions containing the p53 and mamose-6-phosphate/insulin-like growth factor II receptor tumor suppressor genes and the generally low frequency of allelic imbalances in these tumors, suggests that LOH and allelic imbalances play a less significant role in the molecular pathogenesis of HCC in rats than humans.

Molecular genetic evidence supporting a novel human hepatocellular carcinoma tumor suppressor locus at 13q12.11

“…They concluded that the expression of RB1 and BRCA2 in vitro and in vivo is insufficient and required at least another TSG on chromosome 13 to suppress tumorigenicity in rat GN6TF liver cells. Finally, the syntenic regions of human 13q12.11 were also reported to be a frequent LOH region in rodent models of liver cancers including c‐Myc ‐induced mouse and multiple chemical‐induced rat HCCs (Teeguarden et al, 2000; Wu et al, 2002). In addition, the human 13q12.11 region is a syntenic region of rat HCC susceptibility Hcs7 or QTL 7 (quantitative trait locus 7) that shows positive linkage to the volume fraction of nonremodeling lesions in CFF2 rats (De Miglio et al, 2004).…”

Frequent Loss of Heterozygosity At The Hcr1 ( Hepatocarcinogenesis Resistance ) Locus on Chromosome 10 in Primary Hepatocellular Carcinomas From Lff1 Rat Strain

“…6 Our results contrast with recent evidence of infrequent LOH on chromosomes 1 and 8, in HCCs induced by the IPP protocol in Wistar-FurthxF344 rats, in which no LOH was seen on chromosomes 7 and 10. 19 Maybe this depends on differences between rat strains, experimental models, and developmental stage of lesions. Interstrain differences in AI were seen in mouse and rat liver carcinogenesis 11,[13][14][15]17 (also a finding of our present work).…”

“…43,46 However, frequent breakage of chromosomes 1, 6, 7, and 12 has been observed in preneoplastic liver and HCCs of c-myc/Tgf␣ double-transgenic mice. 47 No differences in LOH rate, linked to histologic grade, have been found in HCCs induced in WistarFurthxF344 rats by the IPP proto-col. 19 Finally, the importance of the carcinogenic protocol is underlined by the presence of allelic deletions in neoplastic hepatocytes, induced by the IPP protocol (DENA ϩ phenobarbital ϩ ethylnitrosourea), but not in those induced by the IP protocol (DENA ϩ phenobarbital). 20 In conclusion, our data show a high AI rate in rat HCCs, in vivo, at least on chromosomes 7 and 10.…”

Dissection of Signal Transduction Pathways as a Tool for the Development of Targeted Therapies of Hepatocellular Carcinoma

“…Frequent breakages of chromosomes 1, 6, 7, and 12 have been observed in preneoplastic lesions and HCCs from c-Myc/Tgf-double transgenic mice [69]. Allelic imbalances have also been found [70,71] on various chromosomes in HCCs induced in susceptible rats generated by crossing Wistar-Furth or Long-Evans with F344 rats. Interestingly, detailed deletion mapping of chromosome 10, in HCCs of the hybrid (LongEvans x F344)F1 strain led to the localization of a putative suppressor Hcr1 gene [71,72].…”