Suppression of tumorigenicity of rat liver tumor cells by human chromosome 13: Evidence against the involvement of pRb and BRCA2

Abstract

Aberrations of chromosome 13, including large-scale deletions and rearrangements, have been implicated in the development of a significant fraction of human hepatocellular carcinomas, suggesting that liver tumor suppressor genes may be located on this chromosome. In this study, we have employed a microcell hybrid-based model system to investigate the presence of liver tumor suppressor loci on human chromosome 13. The parental GN6TF rat liver epithelial tumor cells are highly tumorigenic in vivo and exhibit altered cellular morphology and growth characteristics in vitro. The GN6TF cells form tumors in 100% of syngeneic animals with short latency, are not contact inhibited or anchorage-dependent in cell culture, and do not express mRNAs for rat Rb1 and BRCA2. Microcell-mediated introduction of human chromosome 13 into the rat liver tumor cell line GN6TF resulted in the generation of clonal microcell hybrid (MCH) cell lines that differentially exhibited tumor suppression and/or alteration of other transformation-associated phenotypes in vitro. Two GN6TF-13neo MCH lines exhibited characteristics indicative of suppression by the human chromosome, including a normalized cellular morphology and growth pattern, loss of anchorage-independent growth potential, partial restoration of contact inhibition, reduction in tumorigenic potential in vivo, and dramatic elongation of tumor latency. In contrast, three GN6TF-13neo MCH cell lines were minimally affected by the introduction of the human chromosome and were nearly indistinguishable from the parental GN6TF tumor cells, exhibiting a highly aggressive tumorigenic phenotype in vivo. Both suppressed and non-suppressed GN6TF-13neo MCH cell lines express Rb1 and BRCA2 mRNA in vitro, and tumors derived from the non-suppressed GN6TF-13neo MCH cell lines continue to express Rb1 and BRCA2 mRNA in vitro, and express pRb in vivo. The results suggest that: i) human chromosome 13 contains a liver tumor suppressor locus, ii) expression of Rb1 and/or BRCA2 is insufficient to produce tumor suppression in this rat liver tumor cell line, and iii) that the human chromosome 13 liver tumor suppressor may represent a novel tumor suppressor gene, distinct from Rb1 and BRCA2.