Identification of candidate liver tumor suppressor genes from human 11p11.2 by transcription mapping of microcell hybrid cell lines.

Our previous studies utilized a microcell hybrid (MCH) cell line-based functional model of tumor suppression to localize a liver tumor suppressor to human chromosome 11, map the suppressor locus to a <1-Mb region within human 11p11.2, and identify a number of expressed sequence tags (ESTs) and genes that represent candidate liver tumor suppressor genes. The Human Genome Project has recently positioned a number of additional genes, ESTs, and predicted genes within the human 11p11.2 liver tumor suppressor region. In this study, we analyzed 26 ESTs and genes (known and predicted) that have been localized to human 11p11.2. Four of these ESTs/genes (FLJ23598, FLJ10450, KIAA1580, SYT13) mapped to the minimal tumor suppressor region of human 11p11.2, the smallest region conferring suppression of tumorigenicity in the MCH cell lines. Each of these ESTs/genes were expressed among an index panel of suppressed MCH cell lines (derived from GN6TF rat liver tumor cells), suggesting that these ESTs/genes represent excellent candidates for the human 11p11.2 liver tumor suppressor gene. To verify the candidate status of these sequences, 8 additional MCH cell lines (derived from GN3TG and GP10TA rat liver tumor cells) were analyzed. Three ESTs/genes (FLJ23598, FLJ10450, KIAA1580) proved to be less than ideal candidates, based upon their loss from suppressed MCH cell lines (DNA deletion), and/or their retention and expression in a non-suppressed MCH cell line. In contrast, SYT13 is present in the DNA from all suppressed MCH cell lines (n=10), and is deleted in a non-suppressed MCH cell line. Furthermore, SYT13 mRNA is expressed in 100% of suppressed cell lines, and is not expressed in the non-suppressed MCH cell line or in MCH-derived tumor cell lines (n=6). These results suggest that SYT13 is an excellent candidate for the human 11p11.2 liver tumor suppressor gene based upon its: i) location within the human 11p11.2 liver tumor suppressor region; ii) loss from the DNA of a non-suppressed MCH cell line that lacks the human 11p11.2 liver tumor suppressor region; iii) expression among suppressed MCH cell lines; and iv) lack of expression by MCH-derived tumor cell lines.

Identification of three 11p11.2 candidate liver tumor suppressors through analysis of known human genes.

We have previously mapped a liver tumor suppressor locus to human chromosome 11p11.2-p12 using a functional model of tumor suppression. Using this model system, we have employed a candidate gene approach to identify potential liver tumor suppressor genes. Thirty-eight known genes have been positioned in human 11p11.2-p12 by the Human Genome Project. Here we show that four of these genes (guanine nucleotide binding protein gamma 3; mitochondrial carrier homolog 2; p53-induced protein (PIG11), and pRDI-BF1-rIZ1 domain containing 11) localized to the minimal liver tumor suppressor region within 11p11.2-p12. In fact, all of these genes mapped to human 11p11.2, allowing refinement of the liver tumor suppressor region to this cytogenetic band. Three of the four genes (mitochondrial carrier homolog 2, PIG11, and pRDI-BF1-rIZ1 domain containing 11) were uniformly expressed by an index panel of suppressed microcell hybrid cell lines, identifying them as candidate liver tumor suppressor genes. In a preliminary analysis of four human hepatocellular carcinoma cell lines (HepG2, Hep3B, SNU398, and SNU449), the transcript for PIG11 was lost or significantly decreased in two of these cell lines (HepG2 and Hep3B), suggesting the potential involvement of PIG11 in some human hepatocellular carcinomas. The results of this study extended our previous knowledge of genes located in the minimal liver tumor suppressor region of human 11p11.2 and identified several candidate liver tumor suppressor genes from this region. Further characterization of these candidates will provide new insight into the role of human 11p11.2 in the molecular pathogenesis of human liver cancer.

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

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.

Identification of candidate liver tumor suppressor genes from human 11p11.2-p12.

We have previously described a functional model for identification of human liver tumor suppressor genes in which human chromosome 11 was introduced into rat liver epithelial tumor cell lines via microcell-mediated chromosome transfer, producing microcell hybrid (MCH) cell lines that exhibit suppression of tumorigenicity in vivo. Chromosome deletion mapping studies identified a 950-kb region of 11p11.2-p12 that was retained in all suppressed MCH cell lines, suggesting that this region may harbor one or more genes with liver tumor suppressor function. In this study, we generated a comprehensive transcription map of the 11p11.2-p12 liver tumor suppressor region through examination of 142 expressed sequence tag (EST) markers among a group of suppressed MCH cell lines. Of 142 ESTs examined, 19 were localized within the 11p11.2-p12 liver tumor suppressor region. RT-PCR analysis of gene expression for these 19 ESTs among an index panel of suppressed MCH cell lines (n = 3) identified 11 potential candidate liver tumor suppressor genes. Examination of candidate gene expression among six additional suppressed MCH cell lines reduced the number of potential candidate genes to three (stSG30184, stSG10014, and stSG29748). Northern blot analysis of suppressed MCH cell lines and derived tumor cell lines suggested stSG30184 as the best candidate liver tumor suppressor gene. The 3.7 kb stSG30184 transcript was expressed by all suppressed MCH cell lines, but expression was extinguished coordinately with reexpression of tumorigenicity by these cells, consistent with a tumor suppressor gene. Subsequent characterization of this EST indicates that it is a novel transcript with expression in a broad range of tissue types. Further characterization of the genes identified in this study will provide a greater understanding of their role in the molecular pathogenesis of neoplastic liver disease.