Genetic analysis of indefinite division in human cells: identification of four complementation groups.

Abstract

Hybrids obtained following fusion of normal human diploid fibroblasts with different immortal human cell lines exhibited limited division potential. This led to the conclusion that the phenotype of cellular senescence is dominant and that immortal cells arise as a result of recessive changes in the growth control mechanisms of the normal cell. We have exploited the fact that immortality is recessive and, by fusing immortal human cell lines with each other, assigned 21 cell lines to at least four complementation groups for indefinite division. A wide variety of cell lines was included in the study to determine what parameters, if any, would affect complementation group assignment. The results indicate that cell type, embryonal layer of origin, and type of tumor do not affect group assignment. There does not appear to be any correlation between expression of an activated oncogene and group assignment. However, all of the immortal simian virus 40-transformed cell lines studied (with the exception of one xerodermapigmentosum fibroblast-derived line) assign to the same group, indicating that this virus immortalizes various human cells by the same processes. The assignment of immortal human cells to distinct'groups provides the basis for a focused approach to determine the genes important in normal growth regnlation that have been modified in immortal cells.

Evidence for the inactivation of multiple replicative lifespan genes in immortal human squamous cell carcinoma keratinocytes

“…There was no LOH at any of the genetic loci investigated in neoplastic keratinocytes which were senescent in vitro, consistent with the notion that the genes mapping to chromosomes 3, 4 and 7 may be involved in the control of keratinocyte replicative lifespan but with the reservation that the neoplastic keratinocytes which senesce show a very low frequency of allele loss overall (Edington et al, 1995). It has been suggested that multiple genetic routes to senescence exist (Pereira-Smith and Smith, 1988;Sasaki et al, 1994) and di erent recessive mutations are predicted to exist within the same tumor type (Pereira-Smith and Smith, 1988). These mutations may be mutually exclusive (Pereira-Smith and Smith, 1988) or additive (Sasaki et al, 1994).…”

“…Several human chromosomes have been shown to harbour antiproliferative genes which have been mapped to 1q25 (Karlsson et al, 1996), 1q42 (Karlsson et al, 1996), 2 (Uejima et al, 1995), 3p21 (Rimessi et al, 1994), 4q32-q34 (Ning et al, 1991; O Pereira-Smith ± personal communication), 6q14-q21 (Sandhu et al, 1996), 6q21-qter (Sandhu et al, 1994), 7q31-32 (Ogata et al, 1993), 11p15 (Koi et al, 1993), 17 (Casey et al, 1993), 18 (Sasaki et al, 1994) and Xp11-ter respectively. Genetic complementation groups for the immortal phenotype have been identi®ed (Pereira-Smith and Smith, 1988) and although the complementation group A locus is unknown, genes assigned to chromosomes 4 (Ning et al, 1991), 1 (Hensler et al, 1994) and 7 (Ogata et al, 1993) are thought to represent complementation groups B, C and D respectively. The chromosome 1 gene has been mapped to either the p arm or to 1q13-q31 , making the antiproliferative gene at 1q25 (Karlsson et al, 1996) a likely candidate.…”

SEN6, a locus for SV40-mediated immortalization of human cells, maps to 6q26-27

“…Immortalization of human tumors and cell lines in culture is due to a limited number of genetic alterations as Pereira-Smith and Smith (1988) reported that 20 tumors could be categorized into four complementation groups (A±D). Since many SV40-immortalized cell lines have been shown to fall into complementation group A (Pereira-Smith and Smith, 1988), they share a chromosome 6 rearrangement and they are suppressed by introduction of a normal chromosome 6, it would appear that the group A gene is on chromosome 6, i.e.…”

“…Furthermore, we expect that its reexpression will inhibit cell proliferation of other immortal tumor cell lines, i.e. those which show evidence of LOH consistent with SEN6 and/or those which do not complement the immortal phenotype in cell hybrids with SV/HF-5/39 (`clone 39') and other members of the A group reported by Pereira-Smith and Smith (1988). Current e orts are directed to clone the sequences in 6q26-27 encoding SEN6 to test this hypothesis.…”

“…Hence, one might also consider senescence to be a mechanism of tumor suppression (Sager, 1989). Genetic studies have shown that the immortal phenotype is recessive in cell hybrids (Pereira-Smith and Smith, 1983) and that human tumors can be assigned to a limited number of complementation groups by pairwise cell fusion analysis of cell lines (Pereira-Smith and Smith, 1988;Duncan et al, 1993). Members of four complementation groups (A±D) have been con®rmed to contain unique alterations since distinct individual normal human chromosomes can suppress cell growth in representative cell lines (Ning et al, 1991;Ogata et al, 1993;Sagawara et al, 1990;Sandhu et al, 1994).…”