Abstract
Normal human diploid fibroblasts have limited life span in culture and undergo replicative senescence after 50-60 population doublings. On the contrary, cancer cells typically divide indefinitely and are immortal. Expression of SV40 large T and small t antigens in human fibroblasts transiently extends their life span by 20-30 population doublings and facilitates immortalization. We have identified a rearrangement in chromosome 6 shared by SV40-transformed human fibroblasts. Rearrangements involving chromosome 6 are among the most frequent in human carcinogenesis. In this paper, we extend analysis of the 6q26-q27 region, a putative site for a growth suppressor gene designated SEN6 involved in immortalization of SV40-transformed cells. Detailed molecular characterization of the rearranged chromosomes (6q*, normal appearing; and 6q(t), translocated) in the SV40-immortalized cell line HALneo by isolating each of these 2 chromosomes in mouse/HAL somatic cell hybrids is presented. Analysis of these mouse/HAL somatic cell hybrids with polymorphic and nonpolymorphic markers revealed that the 6q* has undergone a chromosomal break in the MLLT4 gene (alias AF6). This result in conjunction with previous published observations leads us to conclude that SEN6 lies between MLLT4 and TBP at chromosomal region 6q27. Examination of different genes (MLLT4, DLL1, FAM120B, PHF10) located within this interval that are expressed in HS74 normal fibroblast cells reveals that overexpression of epitope-tagged truncated PHF10 cDNAs resulted in reduced cell proliferation in multiple cell lines. Paradoxically, down-regulation of PHF10 by RNAi also resulted in loss of cell proliferation in normal fibroblast cells, indicating PHF10 function is required for cell growth. Taken together, these observations suggest that decreased cell proliferation with epitope-tagged truncated PHF10 proteins may be due to dominant negative effects or due to unregulated expression of these mutant proteins. Hence we conclude that PHF10 is not SEN6 but is required for cell growth.