Chromosome 13 transfer provides evidence for regulation of RB1 protein expression

The human retinoblastoma susceptibility gene (RB1) located on chromosome 13 has been shown to function as a growth/tumor suppressor gene in a large number of human cancers. Although constitutive expression has been observed in most cultured cells and normal tissues, overexpression of RB1 protein has not been well documented. Perhaps regulating the level of normal RB1 protein expression is one of several ways of controlling its function. To test this hypothesis, we transferred normal copies of chromosome 13 via microcell fusion into the human fibrosarcoma cell line HT1080. Microcell hybrids were generated that contained one, two, or three extra copies of the transferred fibroblast chromosome 13. Compared to the parental cell line, the hybrids were completely unaltered with respect to several properties in vitro and in vivo, including morphology, growth rate, and tumor formation. Northern blot analysis revealed a stepwise increase in RB1 mRNA expression which increased in proportion to the number of alleles present in each cell line. Although RB1 protein exhibited correct nuclear localization and was phosphorylated in a normal cell cycle-dependent manner in the hybrids, the increased level of protein expression in each hybrid was nearly identical and did not increase beyond a threshold amount, although mRNA expression continued to increase. These results demonstrate that HT 1080 cells can tolerate an increase level of RB1 protein, but that expression beyond a certain level may be down-regulated. These transfer studies provide evidence for regulation of RB1 protein expression and may suggest an alternative form of monitoring and controlling normal RB1 functioning.