PERTURBATION OF TSG101 PROTEIN AFFECTS CELL-CYCLE PROGRESSION

tsg101 was recently identified as a tumor susceptibility gene by funct ional inactivation of allelic loci in mouse 3T3 fibroblasts. Although previous studies suggested that homozygous intragenic deletion of TSG1 01 is rare in breast cancer cells and specimens, the neoplastic phenot ype caused by tsg101 inactivation implicated that tsg101 may play a si gnificant role in cell growth control. Here, we characterize mouse pol yclonal and monoclonal antibodies that specifically recognize the TSG1 01 protein (molecular mass, 46 kDa) in whole-cell lysates by straight Western blot analysis. By indirect immunofluorescence staining, TSG101 was found to be localized in the cytoplasm throughout the entire cell cycle, However, the nuclear staining increases from G(1) to S phase a nd becomes dominant in late S phase. TSG101 is mainly distributed surr ounding the chromosomes during M phase. The expression level of TSG101 is not cell cycle dependent. It is possible that the relocalization o f TSG101 from the cytoplasm into the nucleus may be relevant to its fu nction. Microinjection of both polyclonal and monoclonal antibodies sp ecific to TSG101 into cells during G, or S phase results in cell cycle arrest. Furthermore, overexpression of TSG101 leads to cell death, su ggesting that the appropriate amount of TSG101 is critical for cell cy cle progression. Taken together, these results suggest that neoplastic transformation caused by TSG101 deficiency may result from bypassing of the cell cycle checkpoints.