Targeted deletion of the Tsg101 gene results in cell cycle arrest at G₁/S and p53-independent cell death

The tumor susceptibility gene 101 (Tsg101) was originally discovered in a screen for potential tumor suppressors using insertional mutagenesis in immortalized fibroblasts. To investigate essential functions of this gene in cell growth and neoplastic transformation, we derived primary mouse embryonic fibroblasts from Tsg101 conditional knockout mice. Expression of Cre recombinase from a retroviral vector efficiently down-regulated Tsg101. The deletion of Tsg101 caused growth arrest and cell death but did not result in increased proliferation and cellular transformation. Inactivation of p53 had no influence on the deleterious phenotype, but Tsg101^-/- cells were rescued through expression of exogenous Tsg101. Fluorescence-activated cell sorting, proliferation assays, and Western blot analysis of crucial regulators of the cell cycle revealed that Tsg101 deficiency resulted in growth arrest at the G₁/S transition through inactivation of cyclin-dependent kinase 2. As a consequence, DNA replication was not initiated in Tsg101-deficient cells. Our results clearly demonstrate that Tsg101 is not a primary tumor suppressor in mouse embryonic fibroblasts. However, the protein is crucial for cell proliferation and cell survival.