Bleomycin resistance in mammalian cells expressing a genetic suppressor element derived from the SRPK1 gene.

Abstract

The genetic suppressor element (GSE) approach allows identification of genes essential for certain cell phenotypes. To identify genes controlling the cell response to cytotoxic agents, a normalized retroviral library of randomly fragmented cDNAs from the Chinese hamster cell line DC-3F was screened for GSEs conferring resistance to bleomycin. One of these GSEs, GSE(BLM), conferring an approximately 2-fold bleomycin resistance in DC-3F cells, displayed 98% identity with an amino acid sequence located in the functional domain of human SRPK1. Using GSE(BLM) as a probe, we cloned a cDNA with a nucleotide sequence that was 76.7% identical to that of human SRPK1, whereas the corresponding amino acid sequence was 92.6% identical to that of this enzyme. When GSE(BLM), inserted in the retroviral vector pLNCX, was transduced in HeLa cells, its expression resulted in a 5-10-fold bleomycin resistance, which was abolished when these cells were further transfected with SRPK1 cDNA. In our experimental conditions, DC-3F or HeLa cells expressing GSE(BLM) did not show any detectable cross-resistance to other cytotoxic agents with various mechanisms of action. GSE(BLM), which is sense oriented in the vector, is likely to be translated in a peptide active as a dominant-negative inhibitor of SRPK1. SRPK1 is a protein serine kinase that regulates the activity of RS-proteins (arginine-serine-rich proteins), a group of nuclear factors controlling various physiological processes.