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Bleomycin Resistance in Mammalian Cells Expressing a Genetic Suppressor Element Derived from the SRPK1 Gene¹

CNRS UMR 8532, Institut Gustave Roussy, 94805 Villejuif, France [G. S., L. M.], and Laboratoire de Pharmacologie des Agents Anticancéreux, Institut Bergonié, 33076 Bordeaux Cedex, France [A. J-S.]

The genetic suppressor element (GSE) approach allows identification of genes essential for certaincell 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 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.

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