Induction of Apoptosis and Inhibition of Cell Proliferation bysurvivin Gene Targeting

Abstract

Survivin is a new IAP apoptosis inhibitor expressed during development and in human cancer in vivo. The coding strand of the survivin gene was extensively complementary to that of effector cell protease receptor-1 (EPR-1), prompting the present investigation on the origin and functional relationship of these two transcripts. Southern blots of genomic DNA were consistent with the presence of multiple, evolutionarily conserved, EPR-1/Survivin-related genes. By pulsed field gel electrophoresis and single- and two-color fluorescence in situ hybridization, these were contained within a contiguous physical interval of 75-130 kilobases (kb) on chromosome 17q25. In Northern blots, a single strand-specific probe identified a 1.3-kb EPR-1 mRNA broadly distributed in normal adult and fetal tissues, structurally distinct from the 1.9-kb Survivin transcript expressed in transformed cell lines. Transient co-transfection of an EPR-1 cDNA potentially acting as a Survivin antisense with a lacZ reporter plasmid resulted in loss of viability of HeLa cells. In contrast, co-transfection of an antisense cDNA of intercellular adhesion molecule-1 or a sense-oriented Survivin cDNA was without effect. In stably transfected HeLa cells, ZnSO4 induction of an EPR-1 mRNA under the control of a metallothionein promoter suppressed the expression of endogenous survivin. This resulted in (i) increased apoptosis as detected by analysis of DNA content and in situ internucleosomal DNA fragmentation and (ii) inhibition of cell proliferation as compared with induced vector control transfectants. These findings suggest the existence of a potential EPR-1/survivin gene cluster and identify survivin as a new target for disrupting cell viability pathways in cancer.