Antisense RNA-mediated deficiency of the calpain protease, nCL-4, in NIH3T3 cells is associated with neoplastic transformation and tumorigenesis

We previously have described the use of an antisense RNA strategy termed ra ndom homozygous knock-out (RHKO) to identify negative regulators of cell pr oliferation, Here we report the discovery that RHKO-mediated deficiency of the nCL-4 calpain protease results in cellular transformation of and tumori genesis by murine NIH3T3 fibroblasts. We isolated cell clones able to form colonies on 0.5% soft agar and found that these cells generated tumors when injected subcutaneously into nude mice. The gene inactivated by RHKO was i dentified as nCL-4 by genomic library screening, transcript analysis, and D NA sequencing. Anchorage-independent growth, as indicated by colony formati on on soft agar, was reversed by reversal of antisense-mediated homozygous inactivation, but continued haplo-insufficiency of nCL-4 resulting from ins ertional mutagenesis of one nCL-4 allele was associated with persistent tum origenesis. nCL-4 cDNA expressed in naive 3T3 cells in the antisense, but n ot sense, direction under control of the cytomegalovirus early promoter rep roduced the anchorage-independent growth effects of RHKO. Our results impli cate deficiency of the nCL-4 calpain protease in neoplastic transformation.