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 random homozygous knock-out (RHKO) to identify negative regulators of cell proliferation. Here we report the discovery that RHKO-mediated deficiency of the nCL-4 calpain protease results in cellular transformation of and tumorigenesis 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 identified as nCL-4 by genomic library screening, transcript analysis, and DNA sequencing. Anchorage-independent growth, as indicated by colony formation on soft agar, was reversed by reversal of antisense-mediated homozygous inactivation, but continued haplo-insufficiency of nCL-4 resulting from insertional mutagenesis of one nCL-4 allele was associated with persistent tumorigenesis. nCL-4 cDNA expressed in naive 3T3 cells in the antisense, but not sense, direction under control of the cytomegalovirus early promoter reproduced the anchorage-independent growth effects of RHKO. Our results implicate deficiency of the nCL-4 calpain protease in neoplastic transformation.
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Department of Genetics and Department of Medicine, Stanford University School of Medicine, Stanford, California 94305-5120, USA.
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D016475:3T3 Cells; D000483:Alleles; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002154:Calpain / Q000235:genetics* / Q000502:physiology; D002455:Cell Division; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D003587:Cytomegalovirus / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D005260:Female; D015723:Gene Library; D051379:Mice; D018345:Mice, Knockout; D008819:Mice, Nude; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000235:genetics; D010455:Peptides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016372:RNA, Antisense / Q000737:chemistry* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D010455:Peptides; D016372:RNA, Antisense; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D002154:Calpain