"Antisense RNA-mediated Deficiency of the Calpain Protease, nCL-4, in NIH3T3 Cells Is Associated with Neoplastic Transformation and Tumorigenesis."

Keyi Liu ¹, Limin Li ¹, and Stanley N. Cohen ^{1, 2,*}

From the  ¹ Department of Genetics and ² Department of Medicine, Stanford University School of Medicine, Stanford, California 94305-5120

* To whom correspondence should be addressed: Dept. of Genetics, Rm. M320, Stanford University Medical Center, Stanford, CA 94305-5120. Tel.: 650-723-5315; Fax: 650-725-1536;
E-mail: sncohen@stanford.edu

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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