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Increased Growth of NIH/3T3 Cells by Transfection with Human p120 Complementary DNA and Inhibition by a p120 Antisense Construct¹

Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030

The human nucleolar antigen p120 was detected with an anti-p120 monoclonal antibody in most human malignant tumors but not in most resting human tissues (J. W. Freeman et al., Cancer Res., 48: 1244–1251, 1988) and has been used as a prognostic tumor marker in breast cancer patients (J. W. Freeman et al., Cancer Res., 51: 1973–1978, 1991). After the complementary DNA and gene for the human p120 protein were isolated and sequenced (review: H. Busch, Cancer Res., 50: 4830–4838, 1990), constructs were prepared to study the expression of the sense p120 and its antisense, p021 message. NIH/3T3 cells were transfected by electroporation with pSVX plasmids containing either the p120 complementary DNA (pSVX120) or the antisense, p021 DNA (pSVX021), and clones containing these constructs were selected. The expression of p120 or p021 in these constructs was regulated by Moloney murine leukemia virus long terminal repeats. In pSVX120-transfected NIH/3T3 cells, the expressed human p120 protein was localized to the nucleoli as shown by anti-p120 monoclonal antibody immunofluorescence. Expression of the p120 message and protein was confirmed by Northern (mRNA) and Western (protein) blots. Transfection of the p120 complementary DNA in sense orientation caused malignant transformation of NIH/3T3 cells in vitro and produced rapidly growing tumors in nude mice. Transfection of the antisense p120 constructs markedly delayed the growth of these tumros in vitro and in vivo (L. Perlaky et al., Proc. Am. Assoc. Cancer Res., 32: 1682, 1991). When transformed 3T3/pSVX120 cells were transfected with an inducible antisense p120 construct (pMSG021), dexamethasone induction decreased the growth rate by 62%, and the cell line returned to its normal phenotype. Northern blot analysis showed a decreased level of p120 mRNA, and the immunofluorescence was also markedly reduced.

¹ This work was supported by Cancer Research Center Grant CA10893, P1, awarded by the National Cancer Institute, Department of Human Services, USPHS; the DeBakey Medical Foundation; the H. Leland Kaplan Cancer Research Endowment; the Linda and Ronny Finger Cancer Research Endowment Fund; and the William S. Farish Fund.

² Present address: Sigma Chemical Company, 3500 Dekalb St., St. Louis, MO 63118.

³ Present address: Ohio State University, 446 McCampbell Hall, 1581 Dodd Dr., Columbus, OH 43210.

⁴ To whom requests for reprints should be addressed, at Department of Pharmacology, Baylor College of Medicine, Texas Medical Center, One Baylor Plaza, Houston, TX 77030.

Received 8/12/91. Accepted 11/ 1/91.

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