Identification of rCop-1, a New Member of the CCN Protein Family,as a Negative Regulator for Cell Transformation |
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| Authors: | Rong Zhang Lidia Averboukh Weimin Zhu Hong Zhang Hakryul Jo Peter J. Dempsey Robert J. Coffey Arthur B. Pardee Peng Liang |
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| Affiliation: | Vanderbilt Cancer Center, Department of Cell Biology, Vanderbilt University, Nashville, Tennessee 37232,1. and Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, Boston, Massachusetts 021152. |
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| Abstract: | By using a model system for cell transformation mediated by the cooperation of the activated H-ras oncogene and the inactivated p53 tumor suppressor gene, rCop-1 was identified by mRNA differential display as a gene whose expression became lost after cell transformation. Homology analysis indicates that rCop-1 belongs to an emerging cysteine-rich growth regulator family called CCN, which includes connective-tissue growth factor, CYR61, CEF10 (v-src inducible), and the product of the nov proto-oncogene. Unlike the other members of the CCN gene family, rCop-1 is not an immediate-early gene, it lacks the conserved C-terminal domain which was shown to confer both growth-stimulating and heparin-binding activities, and its expression is lost in cells transformed by a variety of mechanisms. Ectopic expression of rCop-1 by retroviral gene transfers led to cell death in a transformation-specific manner. These results suggest that rCop-1 represents a new class of CCN family proteins that have functions opposing those of the previously identified members.Oncogenic conversion of a normal cell into a tumor cell requires multiple genetic alterations (12). Of particular interest is the fact that mutations in both ras oncogenes (3) and the p53 tumor suppressor gene cooperate in transformation of mammalian cells (11). Mutations in both ras and the p53 gene were also found at high frequencies in a variety of human cancers, including those of the colon, lung, and pancreas (2, 18). It has been proposed that both p53 and Ras function, whether directly or through other signaling molecules, to control expression of genes that are important for cell growth and differentiation (13, 17, 37). To this end, several ras target genes (10) and p53 target genes, including those encoding p21/CIP1/WAF1, an inhibitor of G1 cyclin-dependent kinase (9); Mdm-2, a negative regulator of p53 (1); GADD45, a protein involved in DNA repair (36); and Bax, which promotes apoptosis (28), have been identified. Most of these genes, except p21/CIP1/WAF1, which was cloned by subtractive hybridization, were identified by the candidate gene hypothesis. Recently, more p53 target genes have been isolated by the differential display technique, including those coding for cyclin G (31); MAP4, a microtubule-associated protein negatively regulated by p53 (29); and PAG608, a novel nuclear zinc finger protein whose overexpression promotes apoptosis (14). Functional characterizations of these genes have shed light on the role of p53 in cell cycle control and apoptosis. However, genes that mediate tumor suppression activity by p53 remain elusive.The fact that neither the inactivation of p53 nor the activation of Ras alone is able to transform primary mammalian cells (34), whereas both mutations together can do so, suggests that genes regulated by p53 and Ras cooperate in upsetting normal cell growth control cells (11). Using differential display (22), we set out to identify genes whose expression is altered by both mutant ras and p53 by comparing the mRNA expression profiles of normal rat embryo fibroblasts (REFs) and their derivatives transformed by either a constitutively inactivated or a temperature-sensitive mutant p53 in cooperation with the activated H-ras oncogene (11, 27). In this report we describe the identification and give a functional characterization of rCop-1, a gene whose expression is abolished by cell transformation. By sequence homology, rCop-1 was found to belong to an emerging cysteine-rich growth regulator family called CCN (which stands for connective-tissue growth factor [CTGF], CEF10/Cyr61, and Nov) (4). Here we show that rCop-1 may represent a novel class of CCN family proteins based on its unique cell cycle expression pattern, its lack of the C-terminal (CT) domain conserved in all CCN proteins, its loss of expression in all transformed cells analyzed, and its ability to confer cytotoxicity to the transformed cells. |
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