Detection of genes with a potential for suppressing the transformed phenotype associated with activated ras genes.

Abstract

Seven morphologically nontransformed (flat) revertants with reduced tumorigenicity in vivo have been isolated from populations of Kirsten sarcoma virus-transformed NIH 3T3 cells transfected with a cDNA expression library ofnormal human fibroblasts. Each revertant harbors 1-10 recombinant plis per cell and retains a rescuable transforming virus as well as high level expression of v-Ki-ras-specific RNA and the viral oncogene product, p21v-i-. Transformed phenotypes are suppressed in cell hybrids generated by fusing each revertant to v-Ki-rastransformed NIH 3T3 cells. From two of the revertant lines, plasmids capable ofgiving rise to flat secondary transfectants have been recovered. Thus, in some, if not all, of the revertants, transfected cDNAs seem to be responsible for the suppression of specific transformed phenotypes.Flat revertants from rodent fibroblast cell lines transformed by various agents have been isolated and characterized by several groups of investigators as an approach to define and, ultimately, resolve the mechanism(s) of malignant cell transformation (reviewed in refs. 1 and 2). This approach has been most successfully applied in defining and characterizing those genes that are responsible for the transforming activities of various retroviruses (e.g., see refs. 3 and 4). There are also additional reports describing flat revertants, which appear to have resulted from mutations in specific cellular genes rather than viral-encoded transforming genes (1,(5)(6)(7)(8). Such revertants may be useful in revealing cellular macromolecules that might either directly or indirectly participate in the process of cellular transformation. Previously, we described two nontumorigenic flat revertants isolated from populations of the DT line of Kirsten murine sarcoma virus (Ki-MSV)-transformed cells (7). These revertants seem to harbor dominant mutations in cellular genes, which in some way suppress certain malignant characteristics usually associated with Ki-MSV-induced transformation (7, 9-12). The putative gene or genes responsible for suppression of the malignant characteristics have not yet been identified.In this report, we show that flat revertants with reduced tumorigenicity can be isolated from Ki-MSV-transformed DT cells after transfection of normal human fibroblast cDNA expression library. The properties of some, if not all, of the revertants are consistent with the idea that expression of the introduced cDNA clones contributes to a reversal of the ras-induced transformed phenotype. MATERIALS AND METHODSCell Lines and Viruses. The derivations and methods for maintenance of NIH 3T3, DT (Ki/HGPRT-NIH 3T3), and Ki/TK-NIH have been described (7). DTneoR was derived from DT cells by transfection of pL2 DNA (13) followed by *G418 selection. Rescue for replication-defective sarcoma viruses, focus formation, and soft agar assays were performed as described (7, 9). Growth medium consisted of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS) (KC Biological, Lenexa, KS) and 4 mM...

Expression of an isoform of the novel signal transduction protein ST5 is linked to cell morphology

“…Di erent p70 expressors grew to densities between 6610 4 and 1610 5 cells/cm 2 , while the control transfectants achieved densities of at least 2.5610 5 cells/cm 2 . The saturation density of the p70 expressors was similar to that of contact-inhibited NIH3T3 cells, which typically grow to approximately 1610 5 cells/ cm 2 (Noda et al, 1989). Despite the di erence in saturation density, the initial growth rate of the p70 expressing clones was only slightly slower than that of a b c d Figure 5 Actin organization in NIH3T3-OFF cells and ST5-p70 expressing transfectants.…”

“…The commercially available NIH3T3-OFF line was designed to give inducible expression of proteins placed under the control of a tetracycline sensitive activator protein (Clontech, Palo Alta, CA, USA). This line contained a homogenous population of refractile cells which grew to a considerably greater density than that reported for NIH3T3 (Noda et al, 1989) and displayed some characteristics of the transformed phenotype, including a refractile morphology and high-density growth. In addition, they contained little if any p70 (Figure 3a, lane 1), while NIH3T3 cells contained readily detectable p70 (lane 2).…”

Involvement of the SKP2–p27KIP1 pathway in suppression of cancer cell proliferation by RECK

“…RECK was first isolated as a transformation-suppressor gene by cDNA expression cloning (Noda et al, 1989;Takahashi et al, 1998) and found to encode a membraneanchored regulator of several extracellular metalloproteinases, including MMP2, MMP7, MMP9, MT1-MMP, ADAM10 and CD13 (Takahashi et al, 1998;Oh et al, 2001;Miki et al, 2007;Muraguchi et al, 2007;Omura et al, 2009). These proteases have been implicated in tissue remodeling and cleavage of various extracellular molecules under normal and pathological conditions (Sternlicht and Werb, 2001).…”

Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N)

“…This, together with the observation that Rap1, when overexpressed, can reverse the K-Ras transformed phenotype of NIH3T3 cells , has led to the hypothesis that Rap1 could be a possible antagonist of Ras. Several studies indeed showed this antagonistic action of Rap1 on a variety of Ras-dependent responses and possibly also in the sevenless signal transduction pathway (for references see Bokoch, 1993;Noda, 1993). Despite this, a reversal of the transformed phenotype in a variety of human tumor cell lines upon expression of Rap1A was not observed (Sato et al, 1994).…”