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The met protooncogene product, Met, is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). NIH 3T3 cells express HGF/SF endogenously and become tumorigenic in nude mice via an autocrine mechanism when murine Met is expressed ectopically (Metmu cells) or when human Met and human HGF/SF are coexpressed (HMH cells). Here, we show that Metmu and HMH cells are invasive in vitro and display enhanced protease activity necessary for the invasive phenotype. In experimental and spontaneous metastasis assays, Metmu or HMH cells metastasize to the lung, but lower numbers of subcutaneously injected Metmu and HMH cells produced invasive tumors in the heart, diaphragm, salivary gland, and retroperitoneum. It has been reported elsewhere that Met expression increased with tumor passage in athymic nude mice, and these tumor explants show enhanced activity in the metastasis assays. Autocrine-mediated Met-HGF/SF signal transduction in NIH 3T3 mesenchymal cells may provide an important system for understanding the biological process of metastasis.

Gal4‐p53 fusion constructs demonstrate that wild type p53 is a potent transactivator in human lung cancer cells with the transactivation domain for p53 residing in amino acids 1–42. Strikingly, a variety of lung cancer derived p53 mutations occurring outside this domain disrupt this activity. Temperature sensitive conformational shifts of p53 mutant proteins to the wild type form exist and, with a temperature downshift, several mutants become transcriptionally active. Wild type p53 protein is known to form oligomers with mutant p53 and cotransfection of wild type and mutant genes shows that p53 acts in a transdominant manner that is independent of the DNA binding specificity. Transcription is either increased or decreased depending on whether the wild type is more or less abundant than the mutant form. Finally, lung cancers differ in their ability to support the transactivation related functions, providing evidence of other abnormalities of the p53 system in human cancer.

The Friend-virus-derived mouse erythroleukaemia (MEL) cell lines represent transformed early erythroid precursors that can be induced to differentiate into more mature erythroid cells by a variety of agents including dimethyl sulphoxide (DMSO). There is a latent period of 12 hours after inducer is added, when 80-90% of the cells become irreversibly committed to the differentiation programme, undergoing several rounds of cell division before permanently ceasing to replicate. After DMSO induction, a biphasic decline in steady-state levels of c-myc and c-myb messenger RNAs occurs. Following the initial decrease in c-myc mRNA expression, the subsequent increase occurs in, and is restricted to, the G1 phase of the cell cycle. We sought to determine whether the down-regulation is a necessary step in chemically induced differentiation. Experiments reported here indicate that expression in MEL cells of a transfected human c-myc gene inhibits the terminal differentiation process.

Recent molecular analysis has revealed that L-myc has several domains of extremely conserved anino acid sequence homology with c-myc and N-myc, suggesting similarity of function. We tested the biologic activity of L-myc by using an expression vector containing a cDNA clone coding for the major open reading frame in the 3.9-kilobase mRNA of L-myc under the control of a strong promoter (Moloney long terminal repeat) and found that L-myc complemented an activated ras gene in transforming primary rat embryo fibroblasts. However, the efficiency of transformation was 1 to 10% of that seen with the c-myc and simian virus 40 (SV40) controls. The L-myc/ras transformants initially grew more slowly than c-myc or SV40 transformants, but once established as continuous cell lines, they were indistinguishable from cell lines derived from c-myc/ras or SV40/ras transfectants as determined by morphology, soft-agar cloning, and tumorigenicity in nude mice.

Translation of thymidylate synthase (TS) mRNA is controlled by its own protein product, TS, in an autoregulatory manner. Direct binding of TS protein to two different cis-acting elements on the TS mRNA is associated with this translational regulation. In this study, an immunoprecipitation-reverse transcription-PCR technique was used to identify a TS ribonucleoprotein (RNP) complex in cultured human colon cancer cells. Using antibodies specific for TS protein, we show that TS is complexed in vivo with its own TS RNA. Furthermore, evidence demonstrating a direct interaction between the mRNA of the nuclear oncogene c-myc and TS protein is presented.Recently, there has been an increased interest in the characterization of translational regulatory mechanisms. There are a number of eukaryotic mRNAs whose expression is controlled at the level of translation (18). The regulated synthesis of the iron storage protein ferritin by iron represents one of the best-studied examples of this form of regulation (25). A stem-loop structure located within the 5' untranslated region (UTR) of ferritin mRNA, termed the iron-responsive element (IRE), represents the cis-acting element to which the IRE-binding protein binds (16,23). Recent studies have demonstrated that the redox state in the cell is an important determinant of the binding affinity of this protein to the IRE (15,17).Using an in vitro translation system, we showed that translation of human thymidylate synthase (TS) mRNA is regulated by its own protein product, TS, in a negative autoregulatory manner (7). Although translational autoregulation has been described in prokaryotic systems (2, 5), TS mRNA represents the first eukaryotic mRNA whose regulation is controlled in such a fashion. Furthermore, we demonstrated that incubation of TS protein with either the nucleotide substrate dUMP or the inhibitor 5-fluoro-dUMP repressed its inhibitory effect on TS mRNA translation. during the cell cycle is primarily regulated at the transcriptional level (1,19,30), there is now recent evidence suggesting control at the level of translation (22). These findings, taken together, offer supportive evidence for the model of TS translational autoregulation.The purpose of the present study was to identify a TS ribonucleoprotein (RNP) complex in a cultured cell system. With the use of specific antisera to TS, we show that TS protein is complexed with its corresponding TS RNA in human colon cancer cells. In addition, we present evidence demonstrating a specific interaction between the mRNA of the nuclear oncogene c-myc and TS protein. MATERIALS AND METHODSCell culture. The characteristics of the human colon cancer cell line H630 have been previously described (32). The resistant H630-R1O subline was selected in vitro for resistance to 5-FU by exposure of the parent H630 cell line to stepwise increases in 5-FU and was maintained in medium containing 10 p,. Cell lines were grown in 75-cm2 plastic tissue culture flasks (Falcon Labware, Oxnard, Calif.) in growth medium containing RPMI 1640 with 1...

No abstract

Using an immunoprecipitation-reverse transcription-PCR technique, we characterized a thymidylate synthase (TS) ribonucleoprotein complex in cultured human colon cancer cells that consists of TS protein and the mRNA of the nuclear oncogene c-myc. TS protein is complexed in intact cells with the C-terminal coding region of c-myc mRNA that includes nucleotide positions 1625 to 1790. RNA electrophoretic gel mobility shift assays confirm a specific interaction between TS protein and c-myc mRNA and provide additional evidence that the C-terminal coding region represents an important cis-acting regulatory element. Further evidence demonstrates that the in vitro translational efficiency of c-myc mRNA is inhibited as a result of its direct interaction with TS protein. In addition, the presence of exogenous c-myc mRNA specifically relieves the inhibitory effects of TS protein on TS mRNA translation.