Growth Of NIH3T3 Cells Research Articles

DNA binding domain-independent pathways are involved in EWS/FLI1-mediated oncogenesis.

Specific chromosomal translocations involving the ews gene and one of five members of the ets family of transcription factors create ews/ets fusion genes that are found in approximately 85% of Ewing's family of tumors. ews/ets fusion genes consistently maintain an intact and functional ets DNA binding domain (DBD) in all of these cases. We demonstrate here, however, that EWS/FLI1, the most prevalent EWS/ETS fusion, activates oncogenic pathways independent of its DBD. In in vivo tumor assays, EWS/FLI1 molecules with either point mutations or a large deletion in the ets DBD retain the ability to accelerate tumors in NIH 3T3 cells, whereas they lose the ability to bind DNA in vitro. Additionally, whereas inhibition of DBD functions of EWS/FLI1 with a dominant negative form of FLI1 is sufficient to inhibit anchorage-independent growth in NIH 3T3 cells, it is ineffective in inhibiting tumor growth in SCID mice. Usage of this dominant negative construct in a Ewing's tumor cell line, however, does reduce the rate of tumor formation, supporting the need for a functional DBD in this context. Together, these results suggest that EWS/FLI1 induces both DBD-dependent and DBD-independent oncogenic pathways.

Purification of activins from androgen-independent Shionogi carcinoma cells demonstrates enhanced expression of activin betaB-subunit under androgen-depleted cell conditions in vitro and in vivo.

Here, we report characterization of growth factors secreted from androgen-independent mouse mammary Shionogi carcinoma cells. Previous isolation of fibroblast growth factor 8 (FGF8) from androgen-dependent Shionogi carcinoma SC-3 cells prompted us to characterize growth factors secreted from the androgen-independent cells. After several purification procedures, mitogens for NIH3T3 cells from the androgen-independent cells were identified as activins on the grounds that activin betaA- and betaB-subunits are detected in the active fractions by Western blotting and that the growth-promoting effects by the active fractions are specifically inhibited in the presence of follistatin. In addition, exogenous activins, but not inhibin, stimulated the growth of NIH3T3 cells in a dose-dependent manner. Interestingly, transcripts of activin betaB-subunit were predominantly found in the androgen-independent cells while its betaA-subunit was universally expressed in both androgen-dependent and -independent Shionogi carcinoma cells. In concordant with this in vitro finding, transcripts of activin betaB-subunit were enhanced in murine prostates after castration. Therefore, expression of activin betaB-subunit, but not its betaA-subunit, is likely to be related with androgen-depleted cell conditions in prostates, and possibly in androgen-related cancers.

Ras-dependent cell cycle commitment during G2 phase

Synchronization used to study cell cycle progression may change the characteristics of rapidly proliferating cells. By combining time-lapse, quantitative fluorescent microscopy and microinjection, we have established a method to analyze the cell cycle progression of individual cells without synchronization. This new approach revealed that rapidly growing NIH3T3 cells make a Ras-dependent commitment for completion of the next cell cycle while they are in G2 phase of the preceding cell cycle. Thus, Ras activity during G2 phase induces cyclin D1 expression. This expression continues through the next G1 phase even in the absence of Ras activity, and drives cells into S phase.

Gradient micropattern immobilization of heparin and its interaction with cells

Heparin was immobilized on a polystyrene plate in a graded micropattern by photolithography. The micropattern was confirmed by staining with brilliant green. In the presence of basic fibroblast growth factor (bFGF), the growth of NIH3T3 cells was enhanced only in the heparinimmobilized regions. This result indicated that gradient-micropattern-immobilized heparin activated bFGF for cell growth. In addition, the growth of cells was found to be affected by the surface density of immobilized heparin. The surface density was regulated by a gap length of 2 μm-width stripes immobilized with heparin. Although a high density (a region having short gap length) of immobilized heparin suppressed the cell growth in the absence of bFGF, it enhanced cell growth in the presence of bFGF. The dependence of cell function on the density of immobilized heparin was visualized by gradient-micropattern-immobilization.

A novel germ line juxtamembrane Met mutation in human gastric cancer.

Activating mutations in the Met receptor tyrosine kinase, both germline and somatic, have been identified in human papillary renal cancer. Here we report a novel germline missense Met mutation, P1009S, in a patient with primary gastric cancer. The dosage of the mutant Met DNA was elevated in the tumor when compared to its matched normal DNA. Therefore, as with hereditary renal papillary cancer, the mutant Met allele may also be selectively duplicated in the tumor. Different from previously reported Met mutations, which occur in the tyrosine kinase domain, this missense mutation is located at the juxtamembrane domain, and is not constitutively activated. However, following treatment with HGF/SF, the P1009S mutant Met protein, expressed in NIH3T3 cells, displays increased and persistent tyrosine phosphorylation compared to the wild-type Met. Importantly, these cells also form colonies in soft agar, and are highly tumorigenic in athymic nude mice. A second nucleotide change in this region of Met, T1010I, was found in a breast cancer biopsy and a large cell lung cancer cell line. Although this previously reported 'polymorphism' did not stimulate NIH3T3 cell growth in soft agar, it was more active than the wild-type Met in the athymic nude mice tumorigenesis assay, suggesting that it may have effects on tumorigenesis. Met has been shown to be highly expressed in human gastric carcinoma cell lines, and our results raise the possibility that activating missense Met mutations could contribute to tumorigenesis of gastric cancer.

Platelet-derived Growth Factor (PDGF) Receptor-α Activates c-Jun NH2-terminal Kinase-1 and Antagonizes PDGF Receptor-β-induced Phenotypic Transformation

Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cells. The PDGF B-chain (c-sis proto-oncogene) homodimer (PDGF BB) and v-sis, its viral counterpart, activate both alpha- and beta-receptor subunits (alpha-PDGFR and beta-PDGFR) and mediate anchorage-independent growth in NIH3T3 cells. In contrast, the PDGF A chain homodimer (PDGF AA) activates alpha-PDGFR only and fails to induce phenotypic transformation. In the present study, we investigated alpha- and beta-PDGFR specific signaling pathways that are responsible for the differences between the transforming ability of PDGF AA and BB. To study PDGF BB activation of beta-PDGFR, we established NIH3T3 clones in which alpha-PDGFR signaling is inhibited by a dominant-negative alpha-PDGFR, or an antisense construct of alpha-PDGFR. Here, we demonstrate that beta-PDGFR activation alone is sufficient for PDGF BB-mediated anchorage-independent cell growth. More importantly, inhibition of alpha-PDGFR signaling enhanced PDGF BB-mediated phenotypic transformation, suggesting that alpha-PDGFR antagonizes beta-PDGFR-induced transformation. While both alpha- and beta-receptors effectively activate ERKs, alpha-PDGFR, but not beta-PDGFR, activates stress-activated protein kinase-1/c-Jun NH(2)-terminal kinase-1 (JNK-1). Inhibition of JNK-1 activity using a dominant-negative JNK-1 mutant markedly enhanced PDGF BB-mediated anchorage-independent cell growth, demonstrating an antagonistic role for JNK-1 in PDGF-induced transformation. Consistently, overexpression of wild-type JNK-1 reduced PDGF BB-mediated transformation. Taken together, the present study showed that alpha- and beta-PDGFRs differentially regulate Ras-mitogen-activated protein kinase pathways critical for regulation of cell transformation, and transformation suppressing activity of alpha-PDGFR involves JNK-1 activation.

Deletion of a short, untranslated region adjacent to the polypurine tract in Moloney murine leukemia virus leads to formation of aberrant 5' plus-strand DNA ends in vivo.

Experiments were performed to determine the function of a 28-nucleotide untranslated sequence lying between the envelope gene and the polypurine tract (PPT) sequence in the Moloney murine leukemia virus (Mo-MuLV) genome. A mutant virus carrying a deletion of this sequence (Mo-MuLVDelta28) replicated more slowly than wild-type (wt) virus and reverted by recombination with endogenous sequences during growth in NIH 3T3 cells. We show that this deletion did not affect the level of viral protein expression or genomic RNA packaging. Mo-MuLVDelta28 served as a helper virus as efficiently as the wt virus; in contrast, a retroviral vector harboring this mutation exhibited reduced transduction efficiency, indicating that the mutation acts not in trans but in cis. Analysis of acutely infected cells revealed that reduced levels of viral DNA were generated by reverse transcription of the Mo-MuLVDelta28 RNA as compared to the wt RNA. Analysis of DNA circle junctions revealed that plus-strand DNA of Mo-MuLVDelta28 but not wt virus often retained the PPT and additional upstream sequences. These structures suggest that aberrant 5' ends of plus-strand DNA were generated by a failure to remove the PPT RNA primer and/or by mispriming at sites upstream of the PPT. These data demonstrate that the major role of the sequences immediately upstream of the PPT is specifying efficient and accurate plus-strand DNA synthesis.

Exogenous E2F expression is growth inhibitory before, during, and after cellular transformation.

To gain insight into the tumor suppressor properties of E2F1, we investigated growth inhibition by the E2F family of transcription factors using a tissue culture model system. We first show that exogenous E2F expression causes an 80% decrease in NIH3T3 colony formation and activated c-Ha-Ras-mediated focus formation. Inhibition of Ras-mediated transformation was dependent upon E2F DNA binding activity but did not require amino- or carboxy-terminal E2F1 protein interaction domains. Because E2F upregulation has been suggested to be associated with a neoplastic phenotype, it was possible that increased E2F activity would not be inhibitory to previously transformed cells. However, we found that exogenous E2F was also inhibitory to growth of NIH3T3 cells previously transformed by Ras or Neu. Further characterization revealed that exogenous E2F expression is inhibitory at very early times after transfection, causing dramatic losses in transfected cell populations. Interestingly, those few cells which do establish appear to be unaffected by the overexpressed E2F. Therefore, we propose that increased E2F activity may only be tolerated in a subset of cells which have acquired specific alterations that are dominant over E2F-mediated growth inhibition.

Tunicamycin enhances the anticellular activity of interferon by inhibiting G1/S phase progression in 3T3 cells.

We have shown earlier that the cell growth inhibitory activity of interferon (IFN) is significantly enhanced by tunicamycin (TM) (Maheshwari et al., Science 219, 1339-1341, 1983). In this report, we investigated various regulatory points of synergistic action between TM and IFN-alpha/beta that inhibit cell growth in NIH 3T3 cells. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) viability assays showed a dose-dependent increase in percentage inhibition of the cells when treated with either TM or IFN. When doses of TM and IFN that had no significant inhibition on cell viability were used in combination, there was a pronounced suppression of DNA synthesis (tritiated thymidine incorporation). Flow cytometry studies revealed that individual treatments with either IFN or TM that did not alter the cell cycle profile, when combined, resulted in an impaired cell cycle by inhibiting G1/S progression. The blockage of G1/S transition was associated with reduction of cyclin-dependent kinase (CDK4) activity. The mRNA (analyzed by ribonuclease protection assay) and protein levels (assayed by Western blotting) of cyclins D1, D3, and CDK4 were downregulated by combined treatment with IFN and TM. An increase in the expression of p27/kipl, an inhibitor of CDK4, was observed in cells that were treated with both IFN and TM. These studies suggest that insufficient formation of the active cyclin/CDK complex could possibly be deferring the cells from normal cycling and may be responsible for the ability of TM to enhance cell growth inhibition induced by IFN.

Induction of Anchorage-independent Growth by Transforming Growth Factor-β Linked to Anchorage-independent Expression of Cyclin D1

Transforming growth factor-beta (TGF-beta) was originally identified, characterized, and named on the basis of its ability to induce anchorage-independent growth (phenotypic transformation). This effect has received little attention in recent years, probably because the induction of anchorage-independent growth by TGF-beta has been observed only in a few cell lines, of which NRK fibroblasts are among the best studied. We have previously reported that normal rat kidney cells have lost their normal adhesion requirement for expression of cyclin D1, and we now show that this loss is causal for the induction of anchorage-independent growth by TGF-beta. First, we show that TGF-beta fails to induce anchorage-independent growth in NIH-3T3 cells and human fibroblasts that have retained their adhesion requirement for expression of cyclin D1. Second, we show that TGF-beta complements rather than affects cyclin D-cdk4/6 kinase activity in NRK cells. Third, we show that forced expression of cyclin D1 in suspended 3T3 cells renders them susceptible to transformation by TGF-beta. These results may explain why the induction of anchorage-independent growth by TGF-beta is a rare event and yet also describe a molecular scenario in which the mesenchymal response to TGF-beta could indeed involve the acquisition of an anchorage-independent phenotype.

Inhibition of H-Ras transformation by the PTEN/MMAC1/TEP1 tumor suppressor gene.

The human PTEN/MMAC1/TEP1 (PTEN) tumor suppressor gene encodes a phosphatase with specificity towards the D3 phosphate of phosphatidylinositides. PTEN mutations have been reported in the endometrioid type of uterine tumors which are associated with frequent activations of the Ras oncogenes. In this study, we report the ability of PTEN to potently inhibit H-Ras induced morphological transformation and anchorage-independent growth in NIH3T3 cells. This novel activity of PTEN was correlated more with its ability to suppress the phosphatidylinositol 3-kinase (PI3-K)-dependent signaling cascade, but not the mitogen-activated protein kinase (MAPK) pathway. To define the minimal region in PTEN protein that is responsible for this anti-oncogenic activity, a panel of carboxyl-terminal truncation mutants was generated. While deletions of 4 and 33 amino acids do not have marked effects, removal of up to 68 amino acids drastically reduced the ability of PTEN to inhibit Ras transformation. The propensity of these mutants to suppress Ras transformation is correlated with their relative ability to dephosphorylate inositol (1,3,4,5)-tetrakisphosphate in vitro, and to suppress Akt kinase activity in cultured cells. In addition, we have evidence to suggest that the C-terminal region of PTEN contributes to the stability of the encoded gene product.

Targeting of K-Ras 4B by S- trans, trans-farnesyl thiosalicylic acid

Ras proteins regulate cell growth, differentiation and apoptosis. Their activities depend on their anchorage to the inner surface of the plasma membrane, which is promoted by their common carboxy-terminal S-farnesylcysteine and either a stretch of lysine residues (K-Ras 4B) or S-palmitoyl moieties (H-Ras, N-Ras and K-Ras 4A). We previously demonstrated dislodgment of H-Ras from EJ cell membranes by S- trans, trans-farnesylthiosalicylic acid (FTS), and proposed that FTS disrupts the interactions between the S-prenyl moiety of Ras and the membrane anchorage domains. In support of this hypothesis, we now show that FTS, which is not a farnesyltransferase inhibitor, inhibits growth of NIH3T3 cells transformed by the non-palmitoylated K-Ras 4B(12V) or by its farnesylated, but unmethylated, K-Ras 4B(12) CVYM mutant. The growth-inhibitory effects of FTS followed the dislodgment and accelerated degradation of K-Ras 4B(12V), leading in turn to a decrease in its amount in the cells and inhibition of MAPK activity. FTS did not affect the rate of degradation of the K-Ras 4B, SVIM mutant which is not modified post-translationally, suggesting that only farnesylated Ras isoforms are substrates for facilitated degradation. The putative Ras-recognition sites (within domains in the cell membrane) appear to tolerate both C 15 and C 20 S-prenyl moeities, since geranylgeranyl thiosalicylic acid mimicked the growth-inhibitory effects of FTS in K-Ras 4B(12V)-transformed cells and FTS inhibited the growth of cells transformed by the geranylgeranylated K-Ras 4B(12V) CVIL isoform. The results suggest that FTS acts as a domain-targeted compound that disrupts Ras–membrane interactions. The fact that FTS can target K-Ras 4B(12V), which is insensitive to inhibition by farnesyltransfarase inhibitors, suggests that FTS may target Ras (and other prenylated proteins important for transformed cell growth) in an efficient manner that speaks well for its potential as an anticancer therapeutic agent.

Anabolic effect of aminoterminally truncated fibroblast growth factor 4 (FGF4) on bone

Fibroblast growth factor 4 (FGF4), a member of the FGF family, plays several important roles in bone development during embryogenesis. Systemic administration of FGF4 increases bone mass in rats, which suggests the potential therapeutic usefulness of this growth factor in treatment for osteopenia and in bone regeneration. We investigated the length of FGF4 required to exert its anabolic effects, because this information may be useful in developing new molecules to mimic the effects of FGF4. Because the active site of FGF family molecules is in the carboxylterminal region, we produced aminoterminally truncated recombinant human FGF4s (rhFGF4s) of different sizes. Human FGF4 cDNA containing almost the full length of the coding region (573 bp, 191 amino acid residues) was inserted into pUC18 vector and then deleted from the 5′ end using the ExoIII system. Each of the deleted FGF4 cDNAs was subcloned into a pET29(+) expression vector. Differently sized recombinant proteins were expressed in the BL21(DE3)pLysS Escherichia coli strain and then purified. The growth-stimulative effects on NIH3T3 cells of each recombinant protein were examined by means of MTT colorimetric assay. Full-length and the shortened recombinant proteins, which stimulated NIH3T3 cell growth, were then subcutaneously administered into male ddY mice (6 weeks old) every day for 2 weeks. Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT). The rhFGF4 of 134 amino acid residues, the region homologous to other members of the FGF family, exerted a growth-stimulative effect on NIH3T3 cells comparable to the full-length version of FGF4; however, the shortest version, with 111 amino acid residues, showed a limited growth-stimulative effect. Systemic administration of the rhFGF4 of 134 amino acid residues increased the bone mineral density (BMD) of femurs at a dose of 0.1 mg/kg, which was comparable to that of the full-length rhFGF4. DEXA analysis, pQCT analysis, soft X-ray photos, and contact microradiographs revealed an increase in femoral trabecular bone in FGF4-treated animals; an increase in bone formation was also evident upon histomorphometric analysis. These results indicate that the region of FGF4 that is homologous to other FGF family members provides a sufficient anabolic effect in bone and that this recombinant protein is potentially useful as a therapeutic agent in bone.

The RACK1 Signaling Scaffold Protein Selectively Interacts with the cAMP-specific Phosphodiesterase PDE4D5 Isoform

The WD-repeat protein receptor for activated C-kinase (RACK1) was identified by its interaction with the cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4D5 in a yeast two-hybrid screen. The interaction was confirmed by co-immunoprecipitation of native RACK1 and PDE4D5 from COS7, HEK293, 3T3-F442A, and SK-N-SH cell lines. The interaction was unaffected by stimulation of the cells with the phorbol ester phorbol 2-myristate 3-acetate. PDE4D5 did not interact with two other WD-repeat proteins, beta'-coatomer protein and Gsbeta, in two-hybrid tests. RACK1 did not interact with other PDE4D isoforms or with known PDE4A, PDE4B, and PDE4C isoforms. PDE4D5 and RACK1 interacted with high affinity (Ka approximately 7 nM) [corrected] when they were expressed and purified from Escherichia coli, demonstrating that the interaction does not require intermediate proteins. The binding of the E. coli-expressed proteins did not alter the kinetics of cAMP hydrolysis by PDE4D5 but caused a 3-4-fold change in its sensitivity to inhibition by the PDE4 selective inhibitor rolipram. The subcellular distributions of RACK1 and PDE4D5 were extremely similar, with the major amount of both proteins (70%) in the high speed supernatant (S2) fraction. Analysis of constructs with specific deletions or single amino acid mutations in PDE4D5 demonstrated that a small cluster of amino acids in the unique amino-terminal region of PDE4D5 was necessary for its interaction with RACK1. We suggest that RACK1 may act as a scaffold protein to recruit PDE4D5 and other proteins into a signaling complex.

Suppression of Ras-mediated NIH3T3 transformation by p19ARF does not involve alterations of cell growth properties

The INK4a gene, one of the most frequently disrupted loci in human cancer, encodes two unrelated proteins, p16INK4a and p19ARF, that both block cell proliferation. p16INK4a is a component of the Rb regulatory pathway, while p19ARF has been functionally related to p53. Moreover, p16INK4a is inactivated in many human tumors, while it has been very recently reported that p19ARF null mice develop tumors early in life. We show here that p19ARF is able to inhibit the formation of G418-resistant colonies when transfected into human and mouse cell lines expressing wild-type p53, regardless of p16 status. Moreover its amino terminal domain encoded by exon 1beta is still sufficient to obtain the same effect. We have analysed the ability of p19ARF to interfere with Ras-mediated cellular transformation in the NIH3T3 cell line. Cotransfection of p19ARF together with activated ras potently inhibited the formation of transformed foci in a dose-dependent manner. We have also isolated stable NIH3T3 transfectants expressing p19ARF and we have measured their growth properties as well as their efficiency of transformation by activated ras. Our results suggest that p19ARF can interfere with oncogene-mediated transformation, without significantly affecting NIH3T3 cell growth, at least at the levels of expression achieved in these experiments.

Fragile Sites—Cytogenetic Similarity with Molecular Diversity

The biological significance of chromosomal fragility is not easily generalized among fragile loci. Two of the rare fragile sites on the X chromosome are manifestations of disease (mental retardation)–producing dynamic mutations, whereas the third rare fragile site on this chromosome, FRAXF, is not found within any known gene and does not lead to any obvious phenotypic abnormality. The autosomal fragile sites of this type may be associated with transcriptional silencing of genes in which they might be located. Breakage at fragile sites may be capable of producing chromosomal deletion syndromes, as perhaps occurs when FRA11B breakage leads to Jacobsen syndrome (Jones et al. 1995xAssociation of a chromosome deletion syndrome with a fragile site within the proto-oncogene CBL2. Jones, C, Penny, L, Mattina, T, Yu, S, Baker, E, Voullaire, L, Langdon, WY et al. Nature. 1995; 376: 145–149Crossref | PubMedSee all References1995). It is of interest that none of the rare folate-sensitive fragile sites has been identified in species other than humans, yet repeats that are potentially capable of expansion to fragile sites have been identified in other species.Fragile sites are probably all associated with localized genomic instability; although this instability might lead only to the “gap” seen in cytogenetic preparations, it is also possible that the consequences are more significant. There is growing evidence that some common fragile sites predispose their surrounding region to the localized chromosomal instability seen in certain cancers. FRA3B is one such region of instability, and abnormal transcripts of the FHIT gene in which it is located are found in a range of tumor and normal cell types (Siprashvili et al. 1997xReplacement of FHIT in cancer cells suppresses tumorigenicity. Siprashvili, Z, Sozzi, G, Barnes, LD, McCue, P, Robinson, AK, Eryomin, V, Sard, L et al. Proc Natl Acad Sci USA. 1997; 94: 13771–13776Crossref | PubMed | Scopus (323)See all References1997; Carapeti et al. 1998xAberrant transcripts of the FHIT gene are expressed in normal and leukaemic haemopoietic cells. Carapeti, M, Aguiar, RC, Sill, H, Goldman, JM, and Cross, NC. Br J Cancer. 1998; 78: 601–605Crossref | PubMedSee all References1998; Otterson et al. 1998xProtein expression and functional analysis of the FHIT gene in human tumor cells. Otterson, GA, Xiao, GH, Geradts, J, Jin, F, Chen, WD, Niklinska, W, Kaye, FJ et al. J Natl Cancer Inst. 1998; 18: 426–432CrossrefSee all References1998). The human caveolin genes CAV1 and CAV2 are located in the vicinity of FRA7G, which is frequently deleted in human cancers (Engelman et al. 1998bxGenes encoding human caveolin-1 and -2 are co-localized to the D7S522 locus (7q31.1), a known fragile site (FRA7G) that is frequently deleted in human cancers. Engelman, JA, Zhang, XL, and Lisanti, MP. FEBS Lett. 1998b; 436: 403–410Abstract | Full Text | Full Text PDF | PubMed | Scopus (174)See all References1998b). Caveolin-1 has been shown to have a role in the anchorage-dependent inhibition of growth in NIH 3T3 cells (Galbiati et al. 1998xTargeted downregulation of caveolin-1 is sufficient to drive cell transformation and hyperactivate the p42/44 MAP kinase cascade. Galbiati, F, Volonte, D, Engelman, JA, Watanabe, G, Burk, R, Pestell, RG, and Lisanti, MP. EMBO J. 1998; 17: 6633–6648Crossref | PubMedSee all References1998). The caveolins are therefore candidates for the tumor-suppressor gene presumed to be located in the FRA7G region (Engelman et al. 1998axMolecular genetics of the calveolin gene family: implications for human cancers, diabetes, Alzheimer disease, and muscular dystrophy. Engelman, JA, Zhang, X, Galbiati, F, Volonte, D, Sotgia, F, Pestell, RG, Minetti, C et al. Am J Hum Genet. 1998a; 63: 1578–1587Abstract | Full Text | Full Text PDF | PubMed | Scopus (150)See all References1998a). By similar mechanisms, fragile sites may also lead to gene amplification in tumors. The noncompacted state of the DNA at fragile sites may also facilitate the integration of viruses into the genome.

Stat3 Plays an Important Role in Oncogenic Ros- and Insulin-like Growth Factor I Receptor-induced Anchorage-independent Growth

The role of signal transducers and activators of transcription (STATs) in receptor protein-tyrosine kinase (PTK)-induced cell growth and transformation was investigated using an inducible epidermal growth factor receptor-Ros chimeric receptor called ER2 and a constitutively activated insulin-like growth factor I receptor called NM1, both of which are able to induce anchorage-independent growth of NIH 3T3 cells. ER2 and NM1 receptor PTKs are able to cause Stat3 activation. Co-expressing the dominant negative Stat3 mutant with ER2 or NM1 in transiently or stable transfected cells resulted in a dramatic inhibition of colonies induced by these receptor PTKs and a moderate inhibition of their mitogenicity in monolayer. Therefore, Stat3 is not only important for initiation of transformation, as demonstrated by inhibition of the epidermal growth factor-inducible colony formation of the ER2 cells by the mutant, but it is also required for the maintenance of transformation, as evidenced by reversion of the NM1 transformed cells. The DNA binding and transcriptional activities of the endogenous Stat3 were greatly inhibited in the ER2 and NM1 cells co-expressing the Stat3 mutants. We conclude that activated function of Stat3 is required for the establishment and maintenance of Ros and insulin-like growth factor I receptor PTK-induced cell transformation.

The Role of v-Fgr Myristoylation and the Gag Domain in Membrane Binding and Cellular Transformation

Thev-fgroncogene encodes a chimeric oncoprotein composed of feline sarcoma virus (FeSV)-derived gag and cellular-derived actin and c-Fgr sequences. v-Fgr is myristoylated and membrane bound, two criteria which must be met forsrckinases to induce cellular transformation. Although inhibition of myristoylation resulted in a decreased ability of v-Fgr to sediment with membranes from an NIH-3T3 P100 fraction, deletion of the gag domain caused nearly all of the protein to remain unbound and cytosolic. Systematic deletions within gag indicate that while amino acids 3 through 9 are critical determinants of myristoylation and/or define a domain which directs membrane localization, these residues cooperate with additional gag sequences when anchoring the protein to the plasma membrane. Furthermore, nonmyristoylated and/or cytoplasmic variants of v-Fgr failed to induce anchorage-independent growth of NIH-3T3 cells, indicating that proper subcellular localization of v-Fgr is a key factor in its ability to induce transformation.

RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, inhibits activity of src tyrosine kinases and growth of NIH 3T3 cells.

To isolate and characterize proteins that interact with the unique domain and SH3 and SH2 domains of Src and potentially regulate Src activity, we used the yeast two-hybrid assay to screen a human lung fibroblast cDNA library. We identified RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, as a Src-binding protein. Using GST-Src fusion proteins, we determined that RACK1 binds to the SH2 domain of Src. Coimmunoprecipitation of Src and RACK1 was demonstrated with NIH 3T3 cells. Purified GST-RACK1 inhibited the in vitro kinase activity of Src in a concentration-dependent manner. GST-RACK1 (2 microM) inhibited the activities of purified Src and Lck tyrosine kinases by 40 to 50% but did not inhibit the activities of three serine/threonine kinases that we tested. Tyrosine phosphorylation on many cellular proteins decreased in 293T cells that transiently overexpressed RACK1. Src activity and cell growth rates decreased by 40 to 50% in NIH 3T3 cells that stably overexpressed RACK1. Flow cytometric analyses revealed that RACK1-overexpressing cells do not show an increased rate of necrosis or apoptosis but do spend significantly more time in G0/G1 than do wild-type cells. Prolongation of G0/G1 could account for the increased doubling time of RACK1-overexpressing cells. We suggest that RACK1 exerts its effect on the NIH 3T3 cell cycle in part by inhibiting Src activity.

Ras signals to the cell cycle machinery via multiple pathways to induce anchorage-independent growth.

Several specific cell cycle activities are dependent on cell-substratum adhesion in nontransformed cells, and the ability of the Ras oncoprotein to induce anchorage-independent growth is linked to its ability to abrogate this adhesion requirement. Ras signals via multiple downstream effector proteins, a synergistic combination of which may be required for the highly altered phenotype of fully transformed cells. We describe here studies on cell cycle regulation of anchorage-independent growth that utilize Ras effector loop mutants in NIH 3T3 and Rat 6 cells. Stable expression of activated H-Ras (12V) induced soft agar colony formation by both cell types, but each of three effector loop mutants (12V,35S, 12V,37G, and 12V,40C) was defective in producing this response. Expression of all three possible pairwise combinations of these mutants synergized to induce anchorage-independent growth of NIH 3T3 cells, but only the 12V,35S-12V,37G and 12V,37G-12V,40C combinations were complementary in Rat 6 cells. Each individual effector loop mutant partially relieved adhesion dependence of pRB phosphorylation, cyclin E-dependent kinase activity, and expression of cyclin A in NIH 3T3, but not Rat 6, cells. The pairwise combinations of effector loop mutants that were synergistic in producing anchorage-independent growth in Rat 6 cells also led to synergistic abrogation of the adhesion requirement for these cell cycle activities. The relationship between complementation in producing anchorage-independent growth and enhancement of cell cycle activities was not as clear in NIH 3T3 cells that expressed pairs of mutants, implying the existence of either thresholds for these activities or additional requirements in the induction of anchorage-independent growth. Ectopic expression of cyclin D1, E, or A synergized with individual effector loop mutants to induce soft agar colony formation in NIH 3T3 cells, cyclin A being particularly effective. Taken together, these data indicate that Ras utilizes multiple pathways to signal to the cell cycle machinery and that these pathways synergize to supplant the adhesion requirements of specific cell cycle events, leading to anchorage-independent growth.