Transforming Activity In NIH 3T3 Cells Research Articles

Regulation of Src tumor activity by its N-terminal intrinsically disordered region

The membrane-anchored Src tyrosine kinase is involved in numerous pathways and its deregulation is involved in human cancer. Our knowledge on Src regulation relies on crystallography, which revealed intramolecular interactions to control active Src conformations. However, Src contains a N-terminal intrinsically disordered unique domain (UD) whose function remains unclear. Using NMR, we reported that UD forms an intramolecular fuzzy complex involving a conserved region with lipid-binding capacity named Unique Lipid-Binding Region (ULBR), which could modulate Src membrane anchoring. Here we show that the ULBR is essential for Src’s oncogenic capacity. ULBR inactive mutations inhibited Src transforming activity in NIH3T3 cells and in human colon cancer cells. It also reduced Src-induced tumor development in nude mice. An intact ULBR was required for MAPK signaling without affecting Src kinase activity nor sub-cellular localization. Phospho-proteomic analyses revealed that, while not impacting on the global tyrosine phospho-proteome in colon cancer cells, this region modulates phosphorylation of specific membrane-localized tyrosine kinases needed for Src oncogenic signaling, including EPHA2 and Fyn. Collectively, this study reveals an important role of this intrinsically disordered region in malignant cell transformation and suggests a novel layer of Src regulation by this unique region via membrane substrate phosphorylation.

AKT1 mutations in bladder cancer: identification of a novel oncogenic mutation that can co-operate with E17K

The phosphatidylinositol-3-kinase (PI3 kinase)-AKT pathway is frequently activated in cancer. Recent reports have identified a transforming mutation of AKT1 in breast, colorectal, ovarian and lung cancers. We report here the occurrence of this mutation in bladder tumours. The AKT1 G49A (E17K) mutation was found in 2/44 (4.8%) bladder cancer cell lines and 5/184 (2.7%) bladder tumours. Cell lines expressing mutant AKT1 show constitutive AKT1 activation under conditions of growth factor withdrawal. We also detected a novel AKT1 mutation G145A (E49K). This mutation also enhances AKT activation and shows transforming activity in NIH3T3 cells, though activity is weaker than that of E17K. Enhanced activation of AKT1 when E17K and E49K mutations are in tandem suggests that they can co-operate.

TEL/AML1 shows dominant-negative effects over TEL as well as AML1

The TEL/AML1 chimeric gene is generated by the t(12;21) translocation in pre-B cell acute lymphoblastic leukemia. TEL/AML1 consists of the helix–loop–helix (HLH) dimerization domain from TEL and almost the entire of AML1, but loses the ETS DNA-binding domain from TEL. Dominant-negative effects of TEL/AML1 over wild-type-AML1 are believed to trigger the development of this type of leukemia. However, it could also be possible that TEL/AML1 affects wild-type-TEL’s molecular and tumor suppressive functions through the HLH domain. To test this possibility, we first confirmed that TEL/AML1 associates with wild-type-TEL. TEL/AML1 neither bound to the ETS-binding consensus site nor repressed transcription through it. Regardless, this prevented wild-type-TEL-induced transcriptional repression. Moreover, TEL/AML1 concomitantly inhibited wild-type-TEL-induced growth suppression and wild-type-AML1-mediated transforming activity in NIH3T3 cells. All these data indicate that TEL/AML1 exerts dominant-interfering effects on both AML1 and TEL, and that expression of TEL/AML1 could result in inactivation of TEL’s tumor suppressive functions in t(12;21)-carrying leukemia.

Chondrocyte-derived ezrin-like domain containing protein (CDEP), a rho guanine nucleotide exchange factor, is inducible in chondrocytes by parathyroid hormone and cyclic AMP and has transforming activity in NIH3T3 Cells

Objective The purpose of this study is to investigate stage- and hormone-dependent expression of chondrocyte-derived ezrin-like domain containing protein (CDEP), a putative guanine nucleotide exchange factor (GEF) for Rho in chondrocytes, and demonstrate the guanine nucleotide exchange activity of this protein in vitro, as well as the transforming activity in NIH3T3 cells. Methods The expression of CDEP mRNA in growth plate chondrocytes in vivo and in vitro was examined by RT-PCR Southern analysis. The guanine nucleotide exchange activity was determined using a recombinant CDEP peptide containing the DH and PH domains in Sf9 cell lysates. The transforming activity was examined using NIH3T3 cells transiently transfected with a truncated CDEP cDNA. Results CDEP mRNA was expressed at the highest level in the hypertrophic (terminal) stage of chondrocytes in vivo and in vitro. Parathyroid hormone (PTH) elicited a biphasic increase of CDEP mRNA in chondrocytes. The CDEP mRNA level increased within 1 h, then decreased nearly to the control level at 3 h. Thereafter the mRNA level started to increase at 6 h, reaching a plateau at 24 h. Dibutyryl cyclic AMP had a similar effect on CDEP expression in chondrocytes. The dissociation of [ 3H]GDP from RhoA was stimulated dose-dependently by Sf9 cell lysates containing the CDEP peptide. Furthermore, transfection of a truncated CDEP cDNA induced focus formation in NIH3T3 cultures. Conclusions CDEP is a novel GEF for Rho family GTPases with the transforming activity. CDEP may play a role in mediating or modulating the action of cAMP-elevating hormones on maturing chondrocytes.

MET(PRC) mutations in the Ron receptor result in upregulation of tyrosine kinase activity and acquisition of oncogenic potential.

Ron and Met are structurally related receptor tyrosine kinases that elicit a complex biological response leading to invasive growth. Naturally occurring point mutations activate the Met kinase in papillary renal carcinomas (MET(PRC) mutations). By site-directed mutagenesis, we generated homologous amino acid substitutions in the Ron kinase domain and analyzed the biochemical and biological properties of the mutant receptors. Among the mutations studied, D(1232)H and M(1254)T displayed transforming activity in NIH3T3 cells, inducing focus formation and anchorage-independent growth. The D(1232)H and M(1254)T substitutions resulted in increased Ron autophosphorylation both in vivo and in vitro and constitutive binding to intracellular signal transducers. Both mutations yielded a dramatic increase in catalytic efficiency, indicating a direct correlation between kinase activity and oncogenic potential. Molecular modeling of the Ron D(1232)H mutation suggests that this single amino acid substitution favors the transition of the kinase from the inactive to the active state. These data demonstrate that point mutations can confer transforming activity to the Ron receptor and show that RON is a potential oncogene.

Transforming activity of the human mammary line HBL100 DNA is associated with SV40 large T antigen genetic information integrated in its genome

The HBL100 cell line, established in vitro from milk of an apparently healthy woman, expresses SV40 large T antigen defective in some of its functions. Here we demonstrate that the HBL100 genomic DNA possesses transforming activity in NIH3T3 cells and that this activity is associated with the viral information.