Regulation of mTORC1 signaling by Src kinase activity is Akt1-independent in RSV-transformed cells.

Increased activity of the Src tyrosine protein kinase that has been observed in a large number of human malignancies appears to be a promising target for drug therapy. In the present study, a critical role of the Src activity in the deregulation of mTOR signaling pathway in Rous sarcoma virus (RSV)-transformed hamster fibroblasts, H19 cells, was shown using these cells treated with the Src-specific inhibitor, SU6656, and clones of fibroblasts expressing either the active Src or the dominant-negative Src kinase-dead mutant. Disruption of the Src kinase activity results in substantial reduction of the phosphorylation and activity of the Akt/protein kinase B (PKB), phosphorylation of tuberin (TSC2), mammalian target of rapamycin (mTOR), S6K1, ribosomal protein S6, and eukaryotic initiation factor 4E-binding protein 4E-BP1. The ectopic, active Akt1 that was expressed in Src-deficient cells significantly enhanced phosphorylation of TSC2 in these cells, but it failed to activate the inhibited components of the mTOR pathway that are downstream of TSC2. The data indicate that the Src kinase activity is essential for the activity of mTOR-dependent signaling pathway and suggest that mTOR targets may be controlled by Src independently of Akt1/TSC2 cascade in cells expressing hyperactive Src protein. These observations might have an implication in drug resistance to mTOR inhibitor-based cancer therapy in certain cell types.

Inhibition of MITF transcriptional activity independent of targeting p300/CBP coactivators.

Microphthalmia-associated transcription factor (MITF) activates the expression of melanocyte-specific markers and promotes the survival of embryonic, adult and malignant melanocytes. Although numerous MITF-dependent downstream genes have been identified, the mechanisms by which the MITF activity is coregulated remain elusive. Here we used a non-melanocytic cell line U2-OS as a model in which MITF evokes transcription of a paradigmatic MITF target tyrosinase and show that the adenoviral E1A protein represses the MITF-driven transcription in these cells. The E1A CR1 domain (which alone is insufficient to bind p300) was sufficient for repression, while the N-terminus, through which E1A binds the p300/CBP proteins and other coactivators, was unable to repress. Correspondingly, CR1 inhibited colony formation of MITF-positive, but not MITF-negative, melanoma cells. The repression by CR1 was largely independent of the PCAF-binding motif, previously recognized to be necessary for suppression of muscle-specific enhancer. Interestingly, CR1 conferred transcriptional competence to the MITF-CR1 chimera in which the MITF portion was rendered transcription-deficient. Moreover, MITF mutants defective in binding to p300/CBP in vivo still activated transcription, further supporting a p300/CBP-independent coactivation of MITF targets. MITF is amplified in a subset of melanomas and is thought to be required for sustained proliferation of malignant melanocytes. Our results suggest that understanding how CR1 represses Mitf activity may reveal a route to melanoma therapy.

Regulation of c-Src activity by the expression of wild-type v-Src and its kinase-dead double Y416F-K295N mutant.

Active, wild-type v-Src and its kinase-dead double Y416F-K295N mutant were expressed in hamster fibroblasts. Expression of the active v-Src induced activation of endogenous c-Src and increased general protein-tyrosine phosphorylation in the infected cells. Expression of the kinase-dead mutant induced hypophosphorylation of Tyr416 of the endogenous c-Src. The inactivation of c-Src was reversible, as confirmed by in vitro kinase activity of c-Src immunoprecipitated from the kinase-dead v-Src-expressing cells. Both activation and inactivation of c-Src may be explained by direct interaction of the v-Src and c-Src that may either facilitate transphosphorylation of the regulatory Tyr416 in the activation loop, or prevent it by formation of transient dead-end complexes of the Y416F-K295N mutant with c-Src. The interaction was also indicated by co-localization of v- and c-Src proteins in immunofluorescent images of the infected cells. These results suggest that dimerization of Src plays an important role in the regulation of Src tyrosine kinase activity.

Characterization of four clones derived from human adenocarcinoma cell line, HT29, and analysis of their response to sodium butyrate.

The differentiation of colorectal cancer cells is associated with the arrest of tumor growth and tumor regression. However, the mechanism of such tumor cell differentiation has not yet been elucidated. Several adenocarcinoma cell lines, including HT29 which differentiates only upon stimulation with a differentiation agent, have been used for the study of colorectal cells. Since we had previously obtained variable results during analyses of these cells, we selected several clones of this cell line. In this study, four clones of the parental HT29 cells, H8, G9, G10 and A3, were characterized. All of them differentiated upon treatment with sodium butyrate as the differentiation agent but they appeared different in their response regarding some of the markers of differentiation. As revealed by ultrastructural analysis, H8 and G10 clones formed numerous intercellular cysts with microvilli whereas these structures were found only occasionally in G9 and A3 clones. An elevated level of the indicator of cell differentiation, CEACAM 1, was found in H8 and G10 clones and the activity of alkaline phosphatase, another important marker of colorectal cell differentiation, was up-regulated and highly increased upon butyrate treatment in the H8 clone. Phosphorylation of p38 MAPK was increased in H8 and A3 butyrate-treated clones. According to the levels of cleaved PARP and activated caspase-3, the apoptotic response to butyrate appeared similar in all four clones, while electronoptic analysis revealed that clones G9 and A3 were more perceptive to butyrate-induced apoptosis. In conclusion, our data showed considerable heterogeneity in morphology and some enzymatic activity of the cloned cells. This fact may contribute to the evidence that many HT29 cells possess multipotent information similar to that of stem cells of the normal intestinal crypt.

Expression of beta-catenin is regulated by PI-3 kinase and sodium butyrate in colorectal cancer cells.

beta-catenin has a dual function; it is implicated in intercellular junctions and transcriptional co-activation. Here we examined the regulation of the expression and localization of beta-catenin in HT29 colorectal adenocarcinoma cells. Our results showed that inhibition of PI-3 kinase with wortmannin was accompanied by a considerably reduced expression of beta-catenin. This effect was overcome by butyrate and occurred at the protein level, not at the level of mRNA. Moreover, NaBT significantly increased the phosphorylation of the ribosomal protein, S6, known to participate in the translational control of gene expression. This was accompanied by the increased phosphorylation of p70 S6K and MAPKs, the effector proteins that are upstream of protein S6 in the distinct signaling pathways. These facts indicate that different signaling pathways may be involved in the regulation of beta-catenin synthesis. Modulation of beta-catenin expression induced by NaBT appeared to occur at the level of protein translation, suggesting that NaBT may act as a translational regulator.

Response of HT115, a highly invasive human colorectal adenocarcinoma cell line, to sodium butyrate treatment and glucose deprivation.

Sodium butyrate or glucose deprivation induce a more differentiated phenotype in many cancer cells. The aim of this study was to determine whether the induction effect of butyrate and/or glucose deprivation is dependent, in some way, on the differentiation state of individual cell lines. Sodium butyrate enhanced alkaline phosphatase activity and induced formation of an ultrastructurally more differentiated phenotype in both HT29 and HT115 cell lines. Interestingly, the more invasive HT115 cells responded more strongly to butyrate treatment. On the other hand, the differentiation effect of glucose deprivation was much less prominent in the HT115 cell line in comparison with HT29 cells. Our data confirm the influence of the malignant potential of the cells on their response to treatment with differentiation and apoptosis-inducing agents. Butyrate treatment also enhanced the adhesiveness of HT115 cells. Since E-cadherin was not found in these cells, while the level of CEACAM1 was increased, it is obvious that the CEACAM1 molecules are involved in HT115 cell-cell adhesion.

Sodium butyrate-mediated differentiation of colorectal cancer cells: regulation of PKCbetaII by PI 3-kinase.

The present study focuses on a putative regulation of PKCbetaII by phosphatidylinositol-3 kinase (PI 3-kinase) in colorectal carcinoma cells; little is known about the role and activity of PKCbetaII in these cells. We examined the activity of PI 3-kinase in two adenocarcinoma cell lines, HT29 cells that differentiate only after stimulation with appropriate agents, and Caco-2 cells that can differentiate spontaneously. The activity of PI 3-kinase as well as the activity of PKCbetaII appeared to decrease only in HT29 cells in which differentiation was induced by sodium butyrate. In HT29 cells infected with recombinant adenovirus encoding constitutively active PI 3-kinase, the activity of alkaline phosphatase was almost completely blocked, and this PI 3-kinase significantly potentiated the activity of PKCbetaII in HT29 cells despite the presence of NaBT in the culture medium. On the contrary, in differentiating Caco-2 cells, the activity of PI 3-kinase was not butyrate-sensitive. In agreement with these findings, the alkaline phosphatase activity was not affected by constitutively active PI 3-kinase overexpressed in Caco-2 cells. These observations suggest that PKCbetaII is regulated by PI 3-kinase in HT29 cells and that the mechanisms of spontaneous differentiation versus butyrate-induced differentiation of adenocarcinoma cells may be different.

Transactivation of E-cadherin is not involved in the activity of EGF receptor in colorectal carcinoma cells.

In epithelial cells, the cell surface glycoprotein E-cadherin is a key molecule in the establishment of cell-cell adhesion. In addition to its contribution to cell adhesion, E-cadherin was found to induce ligand-independent activation of the EGF receptor (EGFR), likely as a result of their co-clustering. As it has also been reported that ligand activation of the overexpressed EGFRs disturb E-cadherin-mediated cell-cell adhesion, we analyzed E-cadherin-EGFR interactions and their consequences in A431 cells and in two colorectal cancer cell lines using immunoblotting and analyzes of several protein kinase activities. Activation of the PI3-K/Akt/GSK-3 signaling pathway upon EGF treatment that we observed in the analyzed cells indicates that EGFRs are functional even in the colorectal cancer cells containing a low density of EGFRs. The transactivation of EGFR by E-cadherin did not occur either in the colorectal cancer cells tested or in A431 cells containing a high density of both EGFRs and E-cadherin on their surface. This observation suggests that high amounts of both molecules on the surface of tumour cells did not predetermine ligand-independent activation of EGFRs.

IL2-dependent phosphorylation of 40S ribosomal protein S6 is controlled by PI-3K/mTOR signalling in CTLL2 cells.

Growth factors and hormones activate global and selective protein translation by phosphorylation and therefore activation of p70 S6 kinase through a wortmannin-sensitive phosphoinositide-3 kinase (PI-3K) antiapoptotic pathway and a rapamycin-sensitive signalling pathway of mTOR. Here we demonstrate that the phosphorylation of 40S ribosomal protein S6, a physiological substrate p70 S6 kinase, was highly increased by growth-stimulation of the cytolytic T cells (CTLL2) with interleukin 2 (IL2), which was accompanied with the increased phosphorylation of p70 S6K. The activity of p70 S6K and phosphorylation of the S6 protein was completely blocked by rapamycin and significantly decreased upon treatment of the cells with wortmannin, indicating an involvement of the PI-3K pathway in concert with the signalling pathway of mTOR in IL2-dependent phos-phorylation of ribosomal protein S6. The phosphorylation and activity of PKB/Akt in IL2-stimulated CTLL2 cells were rapamycin-insensitive and reduced upon wortmannin treatment of the cells, confirming a requirement for PI-3K for Akt activity. The data support the hypothesis that Akt may act downstream to PI-3K and upstream to mTOR in an IL2-mediated signal transduction pathway that controls phosphorylation of the regulatory protein S6 in CTLL2 cells.

The v-Src and c-Src tyrosine kinases immunoprecipitated from Rous sarcoma virus-transformed cells display different peptide substrate specificities.

In the cells transformed by Rous sarcoma virus (RSV), two Src proteins are expressed: the ubiquitous tyrosine kinase c-Src and the v-Src, the product of the transforming gene of the virus. Using three synthetic peptide substrates widely used for testing Src kinase activity, we show that they are phosphorylated with different efficiencies by the v-Src and c-Src tyrosine kinases immunoprecipitated from the tumor cell line H19. The v-Src displays higher efficiency (Vmax/Km ratio) toward all three peptides used, but the Vmax of v-Src is much lower than Vmax of c-Src with two peptides out of three. This difference in substrate specificity, if ignored, may cause misestimation of the amounts of active c-Src and v-Src in RSV-transformed cells. On the other hand, the different peptide substrate specificities may also reflect different protein substrate specificities of the v-Src and c-Src kinases in vivo.

Alterations in mitochondria function and morphology in HT29 cells upon conditions inducing differentiation and apoptosis.

Morphological and biochemical studies of HT29 cells treated with sodium butyrate and/or glucose-deprived revealed both apoptotic and differentiation response. The main apoptotic response was accompanied with an increase of floating cells. However, the ultrastructural analysis of adherent cells showed the typical apoptotic character of the nucleus in some of them. In addition, remarkable changes of mitochondria, assumed as early stages starting the apoptotic cascade, were observed. These changes were represented not only by alterations of mitochondrial morphology, but also by the number of mitochondria and their localization.

Initiation factor eIF2B not p70 S6 kinase is involved in the activation of the PI-3K signalling pathway induced by the v-src oncogene.

Our data show that in hamster fibroblasts transformed by Rous sarcoma virus (RSV), the phosphoinositide 3'-kinase (PI-3K)/Akt/glycogen synthase kinase 3 antiapoptotic pathway is upregulated and involved in increased protein synthesis through activation of initiation factor eIF2B. Upon inhibition of PI-3K by wortmannin, phosphorylation of 70-kDa ribosomal protein S6 kinase (p70 S6k) and its physiological substrate, ribosomal protein S6, decreased in the non-transformed cells but not in RSV-transformed cells. Thus PI-3K, which is thought to be involved in regulation of p70 S6k, signals to p70 S6k in normal fibroblasts, but it does not appear to be an upstream effector of p70 S6k in fibroblasts transformed by v-src oncogene, suggesting that changes in the PI-3K signalling pathway upstream of p70 S6k are induced by RSV transformation.

Response of HT29 cells to butyrate depends on time of exposure and glucose deprivation.

The high level of alkaline phosphatase activity in HT29 cells induced after 2 or 5 days of butyrate treatment was decreased during their prolonged exposure (about 30 days) to this agent together with a decrease of sensitivity to apoptosis. However, an enormous additive effect on alkaline phosphatase activity was found after butyrate treatment of glucose-starved cells. In concert with this finding, the substructural analysis revealed a dense brush border, tendency to polarization and morphologically normal mitochondria. It can be concluded that prolonged butyrate treatment of HT29 cells attenuated their response to this agent. On the other hand, glucose deprivation, as another inductor of differentiation, was found to increase the sensitivity of HT29 cells to butyrate.

Increased activity of c-Src and Csk in fibroblasts transformed by v-src oncogene.

When c-Src and v-Src were immunoprecipitated together from hamster fibroblasts transformed by Rous sarcoma virus containing v-src oncogene, the total Src activity was almost threefold higher compared to c-Src activity in the control cells. The activity of v-Src immunoprecipitated separately, however, accounting for only 40% of the total Src activity, indicating that c-Src is activated upon transformation. An increased activity of Csk was also found in RSV-transformed cells. It decreased upon serum stimulation in parallel with an increase in Src kinase activity. In nontransformed cells, serum stimulation induced an enhanced Csk activity, but no changes in c-Src activity were observed. This may suggest that Csk may have more functions in hamster fibroblasts, in addition to its inhibitory effect on c-Src.