Activation of T cells induces rapid tyrosine phosphorylation on the T-cell receptor ; chain and other substrates. These phosphorylations can be regulated by a number of protein-tyrosine kinases (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosinephosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3. 1.3.48). In this study, we demonstrate that phenylarsine oxide can inhibit tyrosine phosphatases while leaving tyrosine kinase function intact. We use this reagent to investigate the effect of tyrosine phosphatase inhibition in a murine T-cell hybridoma. Increasing concentrations of phenylarsine oxide result in an increase in tyrosine phosphate on a number of intracellular substrates in unstimulated T cells, suggesting that a protein-tyrosine kinase is constitutively active in these cells. The effect of phenylarsine oxide on T cells stimulated with an anti-Thy 1 monoclonal antibody is more complex. At low concentrations of drug, there is a synergistic increase in the level of tyrosine phosphate on certain cellular substrates. At higher concentrations, anti-Thy 1-stimulated tyrosine phosphorylation is inhibited. These results indicate that tyrosine phosphorylation in T cells is tightly regulated by tyrosine phosphatases. Partial inhibition of these enzymes results in enhanced substrate phosphorylation. Inhibition of all stimulated tyrosine phosphorylation by high doses of phenylarsine oxide suggests that tyrosine kinase activity is regulated by tyrosine phosphatases.Over the past decade there has been intense study devoted to understanding cellular tyrosine phosphorylation. By investigation of normal and transformed cells, it now appears that protein-tyrosine kinases (PTIK; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) regulate such critical cellular functions as growth, differentiation, and signal transduction (1, 2). Our laboratory has been interested in the function and regulation of tyrosine phosphorylation in T lymphocytes. We demonstrated that activation of T cells by a variety of means results in tyrosine phosphorylation of the T-cell antigen receptor (TCR) on the TCR chain (3-5). Furthermore, we have observed that other cellular substrates are phosphorylated on tyrosine more rapidly than the TCR ; chain (5, 6). Phosphorylation of these proteins can be detected by immunoblotting with specific anti-phosphotyrosine antibodies within seconds of TCR engagement.The PTKs responsible for phosphorylation of TCR r and these other substrates have not been conclusively identified. Since the TCR subunits lack the necessary amino acid sequences that define kinases it has been assumed that the TCR must be activating a nonreceptor kinase such as a member of the src PTK family. In fact, T cells have been shown to express Ick, fyn, and yes PTKs (7,8). The PTK Ick has been demonstrated to be noncovalently associated with the CD4 and CD8 molecules, which are T-cell-specific glycoprot...

The results presented here demonstrate that p53 upregulates estrogen receptor-(ER ) expression in the human breast cancer cell line MCF-7. Two approaches were used to alter the activity of p53 in the cells. In the first approach, stable transfectants expressing an antisense p53 were established. In the stable clones, expression of antisense p53 resulted in a decrease in the expression of ER protein. In the second approach, MCF-7 cells were transiently transfected with wild-type p53. Overexpression of p53 increased the amount of ER . To determine whether the effects of p53 on the expression of ER were due to changes in transcription, deletion mutants of the ER promoter were used. This experimental approach demonstrated that p53 up-regulates ER gene expression by increasing transcription of the gene through elements located upstream of promoter A. Transfection assays using p53 mutants further demonstrated that the p53-induced increase in ER gene transcription was not dependent on the ability of p53 to bind to DNA but on its ability to interact with other proteins.

Ansamycins exert their effects by binding heat shock protein 90 (Hsp90) and targeting important signalling molecules for degradation via the proteasome pathway. We wanted to study the effect of geldanamycin (GA) and its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) on glioblastoma cell lines. We show that these cells are growth inhibited by ansamycins by being arrested in G 2 / M and, subsequently, cells undergo apoptosis. The protein levels of cell division cycle 2 (cdc2) kinase and cell division cycle 25c (cdc25c) were downregulated upon GA and 17-AAG treatment and cdc2 kinase activity was inhibited. However, other proteins involved in the G 2 /M checkpoint were not affected. The cdc2 and cdc25c mRNA levels did not show significant differences upon ansamycin treatment, but the stability of cdc2 protein was reduced. The association of cdc2 and cdc25c with p50 cdc37 , an Hsp90 cochaperone, decreased, but the interaction of cdc2 and cdc25c with the Hsp70 co-chaperone increased after ansamycin treatment. Proteasome inhibitors were able to rescue the cdc2 downregulation, but not the cdc25c reduction. However, calpain inhibitors were able to rescue the cdc25c downregulation, suggesting that cdc25c is proteolysed by calpains in the presence of ansamycins, and not by the proteasome. We conclude that ansamycins downregulate cdc2 and cdc25c by two different mechanisms.

The T cell antigen receptor consists of an antigen-binding heterodimer that is noncovalently associated with at least five CD3 subunits (gamma, delta, epsilon, zeta, and eta). The CD3-zeta chains are either disulfide-linked homodimers (CD3-zeta 2) or disulfide-linked heterodimers with eta (CD3-zeta eta). Variants of a murine antigen-specific T cell hybridoma that express normal amounts of CD3-zeta 2 but decreased amounts of CD3-zeta eta were isolated. When activated, the parental cell line increased both phosphatidylinositol hydrolysis and serine-specific protein kinase activity to a much greater extent than the variants. In contrast, the activation of a tyrosine-specific kinase after stimulation with a cross-linking antibody to CD3 was similar among these cells. There was a positive linear relation between the expression of CD3-zeta eta and phosphoinositide hydrolysis stimulated by the TCR, suggesting a differential coupling of the T cell alpha beta heterodimer to signal transduction mechanisms due to alpha beta association with either CD3-zeta 2 or CD3-zeta eta.

The antitumor activity of the histone deacetylase inhibitors was tested in three well-characterized pancreatic adenocarcinoma cell lines, IMIM-PC-1, IMIM-PC-2, and RWP-1. These cell lines have been previously characterized in terms of their origin, the status of relevant molecular markers for this kind of tumor, resistance to other antineoplastic drugs, and expression of differentiation markers. In this study, we report that histone deacetylase inhibitors induce apoptosis in pancreatic cancer cell lines, independently of their intrinsic resistance to conventional antineoplastic agents. The histone deacetylase inhibitorinduced apoptosis is due to a serine protease -dependent and caspase-independent mechanism. Initially, histone deacetylase inhibitors increase Bax protein levels without affecting Bcl-2 levels. Consequently, the apoptosisinducing factor (AIF) and Omi/HtrA2 are released from the mitochondria, with the subsequent induction of the apoptotic program. These phenomena require AIF relocalization into the nuclei to induce DNA fragmentation and a serine protease activity of Omi/HtrA2. These data, together with previous results from other cellular models bearing the multidrug resistance phenotype, suggest a possible role of the histone deacetylase inhibitors as antineoplastic agents for the treatment of human pancreatic adenocarcinoma. [Mol Cancer Ther 2005; 4(8);1222 -30]

The main goal of our study has been to analyze the efficiency of new anticancer drugs, specifically histone deacetylase inhibitors, in tumor cells bearing a multidrug resistance phenotype. We report that the histone deacetylase inhibitors, Trichostatin A and Suberoylanilide Hydroxamic Acid (SAHA), dramatically reduce cell viability and promote apoptosis in different drug-resistant cells, affecting in a much lesser extent to their parental drug-sensitive counterparts. The differential effects induced by Trichostatin A and SAHA between drug-sensitive and drug-resistant cells are reflected on the main characteristics of the resistant phenotype. Thus, reverse transcription-PCR and Western immunoblots confirm that both histone deacetylase inhibitors promote endogenous down-regulation of P-glycoprotein, which is overexpressed in the drug-resistant cells. Transfection of drugsensitive cells with the P-glycoprotein cDNA ruled out the a priori possible association between apoptosis and down-regulation of P-glycoprotein induced by the histone deacetylase inhibitors. The results suggest a therapeutic potential of histone deacetylase inhibitors in the treatment of cancers with acquired resistance.

The present study of inhibitors shows that the histone deacetylase -induced increase in P-glycoprotein (Pgp) mRNA (MDR1 mRNA) does not parallel either an increase in Pgp protein or an increase in Pgp activity in several colon carcinoma cell lines. Furthermore, studying the polysome profile distribution, we show a translational control of Pgp in these cell lines. In addition, we show that the MDR1 mRNA produced in these cell lines is shorter in its 5 ¶ end that the MDR1 mRNA produced in the MCF-7/Adr (human breast carcinoma) and K562/Adr (human erythroleukemia) cell lines, both of them expressing Pgp. The different size of the MDR1 mRNA is due to the use of alternative promoters. Our data suggest that the translational blockade of MDR1 mRNA in the colon carcinoma cell lines and in wild-type K562 cells could be overcome by alterations in the 5 ¶ end of the MDR1 mRNA in the resistant variant of these cell lines, as in the case of the K562/Adr cell line. This is, to our knowledge, the first report demonstrating that the presence of an additional 5 ¶ untranslated fragment in the MDR1 mRNA improves the translational efficiency of this mRNA. (Mol Cancer Res 2007;5(6):641 -53)

D-amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids generating hydrogen peroxide, a potential producer of reactive oxygen species. In this study, we used a CLytA-DAAO chimera, both free and bound to magnetic nanoparticles, against colon carcinoma, pancreatic adenocarcinoma, and glioblastoma cell lines. We found that the enzyme induces cell death in most of the cell lines tested and its efficiency increases significantly when it is immobilized in nanoparticles. We also tested this enzyme therapy in non-tumor cells, and we found that there is not cell death induction, or it is significantly lower than in tumor cells. The mechanism triggering cell death is apparently a classical apoptosis pathway in the glioblastoma cell lines, while in colon and pancreatic carcinoma cell lines, CLytA-DAAO-induced cell death is a necrosis. Our results constitute a proof of concept that an enzymatic therapy, based on magnetic nanoparticles-delivering CLytA-DAAO, could constitute a useful therapy against cancer and besides it could be used as an enhancer of other treatments such as epigenetic therapy, radiotherapy, and treatments based on DNA repair.