Hyperexpression of Integrin α5ß1 Promotes Resistance of MCF-7 Human Breast Carcinoma Cells to Doxorubicin via ERK Protein Kinase Down-regulation.

In MCF-7 human breast carcinoma cells, α5ß1 integrin hyperexpression, which was accomplished by transduction of a full-length α5 integrin cDNA, increased by about 50-70% the number of cells, survived during 48-72 h cell treatment with doxorubicin. Up-regulation of α5ß1 reduced the level of the apoptogenic p53 protein and p21 cell cycle inhibitor, but enhanced the activity of Akt and mTOR protein kinases. In addition to these findings, we observed a significant decrease in the activity of both isoforms of phosphokinase Erk1/2, which is known to play a key role in cell viability pathways, including pathways alleviating stress damages caused by distinct antitumor drugs. Diminished Erk activity accompanying the rise of drug resistance can be explained by an "atypical" function of this kinase, which, in the cells studied, promotes an enhanced rather than reduced sensitivity to doxorubicin. To verify this suggestion, the effect of a specific Erk inhibitor, PD98059, on the resistance to doxorubicin of control and α5 cDNA-transduced MCF-7 cells was investigated. The data showed that suppression of Erk activity increased the resistance of control cells (transduced with an "empty" vector) to a level higher than that demonstrated by the α5 cDNA-transduced cells. The highest level of resistance was observed in α5ß1-trancduced cells treated with PD98059. Akt and mTOR kinase inhibitors had little if any effect on doxorubicin resistance of α5 cDNA-transduced MCF-7 cells. The data show for the first time that integrin α5ß1 can stimulate drug resistance of tumor cells through a mechanism based on the inhibition of protein kinase Erk. From a more general view, the results of this investigation suggest that signal protein kinases can perform in tumor cells "non-canonical" functions, opposite to those, which are the basis for using kinase inhibitors in targeted cancer therapy. It follows that if a protein kinase is supposed to be used as a target for such therapy, it is important to explore its features in the particular tumor prior to the onset of treatment.

Oct-1 is responsible for the C-33T polymorphism effect in the IL-4 promoter.

IL-4 is a pleiotropic immunoregulatory cytokine secreted by Th2 subset of CD4(+) Th cells. Several transcription factors (TFs) have been determined with various degrees of certainty to bind the IL-4 promoter and to regulate its expression in human. To investigate the mechanisms responsible for phenotypic effects of the C-33T IL-4 promoter polymorphism, we performed a search of TFs binding to this promoter locus and discriminating the -33C and -33T alleles. In silico searches suggest few factors bind this region. Using an electromobility shift assay we found that Jurkat T cells contained proteins which specifically interacted with oligonucleotide probes, corresponding to the -33 region. Considerable binding differences between C and T alleles were demonstrated using competitive conditions, the proteins bound predominantly with -33C allele. We found that the transcription factor Oct-1 produced the major shifted complex. The binding of Oct-1 was not improved using activated nuclear extracts; however, we observed increases in other shifted complexes upon cell activation. We suppose that Oct-1 occupancy may compete for binding of activator proteins to closely or overlapped binding sites. Our findings suggest that the interplay between Oct-1 and unknown TFs may be responsible for the C-33T polymorphism effects.