The role of mitf and tbx3 proteins i̇n hematologi̇cal mali̇gnanci̇es

The development and maintenance of organ systems rely entirely on the presence of healthy and good functioning cells establishing the organs. The proliferation, differentiation, adaptation and responding to environmental cues and even death are all cellular mechanisms that are regulated via transcriptional control at the gene level. Transcription is the process of copying a part of the DNA template carrying the genetic code into messenger RNA and this cellular event is governed by a group of proteins called transcription factors, which are sequence specific DNA binding proteins. Tbx3 and MITF are both transcription factors involved in embryological development. Both of the proteins are capable of regulating expressions of some of the genes that play important roles in cell cycle and therefore can drive cells into proliferation. Although this is rather essential and useful during embryological development, it can be dangerous for the organism under conditions where physiological mechanisms malfunction. Actually, this forms the basis of cancer development, which essentially occurs as a consequence of uncontrolled cell proliferation and differentiation. In accordance with this, both Tbx3 and MITF have been demonstrated to be up-regulated in many forms of cancer. The mechanisms underlying haematological malignancies vary depending on the cell type they originate. However, because the main cellular event is "uncontrolled cell proliferation and differentiation", it is obvious that identification of the genes and the mechanisms they employ in regulation of cell cycle will have an enormous effect both in diagnosis and treatment. In this study, Tbx3 and MITF were analysed in order to reveal their potential roles in expression of PTEN, which is an important gene that has been demonstrated to be inactivated in many different types of cancer. For this purpose, cells obtained from bone marrow of 40 leukaemia patients were analysed for expressions of TBX3, MITF and PTEN genes both at mRNA and protein levels. It was demonstrated that the leukaemia patients had statistically significant increase in Tbx3 and MITF mRNA and protein levels, whereas PTEN gene expression was found to be significantly decreased (p<0,05). In order to reveal the molecular mechanisms underlying this negative correlation, HeLa and HEK cell lines were transfected with a Tbx3 or MITF expressing plasmid and transcription assays were performed to demonstrate the effects of Tbx3 and MITF proteins on PTEN promoter activity. In addition, the endogenous PTEN mRNA and protein levels were determined in response to over-expressed Tbx3 protein. We demonstrated that not MITF but Tbx3 is capable of repressing the PTEN promoter activity, and cause reduction in endogenous PTEN mRNA, therefore protein levels. Furthermore, using EMSA assays the exact binding site of Tbx3 within the PTEN promoter was identified. Moreover it was shown that, with respect to the levels in normal bone marrow blast cells, Tbx3 protein level was increased, while PTEN protein level was decreased in "leukaemia stem cells" that were isolated from the bone marrows of a limited number of patients. Thus, our data not only reveals a new mechanism that may be important in haematological cancer formation, but also suggests that Tbx3 can be used as a potential biomarker both in diagnosis and following up of treatment