Methionine adenosyltransferase (MAT) is a critical enzyme which catalyzes the formation of S-adenosylmethionine (SAM). In mammals, two different genes, MAT1A and MAT2A, encode for two homologous MAT catalytic subunits. MAT1A is expressed only in liver whereas MAT2A is widely distributed. In the past funding cycle we shwed that in adult liver, increased expression of MAT2A is associated with rapid growth or de-differentiation of the liver. Using a cell line model that differ only in the type of MAT expressed, we showed that the type of MAT expressed by the cell influences the rate of cell growth. Cells expressing MAT1A exhibited the slowest rate of cell growth while the opposite was true of MAT2A expression. A change in the SAM level and methylation status mediate this effect. Thus, SAM treatment increased intracellular SAM and global DNA methylation levels and inhibited the rate of growth of HuH-7 cells. To understand transcriptional regulation, we cloned the 5'- flanking region of both human MAT genes. For both Mat genes, methylation of the promoter correlated with decreased expression and cell type-specific transcription factors may regulate their expression. We also have showed increased binding of transcription factors to two key regions of the MAT2A promoter in hepatocellular carcinoma (HCC). To better understand the role of MAT1A in normal liver development and response to injury, we have created a MAT1A knockout mouse model. The following aims represent logical continuation of our studies to: 1. Examine transcriptional regulation of MAT2A- identify key cis-acting elements and trans-activating factors important for the activity of this promoter, determine if the transcriptional activity is regulated by methylation and if there are cell type-specific transcription factors involved in mediating the expression of MAT2A; 2. Examine transcriptional regulation of MAT1A- the same types of analysis for MAT2A will be carried out for MAT1A as well; 3. Elucidate the mechanism of SAM's inhibitory effect on liver cancer cell growth and examine its in vivo applicability- use microarray technology to see whether SAM treatment and MAT expression alter the expression of genes important in cell growth, create an in vivo HCC model and examine the applicability of SAM in treating liver cancer; and 4. Examine the role of MAT1A in normal liver development and response to injury- using the MAT1A knockout mouse, examine normal liver development, the effect of choline deficient diet and thioacetamide on development of liver injury and malignant degeneration, and liver regeneration in response to 2/3 partial hepatectomy.
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