S-adenosylmethionine synthetase (SAMS) catalyzes the formation of S-adenosylmethionine (SAM) and is essential to normal cell function. There are two forms of SAMS, liver-specific and nonliver-specific (often referred to as "kidney"), which are products of two different genes. SAMS isoenzymes differ greatly in kinetic parameters and sensitivity to inhibition by methionine analogs. The current work studied changes in SAMS and their significance in liver cancer. Northern blot analysis showed that while normal liver expresses only liver-specific SAMS, both HepG2 and HuH-7 cells express only nonliver-specific SAMS. Absence of liver-specific SAMS messenger RNA (mRNA) was not because of gene deletion or rearrangement but complete lack of gene transcription. Reverse-transcription polymerase chain reaction (RT-PCR) with liver- and kidney-specific SAMS primers showed that liver-specific SAMS mRNA was absent with only kidney SAMS mRNA present in HepG2, HuH-7, Hep3B, and HuH-1 cells, and four consecutive hepatocellular carcinoma (HCC) specimens. Normal liver tissues from the same patients express both forms of SAMS mRNA. As a result of the change in SAMS expression, SAMS activity was higher in HepG2 and HuH-7 cells at physiologically relevant methionine concentrations but lower at high (mmol/L) methionine concentrations than rat hepatocytes. Treatment with ethionine and seleno-D,L-ethionine, two inhibitors known to have I50 values 50 to 60 times lower against SAMS purified from Novikoff hepatoma cells as compared with SAMS purified from normal rat liver, resulted in increased cell lysis in HepG2 and HuH-7 cells but not cultured rat hepatocytes. These agents did not affect cellular adenosine triphosphate (ATP) levels but inhibited SAMS activity in HepG2 and HuH-7 cells when added to their protein extracts. In summary, expression of SAMS is altered in human liver cancer. This occurrence may provide a potentially exploitable target for cancer chemotherapy.
We describe the molecular cloning of two novel human and murine transcription factors containing the TEA/ ATTS DNA binding domain and related to transcriptional enhancer factor-1 (TEF-1). These factors bind to the consensus TEA/ATTS cognate binding site exemplified by the GT-IIC and Sph enhansons of the SV40 enhancer but differ in their ability to bind cooperatively to tandemly repeated sites. The human TEFs are differentially expressed in cultured cell lines and the mouse (m)TEFs are differentially expressed in embryonic and extra-embryonic tissues in early post-implantation embryos. Strikingly, at later stages of embryogenesis, mTEF-3 is specifically expressed in skeletal muscle precursors, whereas mTEF-1 is expressed not only in developing skeletal muscle but also in the myocardium. Together with previous data, these results point to important, partially redundant, roles for these TEF proteins in myogenesis and cardiogenesis. In addition, mTEF-1 is strongly coexpressed with mTEF-4 in mitotic neuroblasts, while accentuated mTEF-4 expression is also observed in the gut and the nephrogenic region of the kidney. These observations suggest additional roles for the TEF proteins in central nervous system development and organogenesis.
The regions of transcriptional enhancer factor‐1 (TEF‐1) required for its activation function and sequence‐specific DNA binding have been determined. Deletion analysis of a chimera between TEF‐1 and the GAL4 DNA binding domain (DBD) indicated that at least three regions of TEF‐1 were involved in transactivation. However, none of these regions functioned as independent activating domains. Moreover, none of the GAL4 chimeras containing individual TEF‐1 regions interfered with the activity of endogenous HeLa cell TEF‐1, while interference was observed with the GAL4‐TEF‐1 chimeras which functioned as transactivators. These results indicate that there is a general correlation between the abilities of a given GAL4‐TEF‐1 chimera to function in transcriptional activation and interference, thus supporting the idea that transactivation by TEF‐1 is mediated by a limiting transcriptional intermediary factor. In addition, we show experimentally that the TEA/ATTS domain is a novel class of DBD involved in the sequence‐specific DNA binding of TEF‐1 and its Drosophila homologue scalloped. Two other regions of TEF‐1 are also required for DNA binding. These regions are not part of the minimum DBD, but may function by antagonizing the effect of sequences which negatively regulate DNA binding mediated by both the TEF‐1 TEA/ATTS domain and the GAL4 DBD. In addition, analysis of TEF‐1 and scalloped derivatives in which their TEA/ATTS domains have been interchanged further indicates that the TEA/ATTS domain is not the only determinant of DNA binding specificity.
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