Resistance to chemotherapy remains a serious problem in the successful treatment of gastric and esophageal cancers. DNA-damaging agents alter levels of p53 protein in several cell types and it has been speculated that regulation of p53 can be involved in the resistance or sensitivity of cancer cells to some chemotherapeutic drugs, depending on whether cells have mutant or wild-type p53; however, little is known about the relationship of p53 to drug sensitivity in gastric/esophageal cancers. Here we have examined human gastric/esophageal adenocarcinoma cell lines for p53 mutational status, chemosensitivity to 5-fluorouracil, mitomycin C, and cis-dichlorodiammineplatinum(II), alteration in p53 levels following exposure of cells to these drugs, and the mechanisms involved in regulating p53 levels. Our results indicate that wild-type p53 protein levels increase after treatment with each of these drugs via either post-translational and/or translational mechanisms and that this increase in wild-type p53 appears to be required for effective chemotherapeutic growth control of gastric/esophageal adenocarcinoma cells. In contrast, gastric/esophageal cancer cells expressing either mutated p53 protein or no p53 protein are more resistant to the growth-inhibitory effects of these drugs, despite the fact that drug exposure can also increase mutant p53 levels by a translational mechanism. Thus, these data indicate that the mutational status of p53 is predictive of chemosensitivity of gastric and esophageal adenocarcinomas, and suggest a mechanism in which p53 protein contributes to the cellular response to chemotherapy.
Acetylation is the most frequently occurring chemical modification of the a-NH2 group of eucaryotic proteins and is catalyzed by N-acetyltransferase. The yeast enzyme is encoded by the AAAI (amino-terminal a-amino acetyltransferase) gene. A null mutation (aaal-1) created by gene replacement, while not lethal, slows cell growth and results in heterogeneous colony morphology. In comparison with wild-type cells, aaal-l/aaal-l diploids cannot enter stationary phase, are sporulation defective, and are sensitive to heat shock. In addition, the aaal-l mutation specifically reduces mating functions of MATa cells. These results indicate that N' acetylation plays a crucial role in yeast cell growth and mating. a-Amino acetylation is an important cotranslational and posttranslational modification of proteins in procaryotic and eucaryotic cells, and N' acetylation is the most common chemical modification of the a-NH2 group of eucaryotic proteins (reviewed in references 7 and 45). N' acetylation is mediated by at least one Na-acetyltransferase, which catalyzes the transfer of an acetyl group from acetyl coenzyme A to the a-NH2 group of proteins and peptides, and a large number of proteins from various organisms have been shown to possess Na-acetylated NH2-terminal residues (5, 6). N" acetylation plays a role in normal eucaryotic translation and processing (50) and protects against proteolytic degradation (16,33). Further, the rate of protein turnover mediated by the ubiquitin-dependent degradation system apparently depends on the presence of a free a-NH2 group (2, 12), and this dependence indicates that N' acetylation may play a crucial role in impeding protein turnover.Recently, we purified an N-acetyltransferase from Saccharomyces cerevisiae and characterized it as a dimeric protein, whose subunit Mr was 95,000 and which would effectively transfer an acetyl group to various synthetic peptide substrates (including adrenocorticotropin [amino acid residues 1 to 24], human superoxide dismutase [residues 1 to 24], and yeast alcohol dehydrogenase [residues 1 to 24] (20). Further, we demonstrated that this enzyme would not transfer an acetyl group to the e-amino group of lysyl residues in various peptide substrates and histones. By using amino acid sequences of tryptic peptides derived from the purified enzyme, we have cloned its cDNA, demonstrated that the enzyme is encoded by a single gene (AAA], aminoterminal a-amino acetyltransferase), and localized this gene to chromosome IV (21). This yeast DNA forms the basis for elucidating the biological function and regulation of N' acetylation in eucaryotic protein synthesis and degradation. In this paper, we describe the creation of a null mutation (aaal-1) by one-step gene replacement and the testing of various null mutants for entrance into stationary phase, sporulation efficiency, temperature sensitivity, and mating functions. The results indicate that N' acetylation affects one or more proteins involved in each of these vital cell functions. * Corresponding author. MATERIALS AND M...
FSH, the primary trophic hormone for gamete development in mammals, is composed of two protein subunits, alpha and beta. It is known that 17 beta-estradiol (E2) and progesterone (P4) can decrease the secretion and synthesis of FSH in ovine pituitary cultures. Data presented here indicate that E2 and P4 decrease the steady state levels of FSH beta mRNA concomitantly with FSH secretion in ovine pituitary cultures. By 24 h, E2 decreased the steady state levels of FSH beta mRNA and FSH secretion by 68% +/- 5%. P4 also decreased both concomitantly, but by 58% +/- 7% after 24 h. E2 and P4 also decrease steady state levels of alpha mRNA, but at a lower rate. Finally, it is shown that E2 and P4 decrease transcription of the FSH beta by greater than 85% in 2 h; alpha mRNA transcription is decreased by 70% in 12 h. These effects are not altered even when cycloheximide is present to block protein synthesis by 95%. These data further define the mechanisms whereby E2 and P4 inhibit ovine FSH secretion/synthesis directly at the pituitary level. They also provide the first example of negative transcriptional regulation by P4 and the second of two examples now established for E2.
Inhibition of macrophage migration by cryptotanshinone involved inhibition of PI3K activation with consequent reduction of phosphorylation of Akt and ERK1/2.
Pathology, Massachusetts General Hospital, and Departments of Genetics Acetyl-CoA hydrolase, which hydrolyzes acetyl-CoA to acetate and CoASH, was isolated from Succhuromycrs cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked. The enzyme was purified 3 080-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, gel filtration and hydroxylapatite. The molecular mass of the native yeast acetyl-CoA hydrolase was estimated to be 64 f 5 kDa by gel-filtration chromatography. SDSjPAGE analysis revealed that the denatured molecular mass was 65 f 2 kDa and together with that for the native enzyme indicates that yeast acetyl-CoA hydrolase was monomeric. The enzyme had a pH optimum near 8.0 and its PI was approximately 5.8. Several acyl-CoA derivatives of varying chain length were tested as substrates for yeast acetyl-CoA hydrolase. Although acetyl-CoA hydrolase was relatively specific for acetyl-CoA, longer acyl-chain CoAs were also hydrolyzed and were capable of functioning as inhibitors during the hydrolysis of acetyl-CoA. Among a series of divalent cations, Zn2+ was demonstrated to be the most potent inhibitor. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate, a histidine-modifying reagent.The concentration of acetyl-CoA in cells is regulated by its rate of synthesis and its rate of utilization and/or degradation. Acetyl-CoA is primarily synthesized from pyruvate generated from carbohydrates and from amino acids (Ala, Thr, Gly, Ser and Cys), from acetoacetyl-Coh generated from other amino acids (Phe, Tyr, Leu, Lys and Trp), and from p oxidation of fatty acids, although a minor amount is also synthesized by acetyl-CoA synthetase [I]. Utilization of acetyl-CoA occurs during the Krebs cycle or by its conversion to fatty acids or ketone bodies. Furthermore, the acetyltransferase-catalyzed acetylation of proteins and peptides [2 -71, as well as of biological substances other than proteins (e. g. glucosamine, choline, arylamine, arylalkylamine) [8 -1 I], accounts for additional usage of endogenous acetyl-CoA.Acetyl-CoA hydrolase was first identified in pig heart in 1952 [I21 and, subsequently, the enzyme has been found in many mammalian tissues [13 -181. Rat liver has been demonstrated to possess a cytoplasmic and a mitochondrial form of the enzyme. Although only the rat liver cytoplasmic enzyme Correspondence to J. A. Smith,
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