A single novel enzyme, glyoxalase III, which catalyses the conversion of methylglyoxal into D-lactate without involvement of GSH, has been detected in and purified from Escherichia coli. Of several carbonyl compounds tested, only the alpha-ketoaldehydes methylglyoxal and phenylglyoxal were found to be substrates for this enzyme. Glyoxalase III is active over a wide range of pH with no sharp pH optimum. In its native form it has an M(r) of 82000 +/- 2000, and it is composed of two subunits of equal M(r). Glutathione analogues, which are inhibitors of glyoxalase I, do not inhibit glyoxalase III. Glyoxalase III is found to be sensitive to thiol-blocking reagents. The p-hydroxymercuribenzoate-inactivated enzyme could be almost completely re-activated by dithiothreitol and other thiol-group-containing compounds, indicating the possible involvement of thiol group(s) at or near the active site of the enzyme.

In the epithelial compartment of the uterus, estradiol-17β (E 2 ) induces cell proliferation while progesterone (P 4 ) inhibits this response and causes differentiation of the cells. In this study, we identified the mechanism whereby E 2 and P 4 reciprocally regulate the expression of minichromosome maintenance (MCM)-2, a protein that is an essential component of the hexameric MCM-2 to 7 complex required for DNA synthesis initiation. We show in the uterine epithelium that Kruppel-like transcription (KLF) factors, KLF 4 and 15, are inversely expressed; most importantly, they bind to the Mcm2 promoter under the regulation of E 2 and P 4 E 2 , respectively. After P 4 E 2 exposure and in contrast to E 2 treated mice, the Mcm2 promoter displays increased histone 3 (H3) methylation and the recruitment of histone deacetylase 1 and 3 with the concomitant deacetylation of H3. This increased methylation and decreased acetylation is associated with an inhibition of RNA polymerase II binding, indicating an inactive Mcm2 promoter following P 4 E 2 treatment. Using transient transfection assays in the Ishikawa endometrial cell line, we demonstrate that Mcm2 promoter activity is hormonally stimulated by E 2 and that KLF15 inhibits this E 2 enhanced transcription. KLF15 expression also blocks Ishikawa cell proliferation through inhibition of MCM2 protein level. Importantly, in vivo expression of KLF15 in an estrogenized uterus mimics P 4 's action by inhibiting E 2 -induced uterine epithelial MCM-2 expression and DNA synthesis. KLF15 is therefore a downstream physiological mediator of progesterone's cell cycle inhibitory action in the uterine epithelium.endometrium | implantation | menstrual cycle | endometriosis E stradiol-17β (E 2 ) and progesterone (P 4 ) directs uterine preparation for pregnancy. These hormones synthesized cyclically in humans cause a sequential series of proliferative and differentiation events in the uterine stroma and epithelium that result in the epithelium being receptive to the blastocyst for attachment and subsequent implantation (1). Despite this close control of uterine cell proliferation, aberrant proliferative conditions of the human endometrium are common. For example, endometrial polyps and endometriosis are caused by inappropriate proliferation of the uterus, while unopposed estrogen stimulation is associated with menstrual irregularities and endometrial hyperplasia/adenocarcinoma (2). Endometrial cancer is the most common female genital tract malignancy and is responsible for 6% of cancer deaths of women in the United States and more worldwide (3). However, the molecular mechanisms underlying these pathologies are still obscure, as are the molecular mechanisms involved in normal hormonal regulation of cell proliferation in the endometrium, which is essential for successful pregnancy (4).The mouse uterus provides a unique in vivo model to study the regulation of epithelial cell proliferation as the physiological actions of E 2 and P 4 E 2 can be recapitulated in ovariectomized animals by treat...

The effect of methylglyoxal (MG) on the aerobic glycolysis of Ehrlich ascites carcinoma (EAC) cells has been tested. Methylglyoxal inhibited glucose utilization and glucose 6-phosphate (G6P) and L-lactate formation in whole EAC cells. Methylglyoxal strongly inactivated glyceraldehyde 3-phosphate dehydrogenase (GA3PD) of the malignant cells, whereas MG has little inactivating effect on this enzyme from several normal sources. Methylglyoxal also inactivated only the particulate hexominase of the EAC cells, but this inactivation was less pronounced than the effect on GA3PD. Methylglyoxal has little inactivating effect on glucose 6-phosphate dehydrogenase (GIPD), and no effect on L-lactate dehydrogenase (LDH) of the malignant cells. Glucose-dependent L-lactic acid formation of EAC-cell-free homogenate was strongly inhibited by MG, but when GA3PD of normal cells was added to this homogenate, significant lactate formation was observed even in the presence of MG. Methylglyoxal also inhibited the respiration of EAC-cell mitochondria. Respiration of mitochondria isolated from liver and kidney of normal mice, however, remained unaffected. As a consequence of the inhibition of glycolysis and mitochondrial respiration, the ATP level of the EAC cells was drastically reduced. Studies reported herein strongly suggest that the tumoricidal effect of MG is mediated at least in part through the inhibition of mitochondrial respiration and inactivation of GA3PD, and this enzyme may play an important role in the high glycolytic capacity of the malignant cells.o I993 Wilty-Liss, Inc.The growth-inhibitory and carcinostatic actions of MG have been known for a long time. Szent-Gyorgyi (1979), in his pioneering work on the biological role of MG, has put forward strong evidence for the anti-tumor and growth-inhibitory effects of MG. In an extensive study on the effect of MG on tumor growth, Apple and Greenberg (1968) observed that MG inhibited the growth of a wide variety of malignant cells in mice by 90 to 99%.Although MG has been thought for quite a long time to be a normal metabolite, its enzymatic formation and breakdown have not been elucidated till recently. Moreover, the precise biological function of MG has remained obscure.

The effect of methylglyoxal on the oxygen consumption of Ehrlich-ascites-carcinoma (EAC)-cell mitochondria was tested by using different respiratory substrates, electron donors at different segments of the mitochondrial respiratory chain and site-specific inhibitors to identify the specific respiratory complex which might be involved in the inhibitory effect of methylglyoxal on the oxygen consumption by these cells. The results indicate that methylglyoxal strongly inhibits ADP-stimulated alpha-oxo-glutarate and malate plus pyruvate-dependent respiration, whereas, at a much higher concentration, methylglyoxal fails to inhibit succinate-dependent respiration. Methylglyoxal also fails to inhibit respiration which is initiated by duroquinol, an artificial electron donor. Moreover, methylglyoxal cannot inhibit oxygen consumption when the NNN'N'-tetramethyl-p-phenylenediamine by-pass is used. The inhibitory effect of methylglyoxal is identical on both ADP-stimulated and uncoupler-stimulated respiration. Lactaldehyde, a catabolite of methylglyoxal, can exert a protective effect on the inhibition of EAC-cell mitochondrial respiration by methylglyoxal. We suggest that methylglyoxal possibly inhibits the electron flow through complex I of the EAC-cell mitochondrial respiratory chain.

The creatine/creatine kinase (CK) system plays a key role in cellular energy buffering and transport. In vertebrates, CK has four isoforms expressed in a tissue-specific manner. In the process of creatine biosynthesis several other important metabolites are formed. The anticancer effect of creatine had been reported in the past, and recent literature has reported low creatine content in several types of malignant cells. Furthermore, creatine can protect cardiac mitochondria from the deleterious effects of some anticancer compounds. Previous work from our laboratory showed progressive decrease of phosphocreatine, creatine and CK upon transformation of skeletal muscle into sarcoma. It was convincingly demonstrated that prominent expression of creatine-synthesizing enzymes L-arginine: glycine amidinotransferase and N-guanidinoacetate methyltransferase occurs in sarcoma, Ehrlich ascites carcinoma and sarcoma 180 cells; whereas, both these enzymes are virtually undetectable in skeletal muscle. Creatine transporter also remained unaltered in malignant cells. The anticancer effect of methylglyoxal had been known for a long time. The present work shows that this anticancer effect of methylglyoxal is significantly augmented in presence of creatine. On creatine supplementation the effect of methylglyoxal plus ascorbic acid was further augmented and there was no visible sign of tumor. Moreover, creatine and CK, which were very low in sarcoma tissue, were significantly elevated with the concomitant regression of tumor.