Histochemical studies of human breast tumors: Activity of alkaline phosphatase, acid phosphatase and glucose-6-phosphate dehydrogenase.

Histochemical studies of human breast tumors were performed with particular emphasis on the activity of alkaline phosphatase (AIP), acid phosphatase (AcP) and glucose-6-phosphate dehydrogenase (G6PDH). Enzyme activities in benign and malignant lesions were compared. AIP was prominent in normal mammary epithelium, limited to the myoepithelial layer in benign tumors and was absent in cords of malignant cells. AcP activity was faintly detected in normal mammary epithelium, increased in canalicular epithelium of fibroadenomas and was marked in malignant cells. G6PDH exhibited marked activity in neoplastic epithelium and the stroma of nearly all carcinomas studied, whereas in benign tumors, G6PDH activity was strictly limited to the connective tissue. The study suggests a strong correlation between G6PDH activity and malignancy. The different results obtained by various workers in this field are critically reviewed, and discussed in the light of the results of the present study.     

The effects of n-3 polyunsaturated fatty acids by gavage on some metabolic enzymes of rat liver

In this experimental study, the effect of fish n-3 fatty acids was studied on the some important enzymes of carbohydrate metabolism, hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) in rat liver. Wistar albino rats of experimental group (n= 9) were supplemented fish omega-3 fatty acids (n-3 PUFA) as 0.4 g/kg bw. by gavage for 30 days in addition to their normal diet. Isotonic solution was given to the control group (n= 8) by the same way. At 30th day, the rats were killed by decapitation under ether anesthesia, autopsied and liver was removed. Spectrophotometric methods were used to determine the activities of above-mentioned enzymes in the liver. The n-3 PUFA caused increases in the activities of HK, G6PD, LDH, and MDH in comparison with control. These increases were statistically significant (P < 0.01) except 6PGD activity. As a result, n-3 PUFA may regulate the metabolic function of liver effectively by increasing HK, G6PD, 6PGD, LDH, and MDH enzyme activities of rat liver when added in enough amounts to the regular diet.     

2-deoxy-glucose-6-phosphate utilization in the study of glucose-6-phosphate dehydrogenase mosaicism.

The electrophoretic difference between normal glucose-6-phosphate dehydrogenase (G6PD) and two common variants (G6PD A and G6PD A-) has made the G6PD enzyme system very useful for genetic studies and for investigation on the clonal origin of tumors. This approach has not been possible for another common variant, G6PD mediterranean, which has a normal electrophoretic pattern. The different utilization of 2-deoxy-glucose-6-phosphate (2dG6P), an analog of the normal substrate, by the normal enzyme and the Mediterranean variant, allows a convenient determination of the degree of mosaicism in mononuclear cells from heterozygotes.     

Glycolytic cancer cells lacking 6-phosphogluconate dehydrogenase metabolize glucose to induce senescence

We show that knockdown of 6-phosphogluconate dehydrogenase (6PGD) of the pentose phosphate pathway (PPP) inhibits growth of lung cancer cells by senescence induction. This inhibition is not due to a defect in the oxidative PPP per se. NADPH and ribose phosphate production are normal in 6PGD knockdown cells and shutdown of PPP by knockdown of glucose-6-phosphate dehydrogenase (G6PD) has little effect on cell growth. Moreover, 6PGD knockdown cells can proliferate when the PPP is bypassed by using fructose instead of glucose in medium. Significantly, G6PD knockdown rescues proliferation of cells lacking 6PGD, suggesting an accumulation of growth inhibitory glucose metabolics in cells lacking 6PGD. Therefore, 6PGD inhibition may provide a novel strategy to treat glycolyic tumors such as lung cancer.     

Differential inhibition/inactivation of mitochondrial complex I implicates its alteration in malignant cells

Methylglyoxal strongly inhibited mitochondrial respiration of a wide variety of malignant tissues including sarcoma of mice, whereas no such significant effect was noted on mitochondrial respiration of normal tissues with the exception of cardiac cells. This inhibition by methylglyoxal was found to be at the level of mitochondrial complex I (NADH dehydrogenase) of the electron transport chain. L-Lactaldehyde, which is structurally and metabolically related to methylglyoxal, could protect against this inhibition. NADH dehydrogenase of submitochondrial particles of malignant and cardiac cells was inhibited by methylglyoxal. This enzyme of these cells was also inactivated by methylglyoxal. The possible involvement of lysine residue(s) for the activity of NADH dehydrogenase was also investigated by using lysine-specific reagents trinitrobenzenesulfonic acid (TNBS) and pyridoxal 5′ phosphate (PP). Inactivation of NADH dehydrogenase by both TNBS and PP convincingly demonstrated the involvement of lysine residue(s) for the activity of the sarcoma and cardiac enzymes, whereas both TNBS and PP failed to inactivate the enzymes of skeletal muscle and liver. Together these studies demonstrate a specific effect of methylglyoxal on mitochondrial complex I of malignant cells and importantly some distinct alteration of this complex in cancer cells.     

Biochemical studies of growing mouse oocytes: preparation of oocytes and analysis of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities.

Oocytes at several stages of growth were isolated by enzymatic digestion of ovaries from infant mice. These oocytes were free of follicle cells and were obtained in sufficient numbers to permit direct biochemical analysis of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) activities. Both enzymes increase in total activity as the oocyte grows. However, while the specific activity of G6PD remains constant up to an oocyte diameter of 80–85 μm, the specific activity of LDH increases four-fold. The specific activity of G6PD and especially LDH declines in oocytes over 80–85 μm in diameter, suggesting that the synthesis of these enzymes is almost completed at this stage of growth.     

异常激活的葡萄糖-6-磷酸脱氢酶通过上调周期蛋白D1表达促进肾透明细胞癌增殖

葡萄糖6-磷酸脱氢酶（glucose 6-phosphate dehydrogenase,G6PD）为磷酸戊糖途径的调节酶。研究表明,G6PD与多种恶性肿瘤的发生密切相关。然而,G6PD在肾透明细胞癌（clear cell renal cell carcinoma, ccRCC）中的功能及其作用机制却鲜有报道。本研究通过TCGA数据分析发现,G6PD在肾透明细胞癌TNM Ⅲ/Ⅳ期mRNA表达水平显著升高,与患者的性别、原发肿瘤直径、淋巴结转移、远端转移、病灶一侧的偏重性、病理分级以及TNM临床分期密切相关。并且,G6PD异常激活有可能成为评价肾透明细胞癌患者不良预后的分子。细胞系检测结果提示,与对照293T细胞及恶性程度较低的786-O细胞相比,恶性程度较高的Caki-1细胞中的G6PD表达及活性明显增加。基因稳定转染结合CCK8分析结果显示,G6PD过表达或异常激活可显著提高293T及786-O细胞的增殖能力,并且促进786-O细胞中周期蛋白D1基因表达上调。综上,本研究通过TCGA数据库分析和稳定细胞系检测及CCK8分析,结果显示,G6PD在肾透明细胞癌中异常激活,并可上调细胞周期蛋白D1表达,进而促进肿瘤细胞增殖。该研究为进一步揭示肾透明细胞癌分子发病机制以及开发有效的靶向治疗方案提供了借鉴。     

金鱼同工酶的发生遗传学研究——Ⅲ.金鱼同工酶基因座位的加倍与多倍性

以鲫鱼和金鱼为材料,用葡萄糖-6-磷酸脱氢酶(G6PD)、乳酸脱氢酶(LDH)和苹果酸脱氢酶(MDH)同工酶体系作为基因标志,从检测同工酶的多重组合形式来研究基因的加倍与演化。对彩鲫与金鱼G6PD和LDH同工酶的分析结果表明,它们均具有与四倍体鱼类相应的谱带。因而说明了金鱼的G6PD和LDH同工酶基因座位的加倍与染色体的多倍性有关,为金鱼是四倍体的假说提供了证据。而对MDH同工酶的分析却得到了与二倍体鱼类相同的谱带数。这可能与加倍基因发生突变而不表达有关。     

Stimulatory effect of 1,25-dihydroxyvitamin D3 on the glucose-6-phosphate dehydrogenase activity in the MCF-7 human breast cancer cell line

Human breast cancer cell lines have been shown to possess high affinity receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and their growth is inhibited by this steroid. The present study examines the effect of 1,25(OH)2D3 on the activity of glucose-6-phosphate dehydrogenase (G6PD) in cells of a human breast cancer cell line MCF-7. G6PD, an enzyme which controls the hexose monophosphate shunt, is elevated and sensitive to 17 beta-estradiol in breast tumors. G6PD activity was stimulated by 1,25(OH)2D3 in a dose-dependent manner at very low concentrations of steroid (10(-10)-10(-12) M). 1,25(OH)2D3 increased maximum velocity without modifying the affinity constant of the enzyme for glucose-6-phosphate.     

Energy metabolism in the granulation tissue of diabetic rats during cutaneous wound healing

The skin cells chiefly depend on carbohydrate metabolism for their energy requirement during cutaneous wound healing. Since the glucose metabolism is greatly hampered in diabetes and this might affect wound repair process. This prompted us to investigate the intermediate steps of energy metabolism by measuring enzyme activities in the wound tissues of normal and streptozotocin-induced diabetic rats following excision-type of cutaneous injury. The activities of key regulatory enzymes namely hexokinase (HK), phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS) and glucose-6 phosphate dehydrogenase (G6PD) have been monitored in the granulation tissues of normal and diabetic rats at different time points (2, 7, 14 and 21 days) of postwounding. Interestingly, a significant alteration in all these enzyme activities was observed in diabetic rats. The activity of PFK was increased but HK, LDH and CS showed a decreased activity in the wound tissue of diabetics as compared to normal rats. However G6PD exhibited an elevated activity only at early stage of healing in diabetic rats. Thus, the results suggest that significant alterations in the activities of energy metabolizing enzymes in the wound tissue of diabetic rats may affect the energy availability for cellular activity needed for repair process and this may perhaps be one of the factor responsible for impaired healing in these subjects. (Mol Cell Biochem 270: 71–77, 2005)     

A new hepatic protein inactivating glucose 6-phosphate dehydrogenase

Two NADP-cleaving enzymes, namely NADP glycohydrolase and NADP pyrophosphatase, are present in a rat liver extract that inactivates G6PD (glucose 6-phosphate dehydrogenase). The following results suggest that a third G6PD-inactivating protein is present in this extract. (1) Nicotinamide, which selectively inhibits NADP glycohydrolase, enhances the G6PD inactivation under conditions where G6PD activity in control experiments is rather stable. (2) DEAE-cellulose adsorbs the bulk of both NADP glycohydrolase and NADP pyrophosphatase, whereas most of the G6PD-inactivating ability is unadsorbed. (3) Out of 37 liver extracts that were prepared, two were found to lack NADP pyrophosphatase. After removal of NADP glycohydrolase from these extracts by centrifugation, they were still found to inactivate G6PD. (4) Deproteinization of DEAE-cellulose supernatants results in a complete loss of G6PD-inactivating ability; moreover, kinetic experiments performed with the extracts lacking pyrophosphatase strongly support the view that the inactivating protein is an enzyme, although its mechanism is not clear. (5) NADP protects G6PD from inactivation and also reactivates the enzyme completely, thus supporting the view of some action of the inactivating protein on the G6PD-bound NADP.     

First Evaluation of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency in Vivax Malaria Endemic Regions in the Republic of Korea

Background

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect and affects more than 400 million people worldwide. This deficiency is believed to protect against malaria because its global distribution is similar. However, this genetic disorder may be associated with potential hemolytic anemia after treatment with anti-malarials, primaquine or other 8-aminoquinolines. Although primaquine is used for malaria prevention, no study has previously investigated the prevalence of G6PD variants and G6PD deficiency in the Republic of Korea (ROK).

Methods

Two commercialized test kits (Trinity G-6-PDH and CareStart G6PD test) were used for G6PD deficiency screening. The seven common G6PD variants were investigated by DiaPlexC kit in blood samples obtained living in vivax malaria endemic regions in the ROK.

Results

Of 1,044 blood samples tested using the CareStart G6PD test, none were positive for G6PD deficiency. However, a slightly elevated level of G6PD activity was observed in 14 of 1,031 samples tested with the Trinity G-6-PDH test. Forty-nine of the 298 samples with non-specific amplification by DiaPlexC kit were confirmed by sequencing to be negative for the G6PD variants.

Conclusions

No G6PD deficiency was observed using phenotypic- or genetic-based tests in individuals residing in vivax malaria endemic regions in the ROK. Because massive chemoprophylaxis using primaquine has been performed in the ROK military to kill hypnozoites responsible for relapse and latent stage vivax malaria, further regular monitoring is essential for the safe administration of primaquine.     

The effects of epidermal growth factor and the state of confluence on enzymatic activities of cultured rat liver epithelial cells

In cultured normal rat liver epithelial cells, the specific activity and/or isozyme expression of NADH-diaphorase (NADH-D), pyruvate kinase (PK), glucose-6 phosphate dehydrogenase (G6PD), gamma-glutamyl transpeptidase (GGT), and alkaline phosphatase (AP) were markedly dependent on the growth state of the cultures. Proliferating, preconfluent cells had higher specific activities of PK, NADH-D, and G6PD but lower activities of GGT and AP than did the more stationary confluent cells. Addition of epidermal growth factor [EGF] to the media of proliferating cells enhanced the specific activities of PK, NADH-D, G6PD, GGT, and lactate dehydrogenase (LDH) of these cells, but the specific activity of AP was markedly depressed. The increase in activity of PK and GGT by EGF appeared to involve new protein synthesis, whereas the effect of EGF on AP appeared to involve the EGF-directed suppression of the synthesis of a form of AP that is produced exclusively by cells in confluent cultures. Furthermore, the preconfluent cells were more responsive to the action of EGF on AP than were confluent cells, i.e., the EGF-mediated decrease in AP activity was seen at lower concentration in preconfluent than in confluent cells. Paradoxically, confluent cells exhibited a two-to threefold higher capacity to bind [125 I]EGF because of an increase in surface receptor number. The results of this study indicate that enzymatic or other biochemical studies performed on cultured cells must take into account the growth-state of the cultures. EGF can modulate enzyme activity in growing and nongrowing cells; one effect of EGF is to maintain higher activity of glycolytic enzymes, suggesting that EGF or EGF-like factors may contribute to the high rate of glycolysis in certain neoplasms.     

X chromosome expression during oogenesis in the mouse.

The activities of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) have been assayed in mouse oocytes at several stages of follicle development isolated from XX and XO female mice. Throughout the entire growth period the activity of G6PD was proportional to the number of X chromosomes present in the oocyte, whereas no difference in LDH activity was detected between XX and XO oocytes. It is concluded, therefore, that both X chromosomes are functional throughout oogenesis.     

Rapid release of bound glucose-6-phosphate dehydrogenase by growth factors. Correlation with increased enzymatic activity

Epidermal growth factor (EGF), a mitogen for renal proximal tubule cells, activated the hexose monophosphate (HMP) shunt in renal proximal tubule cells (Stanton, R. C., and Seifter, J. L. (1988) Am. J. Physiol. 254, C267-C271). We therefore evaluated the effect of EGF on the HMP shunt enzymes glucose 6-phosphate dehydrogenase (G6PD, the rate-limiting enzyme) and 6-phosphogluconate dehydrogenase. Rat renal cortical cells (RCC) were incubated with either EGF or platelet-derived growth factor (PDGF) and then assayed for G6PD and 6-phosphogluconate dehydrogenase activities. EGF and PDGF increased G6PD activity by 25 and 27% respectively. Although phorbol myristate acetate (PMA), ionomycin, PMA + ionomycin, and 8-bromo-cyclic AMP had no significant effect on the activity, a 5-min preincubation with PMA potentiated the activation of G6PD by PDGF. Growth factor activation of G6PD was also seen in a fibroblast and epithelial cell line. None of the agents affected 6-phosphogluconate dehydrogenase activity in the RCC or in the cell lines. Further exploration into a possible mechanism for G6PD activation revealed that growth factors caused release of G6PD from a structural element within the cell. Streptolysin O permeabilization of RCC did not cause significant release of G6PD. However, within 1 min of addition of EGF or PDGF to permeabilized cells, G6PD was released into the cell supernatant. The nonhydrolyzable analog of GTP, guanosine 5'-O-(thiotriphosphate), caused a similar release of G6PD. Preincubation with pertussis toxin or guanyl-5'-yl thiophosphate inhibited the PDGF but not the EGF effect. Although the data do not establish a definitive proof linking G6PD release and G6PD activation, these results suggest that they are related. Thus, growth factor stimulation of the HMP shunt likely occurs by a novel mechanism associated with release of bound G6PD.     

Effects of diallyl sulfide and zinc on testicular steroidogenesis in cadmium-treated male rats

Cadmium (Cd) is one of the environmental pollutants that affect various tissues and organs including testis. Harmful effect of cadmium on testis is known to be germ cell degeneration and impairment of testicular steroidogenesis. In the present study, the effect of diallyl sulfide (DAS), a sulfur-containing volatile compound present in garlic, and zinc (Zn) was investigated on cadmium-induced testicular toxicity in rats. Male adult Wistar rats treated with cadmium (2.5 mg/kg body wt, five times a week for 4 weeks) showed decreased body weight, paired testicular weight, relative testicular weight, serum testosterone, luteinizing hormone, follicle-stimulating hormone, and testicular total antioxidant capacity (TAC) and protein levels. Testicular steroidogenic enzymes, such as 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD), and marker enzymes, such as sorbitol dehydrogenase (SDH), lactate dehydrogenase (LDH), acid phosphatase (ACP), alkaline phosphatase (ALP), and glucose-6-phosphate dehydrogenase (G6PD), showed a significant decrease in activities whereas that of gamma-glutamyl transferase was significantly increased after cadmium exposure. The results have revealed that concurrent treatment with DAS or zinc restored key steroidogenic enzymes, SDH, LDH, and G6PD and increased testicular weight significantly. DAS restored the TAC level and increased testosterone level and relative testicular weight significantly. Zinc restored testicular protein level and body weight. It can be concluded that cadmium causes testicular toxicity and inhibits androgen production in adult male rats probably by affecting pituitary gonadotrophins and that concurrent administration of DAS or zinc provides protection against cadmium-induced testicular toxicity.     

Trisomy 10p due to t(5;10)(p15;p11) segregating in a large sibship

Summary Three new glucose-6-phosphate dehydrogenase (G6PD) variants, which showed electrophoretically normal mobility and were associated with chronic nonspherocytic hemolytic anemia, were found in Japan. G6PD Ogikubo, found in a 17-year-old male whose red cells contained 3% of normal enzyme activity, had normal Km G6P, normal Km NADP, normal utilization of deamino-NADP, decreased heat stability, and a normal pH curve. G6PD Yokohama, characterized from a 15-year-old male, had 1.9% of normal enzyme activity, normal Km G6P, normal Km NADP, low Ki NADPH, normal utilizations of both 2-deoxy-G6P and deamino-NADP, decreased heat stability, and normal pH curve. G6PD Akita, characterized from a 56-year-old male, had an undetectably low activity when hemolysate was examined, normal Km G6P, normal Km NADP, normal Ki NADPH, normal utilizations of both 2-deoxy-G6P and deamino-NADP, decreased heat stability, and normal pH curve.The degree of hemolytic anemia was moderate to mild in all three patients.