A simple histochemical assay to detect cancer cells

Oxygen insensitivity of cancer cells and oxygen sensitivity of non-cancer cells in the histochemical assay of glucose-6-phosphate dehydrogenase (G6PD) activity enables detection of cancer cells in unfixed cell smears or cryostat sections of biopsies. The assay is based on reduction of the tetrazolium salt neotetrazolium. It is a cheap assay that is easy to perform. It takes only 30 min at the most. The test discriminates between adenomas and carcinomas of colon and rectum with a certainty higher than 80% and is the best prognosticator of survival of colorectal cancer patients. Pancreatic cancer can be discriminated from pancreatitis with 100% certainty. Therefore, the assay is an excellent tool for the pathologist to provide additional information in difficult cases of diagnosis of cancer and for prognosis.     

Prognostic estimation of survival of colorectal cancer patients with the quantitative histochemical assay of G6PDH activity and the multiparameter classification program CLASSIF1.

Prognosis of colorectal cancer patients that show similar histopathology may vary substantially. An attempt was made to improve prognosis by the self-learning classification program CLASSIF1, based on automated multiparameter analysis of quantitative histochemical and clinical parameters of 64 colorectal carcinomas and adjacent normal mucosae. The histochemical parameters applied were the oxygen-insensitivity assay of glucose-6-phosphate dehydrogenase (G6PDH) activity, a valid discriminator between normal and cancerous mucosae, and related parameters CuZn- and Mn-superoxide dismutase (SOD) levels, and lipid peroxidation (LPO) capacity. Data were processed on the basis of a postoperative follow-up of minimally 32 and maximally 56 months. CLASSIF1 selected the parameters oxygen insensitivity of G6PDH activity, CuZn-SOD and Mn-SOD levels, LPO capacity, lymph node metastasis, Dukes' stage, and age for the highest prognostic value. On the basis of these selected parameters, CLASSIF1 correctly predicted favorable outcome in 100% of the surviving patients and fatal outcome in 64% of the deceased patients. G6PDH activity appeared to be the major information carrier for CLASSIF1. On the basis of G6PDH activity parameters alone, 96% of the surviving patients and 55% of the deceased patients were correctly classified. In comparison, estimation of prognosis on the basis of Dukes' stage alone resulted in 71% correctly classified surviving patients and 61% of patients who died. It is concluded that the self-learning classification program CLASSIF1, on the basis of quantitative histochemical and clinical parameters, is the best prognostic estimator for colon cancer patients yet available.     

磷酸戊糖途径的调节对红豆杉细胞生长及紫杉醇合成的影响

在正常的红豆杉细胞悬浮培养过程,葡萄糖-6-磷酸脱氢酶(G6PDH)活性的变化趋势与生物量的基本相似。而在chitosan处理的细胞中G6PDH活性升高而生物量下降。100 mg·L^-1 chitosan和500mg·L^-1 chitosan均对细胞G6PDH具有诱导作用,且后者的诱导强度较前者的高。乙二醇双2-氨基乙基醚四乙酸(EGTA)的加入降低chitosan对细胞G6PDH的诱导程度,显示chitosan对G6PDH的诱导需要Ca²⁺的参与。谷胱甘肽(GHS)的处理可反馈抑制chitosan对细胞G6PDH的诱导。通过分析调节后G6PDH的各种活性与细胞中紫杉醇产量的关系,认为采用合适的处理方法调节磷酸戊糖途径,有利于红豆杉细胞合成紫杉醇。     

Cytochemical assay of glucose-6-phosphate dehydrogenase in koilocytes

New cervical smears were obtained from 24 patients with a cytologic diagnosis of typical condyloma for a cytochemical assay of glucose-6-phosphate dehydrogenase (G6PDH) activity in the koilocytes that are pathognomonic of this lesion. The smears were air dried and were processed according to Nachlas' modified technique. The controls used were smears from normal cases (which show no G6PDH activity), from dysplasias (which show high levels) and from carcinomas (which show very high G6PDH levels). In the cases of typical condyloma studied, the level of G6PDH was null in 16 (66.7%), very low in 2 (8.3%) and low in 6 (25.0%). If this assay for G6PDH gives the total enzymatic activity of the cell, showing low enzymatic levels in condylomas and high enzymatic levels in dysplasias and carcinomas, an increase in G6PDH activity could indicate the transition of an intraepithelial lesion from condyloma to cervical intraepithelial neoplasia.     

Quantitative comparison between the gel-film and polyvinyl alcohol methods for dehydrogenase histochemistry reveals different intercellular distribution patterns of glucose-6-phosphate and lactate dehydrogenases in mouse liver

Summary The precise histochemical localization and quantification of the activity of soluble dehydrogenases in unfixed cryostat sections requires the use of tissue protectants. In this study, two protectants, polyvinyl alcohol (PVA) and agarose gel, were compared for assaying the activity of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) in normal female mouse liver. Quantification of enzyme activity was determined cytophotometrically in periportal (PP), pericentral (PC) and midzonal (MZ) areas. No coloured reaction product was present in PVA media after the incubation period. In contrast, the agarose gels appeared to be highly coloured after incubation. As a consequence, sections incubated with gel media were less intensely stained than those incubated in PVA-containing media. The specific G6PDH reaction (test minus control) yielded approximately 75% less formazan in sections incubated by the agarose gel method than with the PVA method. Further, the amount of formazan deposits attributable to G6PDH activity was highest in the midzonal and pericentral zones of the liver lobule with PVA media, and Kupffer cells could be discriminated easily because of their high G6PDH activity. Significant zonal differences or Kupffer cells could not be observed when agarose gel films were used for the detection of G6PDH activity. The LDH localization patterns appeared to be more uniform after incubation with both methods: no significant differences in specific test minus control reactions were seen between PP, PC and MZ. However, less formazan production (33%) was detected in sections incubated with agarose gels when compared with those incubated with PVA media. These results clearly show that the gel method is not suitable for the valid demonstration of activity of (partially) soluble enzymes. Furthermore, our results confirm that a greater proportion of G6PDH than of LDH is present in a soluble form in liver cells.     

Glucose-6-phosphate dehydrogenase regulation during hypometabolism

Glucose-6-phosphate dehydrogenase (G6PDH) from hepatopancreas of the land snail, Otala lactea, shows distinct changes in properties between active and estivating (dormant) states, providing the first evidence of pentose phosphate cycle regulation during hypometabolism. Compared with active snails, G6PDH Vmax increased by 50%, Km for glucose-6-phosphate decreased by 50%, Ka Mg x citrate decreased by 35%, and activation energy (from Arrhenius plots) decreased by 35% during estivation. DEAE-Sephadex chromatography separated two peaks of activity and in vitro incubations stimulating protein kinases or phosphatases showed that peak I (low phosphate) G6PDH was higher in active snails (57% of activity) whereas peak II (high phosphate) G6PDH dominated during estivation (71% of total). Kinetic properties of peaks I and II forms mirrored the enzyme from active and estivated states, respectively. Peak II G6PDH also showed reduced sensitivity to urea inhibition of activity and greater stability to thermolysin protease treatment. The interconversion of G6PDH between active and estivating forms was linked to protein kinase G and protein phosphatase 1. Estivation-induced phosphorylation of G6PDH may enhance relative carbon flow through the pentose phosphate cycle, compared with glycolysis, to help maintain NADPH production for use in antioxidant defense.     

Age-related changes of glucose-6-phosphate dehydrogenase activity in mouse oocytes

Summary Glucose-6-phosphate dehydrogenase (G6PDH) activity was measured in follicular oocytes and in ovulated eggs of prepubertal, adult and aged mice. G6PDH activity in ovulated eggs was 60% of the activity in follicular oocytes in all age groups. The mean G6PDH activity was significantly higher in follicular oocytes of adult mice than in oocytes of both prepubertal and aged mice. In aged mice, the decreased mean activity in follicular oocytes as well as in ovulated eggs was mainly due to a high percentage of cells with extremely low activity (25 and 18%, respectively). The percentage of preovulatory oocytes with low activity in prepubertal mice was 9% and in adult mice 0.3%. For ovulated eggs these percentages were 0% for both prepubertal and adult mice. In every age group, all ovulated eggs showed a normal morphology. When ovulated eggs with extremely low G6PDH activity can still be fertilized, it can be questioned whether this loss of activity could cause disturbances in development of (preimplantation) embryos. Our findings emphasize the potentialities of investigating intact single oocytes for changes in enzyme activities, which could be applied as parameters for quality control of these cells.     

Loss of peroxisomes causes oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase activity to detect cancer cells.

Oxygen insensitivity of carcinoma cells and oxygen sensitivity of non-cancer cells in the histochemical assay of glucose-6-phosphate dehydrogenase (G6PD) enables detection of carcinoma cells in unfixed cell smears or cryostat sections of biopsies. The metabolic background of oxygen insensitivity is still not understood completely. In the present study, rat hepatocytes, rat hepatoma cells (FTO-2B), and human colon carcinoma cells (HT29) were used to elucidate these backgrounds. The residual activity in oxygen was 0%, 55%, and 80% in hepatocytes, hepatoma cells, and colon carcinoma cells, respectively. N-ethylmaleimide (NEM), a blocker of SH-groups, did not affect G6PD activity in both carcinoma cell types but reduced G6PD activity in hepatocytes by 40%. Ultrastructural localization of G6PD activity was exclusively in the cytoplasm of carcinoma cells, but in hepatocytes both in cytoplasm and peroxisomes. NEM abolished peroxisomal G6PD activity only. Histochemical assay of catalase activity demonstrated absence of peroxisomes in both carcinoma cell lines. It is concluded that absence of SH-sensitive G6PD activity in peroxisomes in cancer cells is responsible for the oxygen-insensitivity phenomenon.     

High activity of glucose-6-phosphate dehydrogenase in Kupffer cells isolated from rat liver

Summary Sinusoidal cells in the rat liver react intensively for G6DPH activity after appropriate incubation (Rieder et al. 1978). After isolation and purification of the sinusoidal Kupffer and endothelial cells, it was demonstrated that Kupffer cells exhibit a 5–8 times higher G6PDH activity on a per cell basis by comparison with endothelial cells, while the specific G6PDH activity was 3–4 times higher in Kupffer cells. The Kupffer cells can be divided into two groups which differ significantly in G6PDH activity calculated on a per cell basis. In histochemical studies, G6PDH can be used as a marker for Kupffer cell identification.     

Importance of glucose-6-phosphate dehydrogenase in taxol biosynthesis in <Emphasis Type="Italic">Taxus chinensis</Emphasis> cultures

The roles of glucose-6-phosphate dehydrogenase (G6PDH) in paclitaxel production were investigated in cell suspension cultures of Taxus chinensis. In the normal cultures, the trend of G6PDH activity was similar to that of cell growth. Addition of glutamate increased G6PDH activity, while dehydroepiandrosterone (DHEA) decreased G6PDH activity. In elicitor-treated cultures, cell growth was depressed, while G6PDH activity and taxol production were enhanced compared with the control. Glutamate recovered the depression of cell growth, and resulted in further increase in G6PDH activity and taxol production. Contrarily, DHEA exacerbated the depression of cell growth, and decreased G6PDH activity and taxol production induced by fungal elicítor. The results indicated that G6PDH played a critic role of taxol production by affecting cell viability.     

Effects of dehydroepiandrosterone on obesity and glucose-6-phosphate dehydrogenase activity in the lethal yellow mouse (strain 129/Sv-Ay/Aw)

We investigated the anti-obesity effects of the adrenal androgen, dehydroepiandrosterone (DHEA), on genetically predisposed obese lethal yellow mice (Ay/Aw). Secondly, we tested the hypothesis that DHEA promotes its anti-obesity effects by decreasing the activity of glucose-6-phosphate dehydrogenase (G6PDH). We subjected four genotype-sex combinations of yellow and agouti (control) mice to four dietary treatments and determined weight changes, food consumption, and G6PDH activity. Although G6PDH activities of yellow mice were considerably decreased in the 0.4% DHEA treatment group, they were elevated in the 0.0 and 0.1% DHEA treatment groups. In contrast, G6PDH activities of DHEA-treated control agouti mice remained relatively constant. These studies confirm that DHEA prevents the Ay gene from promoting excess fat deposition via some mechanism(s) other than reduced dietary intake. However, the overall absence of agreement between weight change (gain or loss) and G6PDH activity suggests that the anti-obesity activity of DHEA is not mediated via G6PDH. Since yellow obese (Ay/Aw) mice were found to be more susceptible to DHEA's effects than their agouti (Aw/Aw) littermates, Ay appears to induce an altered metabolism in Ay/Aw mice which is more susceptible to the effects of DHEA than the normal metabolism of Aw/Aw mice.     

Quantitative aspects of the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetranitro BT studied in a model system of polyacrylamide films

Summary The cytochemical determination of the activity of glucose-6-phosphate dehydrogenase (G6PDH) with tetranitro blue tetrazolium (TNBT) was studied with model films of polyacrylamide gel incorporating purified enzyme. This model system enabled a quantitative study to be made of different parameters involved with the cytochemical assay as it is applied to sections or smears. The enzyme activity of G6PDH incorporated in the model films was also assayed biochemically. Optimal conditions for retaining the maximum amount of enzymic activity are described. The behaviour of G6PDH towards enzyme inhibitors was found to be similar in model films and in solution. With TNBT, absorbance measurements at a single wavelength (535 nm) were used to estimate the enzyme activity quantitatively. When carried out under standardized conditions, both the cytochemical and biochemical assay showed a linear relation with the time of incubation and obeyed the Beer-Lambert law. The correlation between biochemical and cytochemical data was very high, which enabled cytochemical data to be converted into absolute units of enzyme activity. The data obtained in this way closely resembled the data of enzyme activity calculated from the absorbance of formazan produced inside polyacrylamide model films and afterwards extracted into a suitable solvent.     

Mir-335-5p靶向G6PD对结肠癌细胞增殖、凋亡的影响

目的:探讨Mir-335-5p通过靶向G6PD对结肠癌细胞增殖、凋亡的影响.方法:设置正常结肠细胞组、空白对照组、NC组、miRNA-335-5p mimic组;体外培养结肠上皮细胞(IEC)和人源性结肠癌细胞SW480,并对NC组、miRNA-335-5p mimic组细胞进行转染;采用RT-qPCR检测各组细胞中m...     

Semiquantitative cytochemical estimation of glucose-6-phosphate dehydrogenase activity in benign diseases and carcinoma of the breast

The level of glucose-6-phosphate dehydrogenase (G6PDH) activity was semiquantitatively evaluated in fresh imprints of infiltrative ductal carcinoma, fibrocystic disease and fibroadenoma of the breast. A significantly higher level of G6PDH activity was found in the carcinomas. The results suggest that the estimation of G6PDH activity could be a valuable method for evaluating the cells in benign and malignant breast lesions. It is possible that the intensification of G6PDH activity in carcinomas is a sign of the shift of the carbohydrate metabolism from an aerobic path or that the activity of the pentose shunt is higher because of the increased need for nucleic acid precursors in tissues with faster growth rates.     

Ploidy class-dependent metabolic changes in rat hepatocytes after partial hepatectomy

Glucose-6-phosphate dehydrogenase (G6PDH), succinate dehydrogenase (SDH) activity and the single-stranded RNA (ssRNA) content of isolated hepatocytes of different ploidy classes from adult male rats have been studied after partial hepatectomy using quantitative cytochemical means. The SDH activity and ssRNA content in all classes of hepatocytes are decreased during the first hours after operation followed by an increase above control values. The increase of both SDH activity and ssRNA content is significant only in the mononuclear diploid (MD) cells but not in the hepatocytes of higher ploidy classes and is related with the mitotic wave at 32 h after hepatectomy. After the mitotic wave, the values quickly return to normal levels. The G6PDH activity does not show any significant change in hepatocytes other than MD cells. In MD cells the G6PDH activity is elevated on a highly significant level up to a maximum value of 3.5 times the control value at 48 h after operation. The G6PDH activity in MD cells is returned to normal values within 14 days after operation. It is concluded that: 1. The MD cells show a distinct metabolic behaviour due to their function as stem cells of liver parenchyma and retain at least some of their fetal characteristics. 2. G6PDH activity is not a transformation-linked discriminant for neoplastic metabolism.     

Glucose-6-phosphate dehydrogenase activity in individual rat hepatocytes of different ploidy classes. I. Developments during postnatal growth

The glucose-6-phosphate dehydrogenase (G6PDH) activity of isolated male rat hepatocytes has been investigated in relationship to the ploidy classes of the cells during the first 20 weeks of postnatal growth. The G6PDH activity in the individual cells was measured with an improved quantitative cytochemical method. The data obtained showed that throughout the whole period of postnatal growth there existed a proportional relationship between the genome copies per cell and the amount of G6PDH activity per cell for binuclear diploid (BD), mononuclear tetraploid (MT) and binuclear tetraploid (BT) cells but not for mononuclear diploid (MD) cells. In the MD cells, which are the stem cells of the liver parenchyma, the activity measured was 1.5 times higher than expected. Furthermore, during postnatal growth, the G6PDH activity per hepatocyte was low at the age of 2 weeks, increased somewhat after weaning (5 weeks) and then more dramatically after 8 weeks to reach a maximum between 12 and 16 weeks. This development occurred in MT and BT cells at an earlier age than in MD and BD cells, in which the increase in enzyme activity followed some 3 weeks later. Castration of the rats before puberty did not influence the development of the amount of G6PDH activity per cell of any of the ploidy classes.     

Coordination of Chloroplastic Metabolism in N-Limited Chlamydomonas reinhardtii by Redox Modulation (I. The Activation of Phosphoribulosekinase and Glucose-6-Phosphate Dehydrogenase Is Relative to the Photosynthetic Supply of Electrons)

Extraction of Chlamydomonas reinhardtii CW-15 cells by rapid freezing and thawing demonstrates that the in vivo activity of the algal glucose-6-phosphate dehydrogenase (G6PDH) is inhibited by the presence of light and activated in the dark, whereas phosphoribulosekinase (PRK) is light activated and inhibited in the dark. The effects of darkening are reversed by incubation with dithiothreitol (DTT) and mimicked by chemical oxidants, indicating that, as in higher plants, reduction via the ferredoxin-thioredoxin system likely regulates these enzymes. The two enzymes varied in their sensitivity to reduction; the inclusion of 0.5 mM DTT during extraction inhibited G6PDH, whereas PRK required treatment with 40 mM DTT for 1 h to reach maximum activation. The activation change for both enzymes was nearly complete within the 1st min after cells were transferred between light and dark, but the level of activation was relative to the incident light at low intensities; G6PDH activity decreased with increasing light, whereas PRK became more active. The reductive inhibition of G6PDH saturated at very low light, whereas PRK activation kinetics closely followed the increase in photosynthetic oxygen evolution. These results indicate that light-driven redox modulation of G6PDH and PRK is more than an on/off switch, but acts to optimize the reduction and oxidation of carbon in the chloroplast in accordance with the supply of electrons.     

Release of glucose-6-phosphate dehydrogenase from cortex of Spisula eggs at fertilization and its recombination after meiosis

Changes in subcellular distributions of glucose-6-phosphate dehydrogenase (G6PDH) were observed after fertilization or artificial (KCl) activation of Spisula eggs. Though the total activity of G6PDH did not change during early stages, that in the 100,000g supernatant fraction increased after fertilization, attained a maximum at the first meiotic metaphase, and then decreased. This change of activity in the supernatant was accompanied by a mirror-image change of activity in the pellet. Most of the G6PDH was localized in the 3000g pellet fraction; furthermore, the activity in isolated cortices showed fluctuations during meiosis similar to that of the 3000g pellet fraction. Conditions for the release and binding of the NADP-specific G6PDH from the pellet fraction were investigated in vitro. NADP⁺ or NADPH can induce release of G6PDH, although NADPH is three to four times more efficient than NADP⁺. NAD⁺ does not affect release. High concentrations of salts (ionic strength >0.3) caused complete G6PDH release from the pellet. Although raising the pH alone showed only a slight releasing effect, increase of pH to pH 7 or above considerably augmented release due to NADP⁺ or NADPH. The release of G6PDH from the pellet fraction was shown to be reversible. These results suggest that the reversible association of G6PDH with particulate components of the cytoplasm may play an important role in regulation of G6PDH activity in marine eggs and that the cortex is one of the sites which may be involved in such regulation. The mechanism of recombination of G6PDH with its sites remains to be elucidated.     

Syndecan-2 mediates adhesion and proliferation of colon carcinoma cells

Syndecan-2 is a transmembrane heparan sulfate proteoglycan whose function at the cell surface is unclear. In this study, we examined the function of syndecan-2 in colon cancer cell lines. In several colon cancer cell lines, syndecan-2 was highly expressed compared with normal cell lines. In contrast, syndecan-1 and -4 were decreased. Cell biological studies using the extracellular domain of recombinant syndecan-2 (2E) or spreading assay with syndecan-2 antibody-coated plates showed that syndecan-2 mediated adhesion and cytoskeletal organization of colon cancer cells. This interaction was critical for the proliferation of colon carcinoma cells. Blocking with 2E or antisense syndecan-2 cDNA induced G(0)/G(1) cell cycle arrest with concomitantly increased expression of p21, p27, and p53. Furthermore, blocking of syndecan-2 through antisense syndecan-2 cDNA significantly reduced tumorigenic activity in colon carcinoma cells. Therefore, increased syndecan-2 expression appears to be a critical for colon carcinoma cell behavior, and syndecan-2 regulates tumorigenic activity through regulation of adhesion and proliferation in colon carcinoma cells.