ARTICLE |
Correspondence to: Wilma M. Frederiks, Dept. of Cell Biology and Histology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail: w.m.frederiks@amc.uva.nl
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Summary |
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Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) is the key regulatory enzyme of the pentose phosphate pathway and produces NADPH and riboses. In this study, the kinetic properties of G6PD activity were determined in situ in chemically induced hepatocellular carcinomas, and extralesional and control parenchyma in rat livers and were directly compared with those of the second NADPH-producing enzyme of the pentose phosphate pathway, phosphogluconate dehydrogenase (PGD). Distribution patterns of G6PD activity, protein, and mRNA levels were also compared to establish the regulation mechanisms of G6PD activity. In (pre)neoplastic lesions, the Vmax of G6PD was 150-fold higher and the Km for G6P was 10-fold higher than in control liver parenchyma, whereas in extralesional parenchyma, the Vmax was similar to that in normal parenchyma but the Km was fivefold lower. This means that virtual fluxes at physiological substrate concentrations are 20-fold higher in lesions and twofold higher in extralesional parenchyma than in normal parenchyma. The Vmax of PGD was fivefold higher in lesions than in normal and extralesional liver parenchyma, whereas the Km was not affected. Amounts of G6PD protein and mRNA were similar in lesions and in extralesional liver parenchyma. These results demonstrate that G6PD is strongly activated post-translationally in (pre)neoplastic lesions to produce NADPH. (J Histochem Cytochem 51:105112, 2003)
Key Words: glucose-6-phosphate, dehydrogenase, phosphogluconate, dehydrogenase, (pre)neoplasm, hepatoma, enzyme histochemistry, immunohistochemistry, in situ hybridization, image analysis
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Introduction |
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GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD; EC 1.1.1.49) is a housekeeping enyzme that catalyzes the first step in the pentose phosphate pathway. It produces riboses, which are incorporated into nucleotides and nucleic acids, and NADPH, the major cytoplasmic reducing compound (
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Materials and Methods |
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Induction of Neoplasms in Liver
(Pre)neoplastic lesions were induced in livers of five male Wistar rats weighing 250300 g by administration of 0.01% diethylnitrosamine (DENA; Sigma, St Louis, MO) via drinking water for 9 weeks (
Localization of G6PD and PGD Activity
Cryostat sections were allowed to dry at room temperature (RT) for 5 min and were then incubated for the demonstration of G6PD and PGD activity according to
To test whether PGD activity was involved in the assay for the demonstration of G6PD activity, sections were incubated in the presence of both 10 mM G6P and 10 mM PG. The test minus control reaction of the latter assay was diminished with the test minus control reaction of PGD and was taken as a measure for the actual G6PD activity (
Immunohistochemistry of G6PD Protein
Rat G6PD protein was demonstrated in livers of control rats and DENA-treated rats using a polyclonal rabbit anti-yeast G6PD antibody (Sigma; dilution 1:200) according to
In Situ Hybridization of G6PD mRNA
The procedure to localize G6PD mRNA was performed as described by
Image Analysis and Image Processing
Serial sections of control liver and livers of DENA-treated rats were used to determine Vmax and Km values of G6PD and PGD using image analysis. In each section (three sections per enzyme and per animal), three areas were selected. In each selected area, three measurements were made and mean gray values were calculated. Gray values were converted into absorbance values by using a set of neutral density filters (Kodak, Rochester, NY;
Statistics
Vmax and Km values were expressed as mean ± SD. Student's t-tests were employed for statistical analysis; differences were tested at the 0.05 level of significance. The Vmax and Km values were calculated from plots of substrate concentrations vs the ratio of substrate concentration and reaction velocity according to
y = ax + b
in which 1/a is the Vmax value and b/a is the Km value. Flux rates (ø) of G6PD and PGD with their respective substrates were calculated with the formula
ø = Vmax x [s]
Km + [s]
with an assumed substrate concentration of 100 µM as was calculated for rat liver by
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Results |
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Localization of G6PD Activity
Fig 1A shows the localization of G6PD activity in control rat liver. Low activity was found in liver parenchymal cells. Highest activity was observed in pericentral and midzonal areas. Very high amounts of final reaction product were found in sinusoidal cells in periportal areas, which are Kupffer cells (
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Localization of PGD Activity
PGD activity was exclusively localized in liver parenchymal cells in control rat liver, with a higher activity in pericentral and midzonal areas (Fig 1C). In livers of DENA-treated rats, PGD activity was distinctly higher in (pre)neoplastic lesions than in extralesional parenchyma (Fig 1D).
Kinetic Parameters of G6PD and PGD
We found that amounts of formazan produced in the presence of both G6P and PG were similar to the sum of the amounts of formazan produced in the presence of G6P alone and PG alone. This means that PG produced in the G6PD reaction was hardly converted by PGD and that, in the presence of G6P, only G6PD was active. This is in agreement with previous findings of
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Immunohistochemical Localization of G6PD Protein
Distribution patterns of G6PD protein in control liver are shown in Fig 2C. A homogeneous distribution pattern of G6PD protein was found in liver parenchyma. Higher amounts of G6PD protein were observed in epithelial cells of bile ducts and blood vessel walls in portal tracts. Non-parenchymal sinusoidal cells could not be discriminated from liver parenchymal cells on the basis of G6PD protein content. Fig 2D shows that G6PD protein is homogeneously distributed in livers of DENA-treated rats with similar amounts in (pre)neoplastic lesions and extralesional liver parenchyma. Control incubations resulted in absence of staining in control livers and livers of DENA-treated rats.
In Situ Hybridization of G6PD mRNA
Fig 2E shows the localization of G6PD mRNA in control liver. mRNA levels were similar in all zones of liver parenchyma. G6PD mRNA was also present in epithelial cells of bile ducts and blood vessel walls in portal tracts. Again, non-parenchymal sinusoidal cells could not be discriminated from liver parenchyma on the basis of G6PD mRNA levels. In livers of DENA-treated rats, the distribution pattern of G6PD mRNA was similar to that of G6PD protein: differences between mRNA levels in (pre)neoplastic lesions or extralesional parenchyma were not observed (Fig 2F). Pretreatment of the sections with RNase completely abolished generation of final reaction product.
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Discussion |
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The major findings of the present study are that activity of G6PD and, to a lesser extent, of PGD in (pre)neoplastic lesions in rat livers after chemical induction by the carcinogen DENA was strongly upregulated. G6PD was activated at the post-translational level because G6PD protein and G6PD mRNA levels were similar in (pre)neoplastic lesions, extralesional liver parenchyma, and normal parenchyma. Thus far, induction of G6PD activity was demonstrated at the gene level, e.g., as a consequence of hormone treatment (
The role of G6PD in proliferation has been shown in studies using the non-competitive G6PD inhibitor dehydroepiandrosterone. Proliferation of cancer cells was slowed down by the G6PD inhibitor (
Furthermore, we have shown in the present study that the higher G6PD activity in epithelial cells of bile ducts and blood vessel walls than in liver parenchyma was regulated at the pretranslational level because activity, protein, and mRNA of G6PD were all elevated in a similar way in these cells in comparison with liver parenchyma (Fig 2). In contrast, the high G6PD activity in Kupffer cells was not accompanied by higher levels of protein and mRNA of G6PD, suggesting upregulation of G6PD activity at the post-translational level (see also
Post-translational regulation of G6PD activity in (pre)neoplastic lesions in livers of DENA-treated rats involved both Vmax and Km values (Table 1). Upregulation of G6PD activity, but not of PGD activity, by changes in its kinetic parameters has been found previously in livers of methylcholantrene- and phenobarbital-treated rats (
In conclusion, the consequences of the distinct post-translational upregulation of G6PD and slightly increased activity of PGD in (pre)neoplastic lesions in livers of DENA-treated rats are mainly increased production of NADPH as a reductive power in the cytoplasm.
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Acknowledgments |
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We are grateful to Dr C.-G. Wu for providing the rat specimens, Mr J. Peeterse for photographic work, and Ms T.M.S. Pierik for preparation of the manuscript.
Received for publication April 3, 2002; accepted July 23, 2002.
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