Histochemical localization of NADP-dependent dehydrogenase activity with four different tetrazolium salts.

Noorden, C. J. F. Van; Butcher, R. G.

doi:10.1177/32.9.6747280

Cited by 46 publications

(28 citation statements)

References 21 publications

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“…Increased incubation times give rise to anoxic conditions of the incubation due to the use of oxygen. Furthermore, the relatively low molecular extinction coefficient of NT formazan makes the assay relatively insensitive when compared with the formazan of other tetrazolium salts such as nitro BT and tetranitro BT (Van Noorden and Butcher 1984). Oxygen insensitivity of G6PD activity only seems to be true for adenocarcinoma cells and, for instance, not for fibrosarcoma cells (unpublished data).…”

Section: Discussionmentioning

confidence: 99%

“…Hepatocytes and FTO-2B cells were incubated for 10 min at 37C, and HT29 cells were incubated for 3 min at 37C. Control reactions were incubated in the absence of substrate and coenzyme (Van Noorden and Butcher 1984;Butcher and Van Noorden 1985). Involvement of SH-groups in G6PD activity was investigated by addition of 10 mM N-ethylmaleimide (NEM, BDH; Poole, UK) to the incubation medium .…”

Section: Histochemical Localization Of G6pd Activitymentioning

confidence: 99%

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Loss of Peroxisomes Causes Oxygen Insensitivity of the Histochemical Assay of Glucose-6-Phosphate Dehydrogenase Activity to Detect Cancer Cells

Frederiks

Vreeling-Sindelárová

Noorden

2006

J Histochem Cytochem.

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S U M M A R Y 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. (J Histochem Cytochem 55:175-181, 2007)

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Section: Discussionmentioning

confidence: 99%

Section: Histochemical Localization Of G6pd Activitymentioning

confidence: 99%

Loss of Peroxisomes Causes Oxygen Insensitivity of the Histochemical Assay of Glucose-6-Phosphate Dehydrogenase Activity to Detect Cancer Cells

Frederiks

Vreeling-Sindelárová

Noorden

2006

J Histochem Cytochem.

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“…In their study of the complete (6e-) nitroreduction of 2-nitroimidazole benznidazole by mouse liver Walton and Workman (1987) found that the microsomal enzymes NADPH: cytochrome P-450 (cytochrome c) reductase and cytochrome P-450 mainly were implicated. These enzymes are most active in the centrilobular zone of the liver (Van Noorden & Butcher, 1984). The liver and the gastrointestinal tract are identified as the prime sites for nitroreduction (Chin & Rauth, 1981).…”

Section: Discussionmentioning

confidence: 99%

NAD(P)H nitroblue tetrazolium reductase levels in apparently normoxic tissues: a histochemical study correlating enzyme activity with binding of radiolabelled misonidazole

Cobb¹,

Hacker²,

Nolan³

1990

Br J Cancer

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Hack and Helmy's method for the histochemical identification of NAD(P)H nitroblue tetrazolium reductase activity was employed to pinpoint reductase activity in certain cells in the mouse. High activity was observed in the following: lower airway epithelium, liver (centrilobular zone), eyelid (meibomian and sebaceous glands), vulval gland and parotid gland (striated cells of intralobular ducts). All of these cells had previously been identified as sites of binding of the reactive metabolites formed from the enzymic reduction of misonidazole (MISO) (Cobb et al., 1989). It had previously been thought that MISO binding would only take place in significant amounts in hypoxic tissues (tumour and possibly liver) since in normoxic tissues oxygen should reverse the initial one electron enzymic reduction, thus preventing progressive reduction to reactive species. We suggest that the very high levels of reductase in the above listed, probably normoxic, tissues contribute significantly to the accumulation of bound reactive MISO metabolite(s). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7

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“…Control reactions were incubated in the absence of substrate and coenzyme. 30,31 In situ determination of transketolase activity was performed according to Boren et al 32 Incubation medium contained 18% (w/ v) PVA in 50 mM Tris-HCl buffer, pH 7.6, 5 mM sodium azide, 7.5 mM NAD 1 , 3.7 mM KH 2 PO 4 , 5 mM MgCl 2 , 0.45 mM methoxyphenazine methosulphate, 100 lL substrate mixture (prepared as described above) per mL incubation medium, 0.1 mM TPP and 5 mM NBT. The medium was freshly prepared just before incubation and NBT was added after being dissolved in a heated mixture of DMF and ethanol (1:1) (final dilution of each solvent in the medium was 2% v/v).…”

Section: Cell Sortingmentioning

confidence: 99%

Modulation of pentose phosphate pathway during cell cycle progression in human colon adenocarcinoma cell line HT29

Vizán

Alcarraz-Vizán

Díaz-Moralli

et al. 2009

Intl Journal of Cancer

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Cell cycle regulation is dependent on multiple cellular and molecular events. Cell proliferation requires metabolic sources for the duplication of DNA and cell size. However, nucleotide reservoirs are not sufficient to support cell duplication and, therefore, biosynthetic pathways should be upregulated during cell cycle. Here, we reveal that glucose-6-phosphate dehydrogenase (G6PDH) and transketolase (TKT), the 2 key enzymes of oxidative and nonoxidative branches of the pentose phosphate pathway (PPP), respectively, which is necessary for nucleotide synthesis, are enhanced during cell cycle progression of the human colon cancer cell line HT29. These enhanced enzyme activities coincide with an increased ratio of pentose monophosphate to hexose monophosphate pool during late G1 and S phase, suggesting a potential role for pentose phosphates in proliferating signaling. Isotopomeric analysis distribution of nucleotide ribose synthesized from 1,2-13 C 2 -glucose confirms the activation of the PPP during late G1 and S phase and reveals specific upregulation of the oxidative branch. Our data sustain the idea of a critical oxidative and nonoxidative balance in cancer cells, which is consistent with a late G1 metabolic check point. The distinctive modulation of these enzymes during cell cycle progression may represent a new strategy to inhibit proliferation in anticancer treatments. ' 2009 UICC

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Histochemical localization of NADP-dependent dehydrogenase activity with four different tetrazolium salts.

Abstract: (4A0016) ductase as demonstrated immunocytochemically. BPST did not properly localize dehydrogenase activity, most probably because of the redistribution of formazan, due to its

Cited by 46 publications

References 21 publications

Loss of Peroxisomes Causes Oxygen Insensitivity of the Histochemical Assay of Glucose-6-Phosphate Dehydrogenase Activity to Detect Cancer Cells

Loss of Peroxisomes Causes Oxygen Insensitivity of the Histochemical Assay of Glucose-6-Phosphate Dehydrogenase Activity to Detect Cancer Cells

NAD(P)H nitroblue tetrazolium reductase levels in apparently normoxic tissues: a histochemical study correlating enzyme activity with binding of radiolabelled misonidazole

Modulation of pentose phosphate pathway during cell cycle progression in human colon adenocarcinoma cell line HT29

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