In situ analysis of ischaemia/reperfusion injury in rat liver studied in three different models

Animal models of liver ischaemia and reperfusion are frequently used to study the consequences on liver cells of transient oxygen deprivation. In 3 different rat models we studied ischaemia/reperfusion effects on liver cell membrane integrity, cytoplasmic enzyme proteins and enzyme activities by in situ histochemical techniques. In vivo ischaemia, as well as no-flow hypoxia, or N2-induced hypoxia in isolated perfused livers, reduced the activity of 5′-nucleotidase, a sensitive marker for plasma membrane damage in hepatocytes. As little as 2 minutes of reoxygenation in each model resulted in leakage of soluble enzymes from parenchymal and non-parenchymal liver cells, as shown by decreased protein level and activity of cytoplasmic enzymes. Whereas a multifocal decrease was observed after in vivo reperfusion, a decrease was found in all periportal and midzonal cells after blood-free reoxygenation. As judged by alkaline phosphatase activity and immunohistochemistry, an influx of inflammatory cells was not found in the in vivo model. Our findings indicate that reoxygenation itself, rather than restoration of flow, accounts for the loss of soluble enzymes from liver cells after a period of hypoxia. In situ detection of enzyme protein and activity proved useful for the examination of very early ischaemia/reperfusion effects on rat liver 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 phenomeno

The role of gelatinases in colorectal cancer progression and metastasis

Various proteases are involved in cancer progression and metastasis. In particular, gelatinases, matrix metalloproteinase-2 (MMP-2) and NIMP-9, have been implicated to play a role in colon cancer progression and metastasis in animal models and patients. In the present review, the clinical relevance and the prognostic value of messenger ribonucleic acid (mRNA) and protein expression and proenzyme activation of MMP-2 and MMP-9 are evaluated in relation to colorectal cancer. Expression of tissue inhibitors of MMPs (TIMPs) in relation with MMP expression in cancer tissues and the relevance of detection of plasma or serum levels of MMP-2 and/or MMP-9 and TIMPs for prognosis are also discussed. Furthermore, involvement of MMP-2 and MMP-9 in experimental models of colorectal cancer is reviewed. In vitro studies have suggested that gelatinase is expressed in cancer cells but animal models indicated that gelatinase expression in non-cancer cells in tumors contributes to cancer progression. In fact, interactions between cancer cells and host tissues have been shown to modulate gelatinase expression in host cells. Inhibition of gelatinases by synthetic MMP inhibitors has been considered to be an attractive approach to block cancer progression. However, despite promising results in animal models, clinical trials with MMP inhibitors have been disappointing so far. To obtain more insight in the (patho)physiological functions of gelatinases, regulation of MMP-2 and MMP-9 expression is discussed. Mitogen activated protein kinase (MAPK) signalling has been shown to be involved in regulation of gelatinase expression in both cancer cells and non-cancer cells. Expression can be triggered by a variety of stimuli including growth factors, cytokines and extracellular matrix (ECM) components. On the other hand, MMP-2 and MMP-9 activity regulates bioavailability and activity of growth factors and cytokines, affects the immune response and is involved in angiogenesis. Because of the multifunctionality of gelatinases, it is unpredictable at what stage of cancer development and in which processes gelatinase activity is involved. Therefore, it is concluded that the use of MMP inhibitors to treat cancer should be considered carefully. (C) 2004 Elsevier B.V, All rights reserve

Improved localization of glucose-6-phosphate dehydrogenase activity in cells with 5-cyano-2,3-ditolyl-tetrazolium chloride as fluorescent redox dye reveals its cell cycle-dependent regulation

Since the introduction of cyano-ditolyl-tetrazolium chloride (CTC), a tetrazolium salt that gives rise to a fluorescent formazan after reduction, it has been applied to quantify activity of dehydrogenases in individual cells using flow cytometry. Confocal laser scanning microscopy (CLSM) showed that the fluorescent formazan was exclusively localized at the surface of individual cells and not at intracellular sites of enzyme activity. In the present study, the technique has been optimized to localize activity of glucose-6-phosphate dehydrogenase (G6PD) intracellularly in individual cells. Activity was demonstrated in cultured fibrosarcoma cells in different stages of the cell cycle. Cells were incubated for the detection of G6PD activity using a medium containing 6% (w/v) polyvinyl alcohol, 5 mM CTC, magnesium chloride, sodium azide, the electron carrier methoxyphenazine methosulphate, NADP, and glucose-6-phosphate. Before incubation, cells were permeabilized with 0.025% glutaraldehyde. Fluorescent formazan was localized exclusively in the cytoplasm of fibrosarcoma cells. The amount of fluorescent formazan in cells increased linearly with incubation time when measured with flow cytometry and CLSM. When combining the Hoechst staining for DNA with the CTC method for the demonstration of G6PD activity, flow cytometry showed that G6PD activity of cells in S phase and G2/M phase is 27 +/- 4% and 43 +/- 4% higher, respectively, than that of cells in G1 phase. CLSM revealed that cells in all phases of mitosis as well as during apoptosis contained considerably lower G6PD activity than cells in interphase. It is concluded that posttranslational regulation of G6PD is responsible for this cell cycle-dependent activit

Renal cell carcinoma and oxidative stress: The lack of peroxisomes

Oxidative stress plays an important role in carcinogenesis because of induction of DNA damage and its effects on intracellular signal transduction pathways. Here, we investigated the relationship between the defence against oxidative stress and human renal cell carcinoma that originates from proximal tubular epithelium. Oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase (G6PD) activity is a diagnostic tool for the detection of carcinomas. Its mechanism is based on high G6PD activity, reduced superoxide dismutase activity and reduced numbers of peroxisomes in the cancer cells. Five out of the 8 renal carcinomas studied here demonstrated oxygen insensitivity. These carcinomas showed high G6PD activity, whereas the other 3 carcinomas contained lower G6PD activity and were oxygen sensitive like non-cancer cells. Oxygen insensitivity did not correlate with tumour grade, staging or presence of metastases. Electron microscopy and immunofluorescence of catalase showed large numbers of peroxisomes in epithelial cells of proximal tubules of normal human kidney, whereas these organelles were completely absent in cancer cells of all carcinomas. As a consequence of the absence of peroxisomes in cancer cells, fatty acid metabolism is disturbed in addition to the altered glucose metabolism that is generally observed in cancer cells. Therefore, therapeutic approaches should focus on metabolism in addition to other strategies targeting signal transduction and angiogenesis. (C) 2009 Published by Elsevier Gmb

In situ localization of gelatinolytic activity in the extracellular matrix of metastases of colon cancer in rat liver using quenched fluorogenic DQ-gelatin

Matrix metalloproteinases (MMPs) such as gelatinases are believed to play an important role in invasion and metastasis of cancer. In this study we investigated the possible role of MMP-2 and MMP-9 in an experimental model of colon cancer metastasis in rat liver. We demonstrated with gelatin zymography that the tumors contained MMP-2 and MMP-9, but only MMP-2 was present in the active form. Immunolocalization of MMP-2 showed that the protein was localized at basement membranes of colon cancer cells and in intratumor stroma, associated with extracellular matrix (ECM) components. However, zymography and immunohistochemistry (IHC) do not provide information on the localization of MMP activity. Therefore, we developed an in situ zymography technique using the quenched fluorogenic substrate DQ-gelatin in unfixed cryostat sections. The application of DQ-gelatin in combination with a gelled medium allows precise localization of gelatinolytic activity. Fluorescence due to gelatinolytic activity was found in the ECM of tumors and was localized similarly to both MMP-2 protein and collagen type IV, its natural substrate. The localization of MMP-2 activity and collagen type IV at similar sites suggests a role of MMP-2 in remodeling of ECM of stroma in colon cancer metastases in rat live

In situ localization of transketolase activity in epithelial cells of different rat tissues and subcellularly in liver parenchymal cells

Metabolic mapping of enzyme activities (enzyme histochemistry) is an important tool to understand (patho)physiological functions of enzymes. A new enzyme histochemical method has been developed to detect transketolase activity in situ in various rat tissues and its ultrastructural localization in individual cells. In situ detection of transketolase is important because this multifunctional enzyme has been related with diseases such as cancer, diabetes, Alzheimer's disease, and Wernicke-Korsakoff's syndrome. The proposed method is based on the tetrazolium salt method applied to unfixed cryostat sections in the presence of polyvinyl alcohol. The method appeared to be specific for transketolase activity when the proper control reaction is performed and showed a linear increase of the amount of final reaction product with incubation time. Transketolase activity was studied in liver, small intestine, trachea, tongue, kidney, adrenal gland, and eye. Activity was found in liver parenchyma, epithelium of small intestine, trachea, tongue, proximal tubules of kidney and cornea, and ganglion cells in medulla of adrenal gland. To demonstrate transketolase activity ultrastructurally in liver parenchymal cells, the cupper iron method was used. It was shown that transketolase activity was present in peroxisomes and at membranes of granular endoplasmic reticulum. This ultrastructural localization is similar to that of glucose-6-phosphate dehydrogenase activity, suggesting activity of the pentose phosphate pathway at these sites. It is concluded that the method developed for in situ localization of transketolase activity for light and electron microscopy is specific and allows further investigation of the role of transketolase in (proliferation of) cancer cells and other pathophysiological processe

Visualization of early events in tumor formation of eGFP-transfected rat colon cancer cells in liver

Colon cancer preferentially metastasizes to the liver. To determine cellular backgrounds of this preference, we generated an enhanced green fluorescent protein (eGFP)-expressing rat adenocarcinoma cell line (CC531s) that forms metastases in rat liver after administration to the portal vein. Intravital videomicroscopy (IVVM) was used to visualize early events in the development of tumors in livers of live animals from the time of injection of the cancer cells up to 4 days afterward. Based on information obtained with IVVM, tissue areas were selected for further analysis using confocal laser scanning microscopy (CLSM), electron microscopy (EM), and electron tomography. It was shown that initial arrest of colon cancer cells in sinusoids of the liver was due to size restriction. Adhesion of cancer cells to endothelial cells was never found. Instead, endothelial cells retracted rapidly and interactions were observed only between cancer cells and hepatocytes. Tumors developed exclusively intravascularly during the first 4 days. In conclusion, initial steps in the classic metastatic cascade such as adhesion to endothelium and extravasation are not essential for colon cancer metastasis in live

NADPH production by the pentose phosphate pathway in the zona fasciculata of rat adrenal gland

Biosynthesis of steroid hormones in the cortex of the adrenal gland takes place in smooth endoplasmic reticulum and mitochondria and requires NADPH. Four enzymes produce NADPH: glucose-6-phosphate dehydrogenase (G6PD), the key regulatory enzyme of the pentose phosphate pathway, phosphogluconate dehydrogenase (PGD), the third enzyme of that pathway, malate dehydrogenase (MDH), and isocitrate dehydrogenase (ICDH). However, the contribution of each enzyme to NADPH production in the cortex of adrenal gland has not been established. Therefore, activity of G6PD, PGD, MDH, and ICDH was localized and quantified in rat adrenocortical tissue using metabolic mapping, image analysis, and electron microscopy. The four enzymes have similar localization patterns in adrenal gland with highest activities in the zona fasciculata of the cortex. G6PD activity was strongest, PGD, MDH, and ICDH activity was similar to 60%, 15%, and 7% of G6PD activity, respectively. The K-m value of G6PD for glucose-6-phosphate was two times higher than the K-m value of PGD for phosphogluconate. As a consequence, virtual flux rates through G6PD and PGD are largely similar. It is concluded that G6PD and PGD provide the major part of NADPH in adrenocortical cells. Their activity is localized in the cytoplasm associated with free ribosomes and membranes of the smooth endoplasmic reticulum, indicating that NADPH-demanding processes related to biosynthesis of steroid hormones take place at these sites. Complete inhibition of G6PD by androsterones suggests that there is feedback regulation of steroid hormone biosynthesis via G6P