James A. Raleigh

ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth

Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. While much emphasis has been placed on the role of HIF‐1 in this context, the role of additional mechanisms has not been adequately explored. Here we demonstrate that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. Inactivation of ISR signaling by mutations in the ER kinase PERK and the translation initiation factor eIF2α or by a dominant‐negative PERK impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. Collectively, these findings demonstrate that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities.

Evidence that hypoxia markers detect oxygen gradients in liver: pimonidazole and retrograde perfusion of rat liver.

Nitroimidazole markers of tumour hypoxia bind to normoxic liver and the question has been raised whether this is due to low oxygen concentration or microregional activity of specialised nitroreductases. To answer this question, the binding patterns of the 2-nitroimidazole, pimonidazole, were compared following perfusion of surgically isolated rat livers in anterograde and retrograde directions. Perfusion at low flow rates in anterograde or retrograde directions can be used intentionally to alter oxygen gradients without altering enzyme distributions. Perfusion by means of the portal vein (anterograde direction) produced pimonidazole binding in the pericentral region of liver similar to that observed for pimonidazole binding in vivo. A complete reversal of this binding pattern occurred when the isolated liver was perfused by way of the central vein (retrograde direction). In this case, pimonidazole binding occurred in the periportal region. The extent and intensity of binding in the periportal region during perfusion in the retrograde direction was similar to that in the pericentral region during perfusion in the anterograde direction. It is concluded that low oxygen concentration rather than the non-homogeneous distribution of nitroreductase activity is the primary determinant of 2-nitroimidazole binding in liver.

Comparisons among Pimonidazole Binding, Oxygen Electrode Measurements, and Radiation Response in C3H Mouse Tumors

Pimonidazole binding was compared with oxygen electrode measurements and with measurements of the radiobiologically hypoxic fraction in C3H mammary tumors in which oxygenation was manipulated by means of subjecting tumor-bearing CDF1 mice to air breathing, carbogen breathing, oxygen breathing, hydralazine injection or tumor clamping. Hypoxia measured by pimonidazole binding could be correlated with both pO2 (r2 = 0.81) and radiobiologically hypoxic fraction (r2 = 0.85) in this system. The scope and limitation of pimonidazole as an immunohistochemical marker for tumor hypoxia is discussed.

Determination of hypoxic region by hypoxia marker in developing mouse embryos in vivo: A possible signal for vessel development

Hypoxia is a well‐known signal for angiogenesis, but the recent proposal that hypoxia exists in developing embryonic tissues and that it induces vascular development remains to be proven. In the present study, we demonstrate the presence of hypoxia in normal developing embryos by means of a hypoxia marker, pimonidazole, and its associated antibody. Our data clearly show that hypoxia marker immunoreactivity was highly detected in developing neural tubes, heart, and intersomitic mesenchyme at an early stage of organogenesis, suggesting that hypoxia may exist in the early stages of embryo development. We also found that hypoxia inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) were spatiotemporally co‐localized with possible hypoxic regions in embryos. Investigation of platelet endothelial cell adhesion molecule (PECAM) expression provides evidence that endothelial cells proliferate and form the vessels in the hypoxic region in developing organs. Furthermore, we found that hypoxia induced both HIF‐1α and VEGF in F9 embryonic stem and differentiated cells. Thus, we suggest that hypoxia may exist widely in developing embryonic tissues and that it may act as a signal for embryonic blood vessel formation in vivo.

GLUT‐1 and CAIX as intrinsic markers of hypoxia in carcinoma of the cervix: Relationship to pimonidazole binding

The presence of hypoxia in tumours results in the overexpression of certain genes, which are controlled via the transcription factor HIF‐1. Hypoxic cells are known to be radioresistant and chemoresistant, thus, a reliable surrogate marker of hypoxia is desirable to ensure that treatment may be rationally applied. Recently, the HIF‐1‐regulated proteins Glut‐1 and CAIX were validated as intrinsic markers of hypoxia by comparison with pO2 measured using oxygen electrodes. We compare the expression of Glut‐1 and CAIX with the binding of the bioreductive drug hypoxia marker pimonidazole. Pimonidazole was administered to 42 patients with advanced carcinoma of the cervix, 16 hr before biopsy. Sections of single or multiple biopsies were then immunostained for Glut‐1 and CAIX, and the area of staining scored by eye, using a “field‐by‐field” semi‐quantitative averaging system. Using 1 biopsy only, Glut‐1 (r = 0.54, p = <0.001) correlated with the level of pimonidazole binding, and Glut‐1 and CAIX expression also correlated significantly (r = 0.40, p = <0.009). Thus, our study has shown that HIF‐1 regulated genes have potential for future use as predictors of the malignant changes mediated by hypoxia, and warrant further investigation as indicators of response to cancer therapy.

Proliferation and hypoxia in human squamous cell carcinoma of the cervix: First report of combined immunohistochemical assays

PURPOSE To characterize the distribution of hypoxia and proliferation in human squamous cell carcinoma of the cervix via an immunohistochemical approach prior to initiation of therapy. METHODS AND MATERIALS Patients with primary squamous cell carcinoma of the cervix uteri received a single infusion of the 2-nitroimidazole, pimonidazole (0.5 g/m2 i.v.), and 24 h later punch biopsies of the primary tumor were taken. Tissue was formalin fixed, paraffin embedded, and sectioned for immunohistochemistry. Hypoxia was detected by monoclonal antibody binding to adducts of reductively activated pimonidazole in malignant cells. Staining for endogenous MIB-1 and PCNA was detected in tumor cells via commercially available monoclonal antibodies. Point counting was used to quantitate the fraction of tumor cells immunostained for MIB-1, PCNA, and hypoxia marker binding. RESULTS Immunostaining for pimonidazole binding was distant from blood vessels. There was no staining in necrotic regions, and only minimal nonspecific staining, mostly in keratin. In general, cells immunostaining for MIB-1 and PCNA did not immunostain for pimonidazole binding. Cells immunostaining for MIB-1 and PCNA showed no obvious geographic predilection such as proximity to vasculature. Quantitative comparison showed an inverse relationship between hypoxia marker binding and proliferation. CONCLUSIONS Immunohistochemical staining for pimonidazole binding is consistent with the presence of hypoxic cells in human tumors and may be useful for estimating tumor hypoxia prior to radiation therapy. Immunostaining for pimonidazole binding is an ideal complement to immunohistochemical assays for endogenous proliferation markers allowing for comparisons of tumor hypoxia with other physiological parameters. These parameters might be used to select patients for radiation protocols specifically designed to offset the negative impact of hypoxia and/or proliferation on therapy. The inverse relationship between pimonidazole binding and proliferation markers is a preliminary result requiring verification.

Hepatocellular Hypoxia-Induced Vascular Endothelial Growth Factor Expression and Angiogenesis in Experimental Biliary Cirrhosis

We tested the potential role of vascular endothelial growth factor (VEGF) and of fibroblast growth factor-2 (FGF-2) in the angiogenesis associated with experimental liver fibrogenesis induced by common bile duct ligation in Sprague-Dawley rats. In normal rats, VEGF and FGF-2 immunoreactivities were restricted to less than 3% of hepatocytes. One week after bile duct ligation, hypoxia was demonstrated by the immunodetection of pimonidazole adducts unevenly distributed throughout the lobule. After 2 weeks, hypoxia and VEGF expression were detected in >95% of hepatocytes and coexisted with an increase in periportal vascular endothelial cell proliferation, as ascertained by Ki67 immunolabeling. Subsequently, at 3 weeks the density of von Willebrand-labeled vascular section in fibrotic areas significantly increased. Semiquantitative reverse transcription polymerase chain reaction showed that VEGF(120) and VEGF(164) transcripts, that correspond to secreted isoforms, increased within 2 weeks, while VEGF(188) transcripts remained unchanged. FGF-2 mainly consisting of a 22-kd isoform, according to Western blot, was identified by immunohistochemistry in 49% and 100% of hepatocytes at 3 and 7 weeks, respectively. Our data provide evidence that in biliary-type liver fibrogenesis, angiogenesis is stimulated primarily by VEGF in response to hepatocellular hypoxia while FGF-2 likely contributes to the maintenance of angiogenesis at later stages.

Functional Significance of Erythropoietin Receptor Expression in Breast Cancer

Erythropoietin (EPO) is the principal hematopoietic cytokine that regulates mammalian erythropoiesis by binding to its transmembrane receptor EpoR. Recent experimental evidence suggests that the biologic effects of EPO are not limited to the regulation of erythropoiesis. In studies focusing on nonhematopoietic effects of EpoR signaling, we found high levels of EpoR protein expression in human breast cancer cells. The purpose of the present study was to evaluate clinical breast cancer specimens for EPO and EpoR expression, characterize the relationship between EPO expression and tumor hypoxia in biopsies prelabeled with hypoxia marker pimonidazole, analyze breast cancer cell lines for EpoR expression, and study the functional significance of EpoR expression in breast cancer cells in vivo. Immunohistochemical analysis for EPO, EpoR expression, and pimonidazole adducts was performed on 26 tumor biopsies with contiguous sections from 10 patients with breast cancer. High levels of EpoR expression were found in cancer cells in 90% of tumors. EPO expression was found in 60% of tumors and EPO and EpoR colocalization in tumor cells was present in many cases. The expression pattern of EPO with respect to tumor hypoxia was variable, without consistent colocalization of EPO and hypoxia in tumor cells. Human and rat breast cancer tissue culture cells express EpoR mRNA and protein. To study the in vivo function of EpoR expression in breast cancer cells, we used rat syngeneic R3230Ac mammary adenocarcinoma cells in a tumor Z-chamber model (dual porous plexiglass chambers containing fibrin gel, cancer cells, and a putative anti-tumor compound implanted into the subcutaneous tissue of rats). Local, one-time administration of a neutralizing anti-EPO antibody, soluble EPO receptor, or an inhibitor of Jak2, a cytoplasmic tyrosine kinase essential for EPO-mediated mitogenesis, resulted in a delay in tumor growth with 45% reduction in maximal tumor depth in tumor Z-chambers in a dose-dependent manner. These studies demonstrate the expression of functional receptors for EPO in breast cancer cells.

Cyclosporin A increases hypoxia and free radical production in rat kidneys: prevention by dietary glycine

The major side effect of cyclosporin A is severe nephrotoxicity. It is likely that cyclosporin A causes vasoconstriction leading to hypoxia-reperfusion injury; therefore, these experiments were designed to attempt to obtain physical evidence for hypoxia and free radical production in kidney following cyclosporin A. Rats were treated daily with cyclosporin A (25 mg/kg ig) for 5 days, and pimonidazole, a hypoxia marker, was injected 2 h after the last dose of cyclosporin A. A dose of α-(4-pyridyl-1-oxide)- N- tert-butylnitrone (4-POBN) was injected 3 h after cyclosporin A to trap free radicals. Cyclosporin A doubled serum creatinine and decreased glomerular filtration rates by 65% as expected. Pimonidazole adduct binding in the kidney was increased nearly threefold by cyclosporin A, providing physical evidence for tissue hypoxia. Moreover, cyclosporin A increased 4-POBN/radical adducts nearly sixfold in the urine but did not alter levels in the serum. Glycine, which causes vasodilatation and prevents cyclosporin A toxicity, minimized hypoxia and blocked free radical production; however, it did not alter cyclosporin A blood levels. These results demonstrate for the first time that cyclosporin A causes hypoxia and increases production of a new free radical species exclusively in the kidney. Therefore, it is concluded that cyclosporin A causes renal injury by mechanisms involving hypoxia-reoxygenation, effects which can be prevented effectively by dietary glycine.The major side effect of cyclosporin A is severe nephrotoxicity. It is likely that cyclosporin A causes vasoconstriction leading to hypoxia-reperfusion injury; therefore, these experiments were designed to attempt to obtain physical evidence for hypoxia and free radical production in kidney following cyclosporin A. Rats were treated daily with cyclosporin A (25 mg/kg ig) for 5 days, and pimonidazole, a hypoxia marker, was injected 2 h after the last dose of cyclosporin A. A dose of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) was injected 3 h after cyclosporin A to trap free radicals. Cyclosporin A doubled serum creatinine and decreased glomerular filtration rates by 65% as expected. Pimonidazole adduct binding in the kidney was increased nearly threefold by cyclosporin A, providing physical evidence for tissue hypoxia. Moreover, cyclosporin A increased 4-POBN/radical adducts nearly sixfold in the urine but did not alter levels in the serum. Glycine, which causes vasodilatation and prevents cyclosporin A toxicity, minimized hypoxia and blocked free radical production; however, it did not alter cyclosporin A blood levels. These results demonstrate for the first time that cyclosporin A causes hypoxia and increases production of a new free radical species exclusively in the kidney. Therefore, it is concluded that cyclosporin A causes renal injury by mechanisms involving hypoxia-reoxygenation, effects which can be prevented effectively by dietary glycine.

Measurements of hypoxia using pimonidazole and polarographic oxygen-sensitive electrodes in human cervix carcinomas.

BACKGROUND AND PURPOSE The measurement of tumour oxygenation using Eppendorf oxygen-sensitive needle electrodes can provide prognostic information but the method is limited to accessible tumours that are suitable for electrode insertion. In this paper the aim was to study the relationship between such physiological measurements of tumour hypoxia and the labelling of tumours with the hypoxia-specific marker pimonidazole. MATERIALS AND METHODS Assessment of tumour oxygen partial pressure (pO(2)) using an Eppendorf pO(2) histograph and immunohistochemical pimonidazole labelling was carried out in 86 patients with primary cervix carcinomas. Pimonidazole was given as a single injection (0.5 g/m(2) i.v.) and 10-24 h later pO(2) measurements were made and biopsies taken. Tumour oxygenation status was evaluated as the median tumour pO(2) and the fraction of pO(2) values </=10 mmHg (HP(10)), </=5 mmHg (HP(5)) and </=2.5 mmHg (HP(2.5)). Hypoxia was detected by immunohistochemistry using monoclonal antibodies directed against reductively activated pimonidazole. Pimonidazole binding was scored using a light microscope. Each tumour was evaluated by the relative area pimonidazole at highest score and the accumulated area of pimonidazole labelling from score 1 to 4. Necrosis was measured in HE stained sections. RESULTS AND CONCLUSIONS The degree of hypoxia assessed by either pimonidazole binding or invasive electrode measurements varied significantly between tumours. There was a trend that the most hypoxic tumours measured by oxygen electrodes had the highest score of necrosis, and no or little pimonidazole binding. However, this observation was not consistent and there was no correlation between pimonidazole staining expressed in this way and oxygen electrode measurements of hypoxia.