Proteolysis during tumor cell extravasation in vitro: metalloproteinase involvement across tumor cell types.

To test if proteolysis is involved in tumor cell extravasation, we developed an in vitro model where tumor cells cross an endothelial monolayer cultured on a basement membrane. Using this model we classified the ability of the cells to transmigrate through the endothelial cell barrier onto the underlying matrix, and scored this invasion according to the stage of passage through the endothelium. Metalloproteinase inhibitors reduced tumor cell extravasation by at least 35%. Visualization of protease and cell adhesion molecules by confocal microscopy demonstrated the cell surface localization of MMP-2, MMP-9, MT1-MMP, furin, CD44 and αvß3, during the process of transendothelial migration. By the addition of inhibitors and bio-modulators we assessed the functional requirement of the aforementioned molecules for efficient migration. Proteolytic digestion occurred at the cell-matrix interface and was most evident during the migratory stage. All of the inhibitors and biomodulators affected the transition of the tumor cells into the migratory stage, highlighting the most prevalent use of proteolysis at this particular step of tumor cell extravasation. These data suggest that a proteolytic interface operates at the tumor cell surface within the tumor-endothelial cell microenvironment.

Endogenous myoglobin in breast cancer is hypoxia-inducible by alternative transcription and functions to impair mitochondrial activity: a role in tumor suppression?

Recently, immunohistochemical analysis of myoglobin (MB) in human breast cancer specimens has revealed a surprisingly widespread expression of MB in this nonmuscle context. The positive correlation with hypoxia-inducible factor 2α (HIF-2α) and carbonic anhydrase IX suggested that oxygen regulates myoglobin expression in breast carcinomas. Here, we report that MB mRNA and protein levels are robustly induced by prolonged hypoxia in breast cancer cell lines, in part via HIF-1/2-dependent transactivation. The hypoxia-induced MB mRNA originated from a novel alternative transcription start site 6 kb upstream of the ATG codon. MB regulation in normal and tumor tissue may thus be fundamentally different. Functionally, the knockdown of MB in MDA-MB468 breast cancer cells resulted in an unexpected increase of O(2) uptake and elevated activities of mitochondrial enzymes during hypoxia. Silencing of MB transcription attenuated proliferation rates and motility capacities of hypoxic cancer cells and, surprisingly, also fully oxygenated breast cancer cells. Endogenous MB in cancer cells is apparently involved in controlling oxidative cell energy metabolism, contrary to earlier findings on mouse heart, where the targeted disruption of the Mb gene did not effect myocardial energetics and O(2) consumption. This control function of MB seemingly impacts mitochondria and influences cell proliferation and motility, but it does so in ways not directly related to the facilitated diffusion or storage of O(2). Hypothetically, the mitochondrion-impairing role of MB in hypoxic cancer cells is part of a novel tumor-suppressive function.

The influence of hypoxia on bioluminescence in luciferase-transfected gliosarcoma tumor cells in vitro.

Firefly luciferase catalyzes the emission of light from luciferin in the presence of oxygen and adenosine triphosphate. This bioluminescence is commonly employed in imaging mode to monitor tumor growth and treatment responses in vivo. A potential concern is that, since solid tumors are often hypoxic, either constitutively and/or as a result of treatment, the oxygen available for the bioluminescence reaction could be reduced to limiting levels, leading to underestimation of the actual number of luciferase-labeled cells during in vivo experiments. We present studies of the oxygen dependence of bioluminescence in vitro in rat 9 L gliosarcoma cells tagged with the firefly luciferase gene (9L(luc)). We demonstrate that the bioluminescence signal decreases at pO(2)

Graded hypoxia modulates the invasive potential of HT1080 fibrosarcoma and MDA MB231 carcinoma cells.

Spatial and temporal oxygen heterogeneity exists in most solid tumour microenvironments due to an inadequate vascular network supplying a dense population of tumour cells. An imbalance between oxygen supply and demand leads to hypoxia within a significant proportion of a tumour, which has been correlated to the likelihood of metastatic dissemination in both rodent tumour models and human patients. Experimentally, it has been demonstrated that near-anoxic in vitro exposure results in transiently increased metastatic potential in some tumour cell lines. The purpose of this study was to examine the effect of graded low oxygen conditions on the invasive phenotype of human tumour cells using an in vitro model of basement membrane invasion, in which we measured oxygen availability directly at the invasion surface of the transwell chamber. Our results show a relationship between culture vessel geometry and time to achieve hypoxia which may affect the interpretation of low oxygen experiments. We exposed the human tumour cell lines, HT1080 and MDA MB231, to graded normobaric oxygen (5% O(2)-0.2% O(2)) either during or prior to in vitro basement membrane invasion to simulate conditions of intravasation and extravasation. A secondary aim was to investigate the potential regulation of matrix metalloproteinase activity by oxygen availability. We identified significant reductions in invasive ability under low oxygen conditions for the HT1080 cell line and an increase in invasion at intermediate oxygen conditions for the MDA MB231 cell line. There were differences in the absolute activity of the individual matrix metalloproteinases, MMP-2, -9, -14, between the two cell lines, however there were no significant changes following exposure to hypoxic conditions. This study demonstrates cell line specific effects of graded oxygen levels on invasive potential and suggests that intermediate levels of low oxygen may increase metastatic dissemination.

Hypoxia-regulated p53 and its effect on radiosensitivity in cancer cells.

PURPOSE: To determine the response of tumor suppressor p53 to hypoxia in different tumor cell lines and the involvement of p53 activity regulation in the effect of hypoxia on tumor cell sensitivity to radiation and hyperthermia. MATERIALS AND METHODS: Three tumor cell lines with functional p53 were treated with chronic or cyclic hypoxia followed by radiation or hyperthermia to investigate p53 activity and cell survival. Flow cytometry was used to investigate the effect of hypoxia-induced cell cycle arrest on radiosensitivity in KHT-C (mouse fibrosarcoma) cells. Transient transfection was performed to determine the role of altered p53 activity in KHT-C and SCC VII (mouse squamous-cell carcinoma) radiosensitivity. RESULTS: Aerobic radiosensitivity was decreased in KHT-C and SCC VII cells after in vitro chronic or cyclic hypoxia pretreatment, but in HT1080 cells, it was slightly increased after chronic hypoxia, and was unchanged after acute hypoxia pretreatment. Decreased radiosensitivity in hypoxia-pretreated KHT-C and SCC VII cells was unlikely due to hypoxia-induced cell cycle arrest, but rather seemed to be associated with increased expression of Mdm2 (mouse double minute-2) and decreased p53. Furthermore, hypoxia pretreatment inhibited the activation of p53 by radiation. Similar results were observed in hyperthermia treated KHT-C cells. Finally, decreased radiosensitivity was observed in both KHT-C and SCC VII cells transiently transfected with Mdm2 or anti-sense p53 cDNA. CONCLUSION: We demonstrated that hypoxia may decrease tumor cell radiosensitivity through the suppression of p53 activity in some tumor cell lines. These results suggested the response of p53 to hypoxia can be cell type specific and contribute to radiosensitivity of hypoxic cells.

The hypoxic tumour microenvironment and metastatic progression.

The microenvironment of solid tumours contains regions of poor oxygenation and high acidity. Growing evidence from clinical and experimental studies points to a fundamental role for hypoxia in metastatic progression. Prolonged hypoxia increases genomic instability, genomic heterogeneity, and may act as a selective pressure for tumour cell variants. Hypoxia can also act in an epigenetic fashion, altering the expression of genes. Hypoxia-induced changes in gene expression alter non-specific stress responses, anaerobic metabolism, angiogenesis, tissue remodeling, and cell-cell contacts. Experimental studies have demonstrated that inhibition of proteins involved in these processes can modify metastasis formation, suggesting a causal role in metastatic progression. Recent advances in high-throughput screening techniques have allowed identification of many hypoxia-induced genes that are involved in the processes associated with metastasis. Here we review the epigenetic control of gene expression by the hypoxic microenvironment and its potential contribution to metastatic progression.