Deficiencies in tumor blood supply establish gradients of oxygen, glucose, growth regulators, and other substances along diffusion paths radiating from blood vessels. These stress environments represent physiological differences between tumor and normal tissue. SRI researchers have shown that hypoxic stress in human and rodent tumor cells induces the synthesis of a set of proteins called oxygen regulated proteins or ORPs. It is hypothesized that genes regulated by hypoxic stress are important participants in malignant progression. To investigate this hypothesis, SRI proposes to measure the expression of specific ORPs in tumor tissue and to develop a model to test the relationship of expression of an ORP with resistance to therapy. This information will permit the creation of novel strategies for determining the prognosis and treatment of some human cancers. Two approaches will be used to identify hypoxic regions of tumor tissue (FaDu, SQ-20B, and A431 human squamous carcinoma cells) and establish correlations with ORP expression: (1) antibodies to a fluorinated bioreductive drug will be used as a positive control to visualize hypoxic cells in tumor models in vitro, and the expression of selected ORPs (e.g., heme oxygenase-1 [HO-1; ORP 33], metallothionein IIA [MT-IIA; ORP 7], and vascular endothelial growth factor [VEGF]) will be detected by in situ hybridization and immunohistochemistry; and (2) similar experiments will be performed with human tumor xenografts. SRI's preliminary studies indicate that human MT-IIA is a suitable ORP for investigating the development of therapeutic resistance caused by hypoxia and reoxygenation. We will use transient and stable transfectants of human squamous carcinoma cells to create multicellular tumor spheroid models for investigating (1) a hypoxia-responsive MT-IIA 5'-regulatory region and (2) the effect of MT-IIA expression on resistance to cisplatin. These experiments are directed toward creating an in vitro tumor model of the human MT-IIA (ORP 7) 5'-regulatory region and coding sequence for investigation of the effects of hypoxic and glucose stress on a clinically relevant hypoxic stress protein. This model will provide a test system for inducible drug resistance associated with hypoxia and reoxygenation.
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