The p53 tumor suppressor protein negatively regulates hypoxia-inducible factor 1␣ (HIF-1␣). Here, we show that induction of p53 by the small-molecule RITA (reactivation of p53 and induction of tumor cell apoptosis) [2,5-bis(5-hydroxymethyl-2-thienyl) furan] (NSC-652287) inhibits HIF-1␣ and vascular endothelial growth factor expression in vivo and induces significant tumor cell apoptosis in normoxia and hypoxia in p53-positive cells. RITA has been proposed to stabilize p53 by inhibiting the p53-HDM2 interaction. However, induction of p53 alone was insufficient to block HIF-1␣ induced in hypoxia and has previously been shown to require additional stimuli, such as DNA damage. Here, we identify a new mechanism of action for RITA: RITA activates a DNA damage response, resulting in phosphorylation of p53 and ␥H2AX in vivo. Unlike other DNA damage response-inducing agents, RITA treatment of cells induced a p53-dependent increase in phosphorylation of the ␣ subunit of eukaryotic initiation factor 2, requiring PKR-like endoplasmic reticulum kinase activity, and led to the subsequent downregulation of HIF-1␣ and p53 target proteins, including HDM2 and p21. Through the identification of a new mechanism of action for RITA, our study uncovers a novel link between the DNA damage response-p53 pathway and the protein translational machinery.Solid tumors require blood vessels to supply them with oxygen and nutrients in order to grow beyond the macroscopic level or metastasize to other organs. Characteristically, solid tumors contain areas of low oxygen tension (hypoxia). The cellular response to hypoxia is primarily mediated by hypoxiainducible factors (HIFs) hypoxia-inducible factor 1 (HIF-1) and 42). HIFs are transcription factors that are often deregulated in cancer and play a key role in promoting angiogenesis and tumor progression. Hypoxia usually confers tumor resistance to chemotherapy and radiotherapy (8), and HIF-1 is an important determinant for this resistance (7,35,38,49). Thus, targeting HIF has the potential not only to block tumor angiogenesis but also to improve the efficacy of chemotherapy and radiotherapy within a given cancer setting.

SummaryUltrastructural immunocytochemistry requires that minimal damage to antigens is imposed by the processing methods. Immersion fixation in cross-linking fixatives with their potential to damage antigens is not an ideal approach and rapid freezing as an alternative sample-stabilization step has a number of advantages. Rapid freezing at ambient pressure restricts the thickness of well-frozen material obtainable to Ϸ 15 mm or less. In contrast, high-pressure freezing has been demonstrated to provide ice-crystalartefact-free freezing of samples up to 200 mm in thickness.There have been few reports of high-pressure freezing for immunocytochemical studies and there is no consensus on the choice of post-freezing sample preparation. A range of freeze-substitution time and temperature protocols were compared with improved tissue architecture as the primary goal, but also to compare ease of resin-embedding, polymerization and immunocytochemical labelling. Freeze-substitution in acetone containing 2% osmium tetroxide followed by epoxy-resin embedding at room temperature gave optimum morphology. Freeze-substitution in methanol was completed within 18 h and in tetrahydrofuran within 48 h but the cellular morphology of the Lowicryl-embedded samples was not as good as when samples were substituted in pure acetone. Acetone freezesubstitution was slow, taking at least 6 days to complete, and gave blocks which were difficult to embed in Lowicryl HM20. Careful handling of frozen samples avoiding rapid temperature changes reduced apparent ice-crystal damage in sections of embedded material. Thus a slow warm-up to freeze-substitution temperature and a long substitution time in acetone gave the best results in terms of freezing quality and cellular morphology. No clear differences emerged between the different freeze-substitution media from immunocytochemical labelling experiments.

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SUMMARYWe have previously demonstrated that purified virgin mouse mammary luminal epithelial and myoepithelial cells promiscuously express cell type-specific cytokeratins when they are cloned in vitro. Changes in cytokeratin expression may be indicators of the loss or change of the differentiated identity of a cell. To investigate the factors that may be responsible for the maintenance of differentiated cellular identity, specifically cell-cell and cell-matrix interactions, we cloned flow-sorted mouse mammary epithelial cells on the extracellular matrix (ECM) derived from the Engelbreth-Holm-Swarm murine sarcoma (EHS matrix). Changes in cell differentiation on EHS, compared with culture on glass, were analyzed by comparing patterns of cytokeratin expression. The results indicate that ECM is responsible for maintenance of the differentiated identity of basal/myoepithelial cells and prevents the inappropriate expression of luminal antigens seen on glass or plastic. Luminal cell identity in the form of retention of luminal markers and absence of basal/myoepithelial antigens, on the contrary, appears to depend on homotypic cell-cell contacts and interactions. The results also show that luminal cells (or a subpopulation of them) can generate a cell layer that expresses only basal cytokeratin markers (and no luminal cytokeratin markers) and may form a pluripotent compartment. To fully understand how the development and differentiation of the mammary gland parenchyma and of the different cell types within it are regulated, it is necessary to be able to study separately the behavior of the individual cell types within the gland. The mature rodent mammary epithelium consists of two cell populations, the luminal epithelial cells and the basal/ myoepithelial cells. The luminal epithelial cells are cuboidal or low columnar cells that line the ducts and alveoli and are responsible for milk secretion. Primary and secondary ducts may be composed of several layers of luminal epithelial cells, which become fewer with higher orders of branching. Most terminal ducts have only one layer of luminal cells (Sekhri et al. 1967). The elongated myoepithelial cells form a monolayer over the ductal luminal epithelial cells. They make a continuous sheath in which the cells are orientated parallel to the duct. In alveoli, the myoepithelial cells form a basket-like network and in the spaces between their cell processes the luminal epithelial cells are in contact with basement membrane (Emerman and Vogl 1986).We have previously described (Smalley et al. 1998) a system for the separation of virgin mouse mammary epithelium by flow cytometry and methods for the clonal culture of such separated cells on tissue culture plastic and glass. We have characterized mouse mammary luminal epithelial and myoepithelial cell-derived clones on the basis of phase-contrast morphology and