Hypoxia contributes to cytotoxic chemotherapy and radiation resistance and may play a role in the efficacy of antiangiogenesis cancer therapy. We have generated a series of cell lines derived from the colon adenocarcinoma models HT29 and HCT116 by exposing cells in vitro to repeated sublethal periods of profound hypoxia. These cell lines have altered sensitivity to hypoxia-induced apoptosis: those derived from HT29 are resistant, whereas those from HCT116 are more susceptible. We used cDNA selected subtractive hybridization to identify novel genes mediating sensitivity to hypoxia-induced apoptosis and isolated macrophage migration inhibitory factor (MIF) from the hypoxia-conditioned cell lines. MIF expression correlates with susceptibility of the cell lines to apoptosis. In hypoxia-resistant cells, the induction of apoptosis by hypoxia can be restored by the addition of exogenous recombinant MIF protein, suggesting that resistance may result in part from down-regulation of MIF production possibly through an autocrine loop. Inhibition of MIF using small interfering RNA in the susceptible lines conferred resistance to hypoxia-induced cell death. The relative expression of MIF in the hypoxia-conditioned cells implanted s.c. in severe combined immunodeficient mice in vivo was similar to that observed in vitro. In an analysis of 12 unrelated colon tumor cell lines, MIF expression and response to hypoxia varied widely. Cell lines in which MIF was inducible by hypoxia were more sensitive to oxaliplatin. In human colon tumor specimens analyzed by immunohistochemistry, MIF expression was similarly variable. There was no detectable expression of MIF in normal colon mucosa or adenoma but positive staining in all carcinomas tested. Taken together, these data indicate that MIF may be a determinant of hypoxia-induced apoptosis in vitro and that its variable expression in human colon cancers may indicate a functional role in vivo. We suggest that MIF expression in colorectal cancer may be a marker of susceptibility to therapies that may depend on induction of hypoxia, possibly including antiangiogenic therapy.Colon cancer is characterized by regions of variable hypoxia, known to be a consequence of disordered vasculature (1). Cells in these hypoxic regions are resistant to radiation and to cytotoxic drugs (2). We have shown previously that hypoxia induces increased expression of genes involved in the detoxification of cytotoxic drugs (3), an effect exerted in part by activation of transcription factors, including activator protein-1 and nuclear factor-nB (4, 5). Hurwitz et al. have shown recently that antiangiogenic therapy adds to the efficacy of chemotherapy in colorectal cancer (6), whereas Willett et al. showed that bevacizumab alone in rectal cancer markedly decreased blood flow to the tumor (7). These findings raise the possibility that apoptosis as a result of oxygen or nutrient withdrawal contributes to the therapeutic effect and prompted an investigation of the resistance of hypoxic cancer cells to apoptos...

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The ability of endometrial stromal cells from nonsensitized rat uteri to undergo decidualization in vitro was investigated. Cells were obtained by enzymatic dispersion from uteri of ovariectomized, steroid-treated rats at the equivalent of day 4, 5 or 6 of pseudopregnancy, or on day 5 from rats treated with 0, 0.3 or 1.0 microgram oestradiol (low, intermediate or high doses of oestradiol, respectively) on day 4, and cultured for 24, 48 or 72 h. Decidualization in vivo, as assessed by uterine mass 5 days after the unilateral intrauterine injection of 100 microliters sesame oil, was maximal for rats receiving the deciduogenic stimulus on day 5 and treated with the intermediate dose of oestradiol. Under control conditions in vitro, alkaline phosphatase (ALP) activity, the increase in ALP activity with time, and prostaglandin E2 (PGE2) accumulation in the medium were greatest for cells from maximally sensitized uteri. Indomethacin, an inhibitor of PG synthesis, reduced PGE2 accumulation to barely detectable amounts, and decreased ALP activity, especially in cells from maximally sensitized uteri, indicating that endogenous PG production contributed to the increase in ALP activity in these cells. The addition of PGE2 with indomethacin increased ALP activities. However, ALP activities were lower for cells derived from nonsensitized uteri when compared with cells from maximally sensitized uteri. These results suggest that endometrial stromal cells from nonsensitized uteri have a reduced capacity to undergo decidualization in vitro, and that this reduced capacity is not explained by differences in PGE2 production.

The rat endometrium undergoes decidualization, a tissue remodeling process, during embryo implantation. Plasminogen activator (PA), particularly the urokinase-type PA (uPA), has been implicated in tissue remodeling. The present study determined whether rat endometrial stromal cells secrete uPA during decidualization in vitro and, if so, whether the secretion is regulated by prostaglandins that are required in decidualization. Endometrial stromal cells were obtained from rats that had been treated with estrogen and progesterone to sensitize their uteri for decidualization, and the cells were cultured for up to 72 h in a serum-free medium. PA activity in the conditioned medium, as determined by a chromogenic assay, increased steadily during the 72-h culture period. PA secretion decreased when endogenous prostaglandin synthesis was inhibited by the addition of indomethacin to the cell cultures. The inhibitory effect of indomethacin on PA secretion was reversed by prostaglandin E2, and much less effectively by prostaglandin F2 alpha. PA activity in the medium was due primarily to uPA because 1) PA activity was inhibited by a uPA-specific inhibitor-amiloride-and by an anti-mouse uPA antibody, and 2) the predominant PA activity in the medium, as identified in zymography, had a molecular mass of approximately 40 kDa, similar to that reported for uPA. Northern blot analyses of RNA from the cultured cells demonstrated that the steady-state levels of mRNA for uPA, but not for tPA and plasminogen activator inhibitor-1, were decreased by indomethacin; this decrease was reversed by prostaglandin E2. Taken together, the data indicate that rat endometrial stromal cells secrete uPA during decidualization in vitro, and that prostaglandin E2 regulates uPA secretion by the decidualizing cells by directly increasing uPA gene transcription and/or stabilizing its transcripts. These findings may help to partially elucidate the mechanism of action of prostaglandin E2 in decidualization.