Despite their origin from the inner cell mass, embryonic stem (ES) cells undergo differentiation to the trophectoderm (TE) lineage by repression of the ES cell master regulator Oct4 or activation of the TE master regulator Caudal-type homeobox 2 (Cdx2). In contrast to the in-depth studies of ES cell self-renewal and pluripotency, few TE-specific regulators have been identified, thereby limiting our understanding of mechanisms underlying the first cell fate decision. Here we show that up-regulation and nuclear entry of AT-rich interactive domain 3a (Arid3a) drives TE-like transcriptional programs in ES cells, maintains trophoblast stem (TS) cell self-renewal, and promotes further trophoblastic differentiation both upstream and independent of Cdx2. Accordingly, Arid3a −/− mouse post-implantation placental development is severely impaired, resulting in early embryonic death. We provide evidence that Arid3a directly activates TE-specific and trophoblast lineage-specific genes while directly repressing pluripotency genes via differential regulation of epigenetic acetylation or deacetylation. Our results identify Arid3a as a critical regulator of TE and placental development through execution of the commitment and differentiation phases of the first cell fate decision.
We studied the effect of the anticancer drug taxol on the cytotoxic mechanism of major histocompatibility complex nonrestricted lymphocytes and their activation with interleukin 2. Unseparated lymphocytes or highly enriched natural killer or T-cells were treated with 0.2-10 micrograms/ml of taxol for various times and tested for cytotoxicity against the K562 cell line and the ovarian cell line, OV-2774. Taxol caused a dose- and time-dependent suppression of lymphocyte cytotoxicity. The most pronounced suppression was noted after treatment with 10 micrograms/ml of taxol for 6 h; a lower but significant decrease in cytotoxicity was observed after treatment with 2 and 5 micrograms/ml of taxol. In addition, taxol inhibited activation of lymphocytes with interleukin 2; however, the cytotoxicity of interleukin 2-preactivated lymphocytes was less sensitive to taxol treatment. Mechanism studies showed that taxol was not directly toxic to lymphocytes and did not alter their ability to form conjugates with target cells. Taxol treatment decreased a rate of kinetics of lysis and lymphocyte recycling ability. The immunofluorescence and electron microscopic analysis showed polymerization of microtubules in taxol-treated lymphocytes. These data demonstrate that taxol impairs lymphocyte cytotoxic function and activation and indicate the role of microtubules in these functions. Clinically, these findings suggest that activation of lymphocytes prior to taxol treatment may increase the therapeutic benefit of this drug.
Abstract Metabolism plays a key role in cell survival and “adaptation” in cancer cells, thus influencing therapeutic resistance and metastasis. As an indication of robust connections between metabolism and cancer, the oncogenic activity of the Myc protein depends on its effects on glucose and glutamine metabolism. We tested the hypothesis that glutamine-dependence for growth in cell culture medium would correlate with metastatic ability of breast cancer cell lines. We chose a variety of cell lines spanning a spectrum from normal to most aggressive: 1) MCF10A immortalized normal breast epithelial cell line, 2) MCF10A/COX-2, a COX-2 transfected premalignant breast cancer cell line, 3) MCF7, a luminal origin estrogen receptor-positive weakly-tumorigenic cell line, 4) MCF7/COX-2, a COX-2 transfected cell line, 5) MDA-MB-231-Luc, a basal origin highly tumorigenic cell line, 6) MDA-MB-231-BSC60, a cell line we established from a bone metastasis after two rounds of selection in female nude mice, 7) SUM149 inflammatory breast cancer (IBC) cell line, and 8) SUM149-FP76, a cell line we established from a fat pad tumor after one round of subcutaneous tumor growth and subsequent culture and inoculation into inguinal fat pad. We determined the long-term growth and survival of cell lines in culture medium without glutamine. Growth of MCF10A, MCF10A/COX-2, MCF7, and MCF7/COX-2 cell lines was not significantly inhibited upon glutamine deprivation; there was an initial modest inhibition (∼50% at day 4), but these cell lines later grew to confluency in culture dishes. In contrast, the metastatic cell lines MDA-MB-231-Luc, MDA-MB-231-BSC60, and SUM149 were severely growth inhibited (∼99% at day 30). Interestingly, although the SUM149-FP76 cell line was severely inhibited (95% at day 5) upon glutamine deprivation, some cells survived and grew to produce small cell colonies within two-weeks. On the other hand, the parental cell line SUM149 largely remained single-celled during the first two-weeks, and only yielded colonies after a much longer time period (2 months). A highly aggressive IBC cell line SUM190 behaved similar to the FP76 variant. We are currently investigating alterations in metabolic networks by whole-genome mRNA and microRNA analysis. In conclusion, 1) there are significant differences in glutamine-dependence between normal, premalignant, poorly metastatic, and metastatic breast cancer cell lines as only the metastatic cell lines exhibit severe dependence on glutamine, and 2) SUM149-FP76, selected to grow as a tumor in nude mice, was able to survive and produce slow-growing small colonies in glutamine-free medium much earlier than the parental SUM149 cell line. This result indicates that growing IBC tumors may involve selection of elastic variant cancer cells containing altered metabolic networks such that they can adjust to glutamine availability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 49.
