We have described a monoclonat antibody that rounds and detaches chick skeletal myoblasts and myotubes from extracellular substrata. The antibody also inhibits the attachment of myogenic cells to a gelatin-coated substratum but has no detectable effect on myoblast fusion. The cellular response to antibody treatment varies wffh differentiation and cell type. Young myoblasts and myotubes are rapidly rounded and detached by the antibody. Older myotubes require longer incubation times or higher antibody titers for rounding and detachment. Chick embryo fibroblasts, cardiac cells, and neurons are not similarly rounded and remain attached. Since the antibody also detaches cells from embryonic muscle tissue explants, the cell-substratum interaction perturbed by the antibody appears relevant to the in vivo interaction of myogenic cells with their extracellular matrices. Binding studies using iodinated antibody revealed 2-4 x 105 sites per myoblast with an apparent Kd in the range of 2-5 x 10 -9 molar. Embryo fibroblasts bind antibody as well and display approximately twice the number of binding sites per cell. The fluorescence distribution of antigen on myoblasts and myotubes is somewhat punctate and particularly bright along the edge of the myotube. The distribution on fibroblasts was also punctate and was particularly bright along the cell periphery and portions of stress fibers. For both cell types the binding was distinctly different than that reported for collagen, fibronectin, and other extracellular molecules. The antigen, as isolated by antibody affinity chromatography, inhibits antibody-induced rounding. SDS PAGE reveals two unique polypeptides migrating in the region of ~120 and 160 kilodaltons (kd). The most straightforward mechanism for the antibody-induced rounding and detachment is the perturbation of a membrane molecule involved in adhesion. The hypothesized transmembrane link between extracellular macromolecules and the cytoskeleton provides an ovbious candidate.

All-trans-retinoic acid (RA) treatment induces remissions in acute promyelocytic leukemia (APL) cases expressing the t(15;17) product, promyelocytic leukemia (PML)͞RA receptor ␣ (RAR␣). Microarray analyses previously revealed induction of UBE1L (ubiquitin-activating enzyme E1-like) after RA treatment of NB4 APL cells. We report here that this occurs within 3 h in RA-sensitive but not RA-resistant APL cells, implicating UBE1L as a direct retinoid target. A 1.3-kb fragment of the UBE1L promoter was capable of mediating transcriptional response to RA in a retinoid receptor-selective manner. PML͞RAR␣, a repressor of RA target genes, abolished this UBE1L promoter activity. A hallmark of retinoid response in APL is the proteasome-dependent PML͞RAR␣ degradation. UBE1L transfection triggered PML͞RAR␣ degradation, but transfection of a truncated UBE1L or E1 did not cause this degradation. A tight link was shown between UBE1L induction and PML͞RAR␣ degradation. Notably, retroviral expression of UBE1L rapidly induced apoptosis in NB4 APL cells, but not in cells lacking PML͞RAR␣ expression. UBE1L has been implicated directly in retinoid effects in APL and may be targeted for repression by PML͞ RAR␣. UBE1L is proposed as a direct pharmacological target that overcomes oncogenic effects of PML͞RAR␣ by triggering its degradation and signaling apoptosis in APL cells.

Core-binding factor beta (Cbfbeta) is the common non-DNA-binding subunit of the Cbf family of heterodimeric transcription factors. Mice deficient in Cbfbeta have a severe block in fetal liver hematopoiesis at the stage of hematopoietic stem cell (HSC) emergence. Here we show that by providing Cbfbeta function in endothelial cells and hematopoietic progenitors we can rescue fetal liver hematopoiesis in Cbfbeta-deficient embryos. The rescued mice die at birth, however, with severe defects in skeletal development, though intramembranous ossification occurs to some extent. Fetal liver hematopoiesis is restored at embryonic day (E) 12.5, but by E17.5 significant impairments in lymphopoiesis and myelopoiesis are observed. Thus, we conclude that the Cbfbeta subunit is required for HSC emergence, bone formation and normal differentiation of lymphoid and myeloid lineage cells.

Tumor hypoxia is a negative prognostic factor that is implicated in oncogenic signal activation, immune escape, and resistance to treatment. Identifying the mechanistic role of hypoxia in immune escape and resistance to immune-checkpoint inhibitors may aid the identification of therapeutic targets. We and others have shown that V-domain Ig suppressor of T-cell activation (VISTA), a negative checkpoint regulator in the B7 family, is highly expressed in the tumor microenvironment in tumor models and primary human cancers. In this study, we show that VISTA and HIF1a activity are correlated in a cohort of colorectal cancer patients. High VISTA expression was associated with worse overall survival. We used the CT26 colon cancer model to investigate the regulation of VISTA by hypoxia. Compared with less hypoxic tumor regions or draining lymph nodes, regions of profound hypoxia in the tumor microenvironment were associated with increased VISTA expression on tumor-infiltrating myeloid-derived suppressor cells (MDSC). Using chromatin immunoprecipitation and genetic silencing, we show that hypoxia-inducible factor (HIF)-1a binding to a conserved hypoxia response element in the VISTA promoter upregulated VISTA on myeloid cells. Further, antibody targeting or genetic ablation of VISTA under hypoxia relieved MDSC-mediated T-cell suppression, revealing VISTA as a mediator of MDSC function. Collectively, these data suggest that targeting VISTA may mitigate the deleterious effects of hypoxia on antitumor immunity.