BRCA1 Functions as a Differential Modulator of Chemotherapy-induced Apoptosis

We have evaluated the role played by BRCA1 in mediating the phenotypic response to a range of chemotherapeutic agents commonly used in cancer treatment. Here we provide evidence that BRCA1 functions as a differential mediator of chemotherapy-induced apoptosis. Specifically, we demonstrate that BRCA1 mediates sensitivity to apoptosis induced by antimicrotubule agents but conversely induces resistance to DNA-damaging agents. These data are supported by a variety of experimental models including cells with inducible expression of BRCA1, siRNA-mediated inactivation of endogenous BRCA1, and reconstitution of BRCA1-deficient cells with wild-type BRCA1. Most notably we demonstrate that BRCA1 induces a 10–1000-fold increase in resistance to a range of DNA-damaging agents, in particular those that give rise to double-strand breaks such as etoposide or bleomycin. In contrast, BRCA1 induces a >1000-fold increase in sensitivity to the spindle poisons, paclitaxel and vinorelbine. Fluorescence-activated cell sorter analysis demonstrated that BRCA1 mediates G2/M arrest in response to both antimicrotubule and DNA-damaging agents. However, poly(ADP-ribose) polymerase and caspase-3 cleavage assays indicate that the differential effect mediated by BRCA1 in response to these agents occurs through the inhibition or induction of apoptosis. Therefore, our data suggest that BRCA1 acts as a differential modulator of apoptosis depending on the nature of the cellular insult.

Derivation of clinically compliant MSCs from CD105+, CD24-differentiated human ESCs

Adult tissue-derived mesenchymal stem cells ( MSCs) have demonstrated therapeutic efficacy in treating diseases or repairing damaged tissues through mechanisms thought to be mediated by either cell replacement or secretion of paracrine factors. Characterized, self- renewing human ESCs could potentially be an invariable source of consistently uniform MSCs for therapeutic applications. Here we describe a clinically relevant and reproducible manner of generating identical batches of hESC- derived MSC ( hESC- MSC) cultures that circumvents exposure to virus, mouse cells, or serum. Trypsinization and propagation of HuES9 or H1 hESCs in feeder- and serum-free selection media generated three polyclonal, karyotypically stable, and phenotypically MSC-like cultures that do not express pluripotency- associated markers but displayed MSC- like surface antigens and gene expression profile. They differentiate into adipocytes, osteocytes, and chondrocytes in vitro. Gene expression and fluorescence- activated cell sorter analysis identified CD105 and CD24 as highly expressed antigens on hESC- MSCs and hESCs, respectively. CD105+, CD24- monoclonal isolates have a typical MSC gene expression profiles and were identical to each other with a highly correlated gene expression profile ( r(2) >.90). We have developed a protocol to reproducibly generate clinically compliant and identical hESC- MSC cultures.