Resistance to tamoxifen in breast cancer patients is a serious therapeutic problem and major efforts are underway to understand underlying mechanisms. Resistance can be either intrinsic or acquired. We derived a series of subcloned MCF7 cell lines that were either highly sensitive or naturally resistant to tamoxifen and studied the factors that lead to drug resistance. Gene-expression studies revealed a signature of 67 genes that differentially respond to tamoxifen in sensitive vs. resistant subclones, which also predicts disease-free survival in tamoxifen-treated patients. High-throughput cell-based screens, in which >500 human kinases were independently ectopically expressed, identified 31 kinases that conferred drug resistance on sensitive cells. One of these, HSPB8, was also in the expression signature and, by itself, predicted poor clinical outcome in one cohort of patients. Further studies revealed that HSPB8 protected MCF7 cells from tamoxifen and blocked autophagy. Moreover, silencing HSBP8 induced autophagy and caused cell death. Tamoxifen itself induced autophagy in sensitive cells but not in resistant ones, and tamoxifen-resistant cells were sensitive to the induction of autophagy by other drugs. These results may point to an important role for autophagy in the sensitivity to tamoxifen.functional screen | estrogen receptor T he two thirds of women with estrogen receptor-(ER) or progesterone receptor-positive breast cancers are excellent candidates for antihormone therapy. Selective ER modulators (SERMs), like tamoxifen, block ER activation and have impacted both therapy and survival. However, the success of tamoxifen therapy is limited by intrinsic and acquired drug resistance. Several pathways have been implicated in antiestrogen resistance, including: the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway, which is implicated in cell survival; the EGFR family; and the RAS/RAF/MEK1/2/ERK1/2 family, which regulate cell proliferation (1, 2). Loss of ER expression or function may also be an important mechanism of de novo resistance to tamoxifen, either through relatively rare ER mutations or changes in coactivators and corepressors (3).Several groups have used gene-expression analysis to identify genes regulated through ER (4) that are affected by SERMs in breast cancer cells (5, 6). Others have used tumor samples to develop gene signatures that can predict clinical responses to tamoxifen (7-10). Genetic strategies have also been used to identify genes that drive tamoxifen resistance. Receptor tyrosine kinases and MAPK signaling were detected using expression of pooled cDNA libraries in ZR-75-1, an approach often biased toward the most abundantly expressed genes and which requires recovery of hits by PCR (11). The analysis of antiestrogen-sensitive and -resistant MCF7 cells by SNP and comparative genomic hybridization pointed to changes in protein abundance rather than somatic genomic changes (12). An RNA interference screen of kinases identified CDK10, CRK7, and MAP2K7, whose knockdown cause tamoxif...
Complications of chemotherapy, such as appearance of multidrug resistance, have persuaded researchers to consider phage therapy as a new method to combat bacterial infections. In vitro experiments were performed to assess the therapeutic value of genetically modified phages for controlling gastrointestinal Escherichia coli O157:H7 cells in Luria-Bertani (LB) media and contaminated cow milk. We constructed a modified nonreplicating M13-derived phage expressing a lethal catabolite gene activator protein (CAP) that is a Glu181Gln mutant of CAP. The modified phagemid was propagated in the lethal CAP-resistant strain XA3DII. Time-kill assay experiments showed a considerable reduction in the number of surviving bacteria in both LB media and contaminated cow milk. Our further study using other test strains demonstrated that the host range of lethal phage is limited to E. coli strains that produce pili. This study provides a possible strategy for the exploitation of genetically engineered nonlytic phages as bactericidal agents by minimizing the risk of release of progeny phages and endotoxins into the environment. The phage was engineered to remain lethal to its bacterial target, but incapable of replicating therein. Furthermore, the addition of an inducer to express the lethal protein is not required.
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