Breast cancer is the most commonly diagnosed cancer among women in Western countries. Currently, there is no effective therapy for malignant estrogen-independent breast cancer. We have screened 38 species of edible mushroom on human estrogen-receptor positive (MCF-7) and estrogenreceptor negative (MDA-MB-231, BT-20) breast cancer cells to select potential agents with broad-spectrum antitumor activity against breast cancer cells. Water-based extracts of three mushroom species, Coprinellus sp., Coprinus comatus, Flammulina velutipes (CME, CCE and FVE respectively), were identified as novel anti-breast cancer agents. The antitumor activities include: 1) marked growth inhibition of both ER + and ERbreast cancer cells; 2) induction of rapid apoptosis on both ER + and ERcells; 3) significant inhibition of MCF-7 tumor colony formation in vitro. The antiproliferative and cytotoxic activities of the three mushroom extracts were dose-dependent, regardless of the hormone receptor status of the cancer cells. The degree of produced cytotoxicity on ERbreast cancer cells was very high, while the IC 50 of mushroom extract CME was found to be as low as 40 μg/ml on MDA-MB-231 cells and the IC 50 of mushroom extract FVE was only 30 μg/ml on BT-20 cells. More interestingly, mushroom extracts CME and FVE induced an exceptionally rapid apoptosis on MCF-7 and MDA-MB-231 detected by Annexin V-FITC within 2 h of treatment and DNA fragment endlabeling assay (TUNEL) in 5 h of treatment. Anchorageindependent growth assays indicated that the MCF-7 tumor colony formation rate was reduced by 60% in CCE-and CME-treated cells and nearly completely inhibited (99%) by FVE treatment. These results suggest that mushroom species Coprinus comatus, Coprinellus sp. and Flammulina velutipes contain potent antitumor compounds for breast cancer. Our finding is important due to the lack of chemotherapeutic and chemopreventive agents for ERhuman breast cancer.

The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA) function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA), the first committed intermediate synthesized during heme synthesis. Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

c‐Myb is a transcription factor and proto‐oncogene that is important for hematopoietic cell differentiation. The regulation of the c‐Myb protein and its down stream targets is well studied while the regulation of expression of the c‐myb gene is not well understood. c‐myb expression is controlled by a conditional block in transcriptional elongation in the first intron of the gene. Previous studies have shown that NF‐kB family members are involved in regulating the activation of c‐myb by binding to Rel‐Related Proteins Binding Elements (RRBE) in the first intron. This leads to the hypothesis that NF‐kB regulates expression of c‐myb during development of hematopoietic cells. Here we show using chromatin immunoprecipitations (ChIPs) and real‐time PCR in murine erythroleukemia cells that the down regulation of c‐myb expression via hexamethylene bisacetamide‐induced differentiation can be over‐come by activation of adenyl cyclase by forskolin. ChIP results demonstrate a correlation between occupancy of the RRBE sites by the p65 subunit of NF‐kB with elevated c‐myb mRNA levels. This study demonstrates for the first time a correlation between NF‐kB occupancy of RRBE sites and c‐myb expression in vivo in hematopoietic cells. Research support provided by a Providence College Committee on Aid to Faculty Research Grant.

c‐Myb is a transcription factor and proto‐oncogene that is important for hematopoietic cell development. This gene is predominately expressed in progenitor cells of adult bone marrow and down regulated during differentiation. Through gene knockout experimentation, the importance for c‐myb in developing cells has been determined. Null mutants show lethality in certain blood cell lines, strengthening the ideas that c‐myb plays an important role in cell line differentiation. Specifically, in murine embryonic cells, the absence of c‐myb results in the disruption of specific blood cell types. Previous studies have shown that NF‐κB family members are involved in regulating the activation of c‐myb by binding to Rel‐Related Proteins Binding Elements (RRBE) in the first intron. This leads to the hypothesis that NF‐kB regulates expression of c‐myb during development of hematopoietic cells. We have set up an in vitro differentiation protocol for differentiating induced pluripotent stem cells into blood cell precursors via a hemangioblast intermediate using defined cytokine and growth factors. Activation of c‐myb and NF‐kB was analyzed using qPCR to analyze the kinetics of c‐myb expression. Work is continuing using mobility shift assays and chromatin immunoprecipitations to determine the localization of NF‐kB to the c‐myb locus. Research support provided by a Providence College Committee on Aid to Faculty Research Grant.