It has been reported that the expression and activity of the interferon-inducible, dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and primary tumor samples. To extend these findings and determine how PKR signaling may affect breast cancer cell sensitivity to chemotherapy, we measured PKR expression by immunohistochemical staining of 538 cases of primary breast cancer and normal tissues. Significantly, PKR expression was elevated in ductal, lobular and squamous cell carcinomas or lymph node metastases but not in either benign tumor specimens or cases of inflammation compared to normal tissues. Furthermore, PKR expression was increased in precancerous stages of mammary cell hyperplasia and dysplasia compared to normal tissues, indicating that PKR expression may be upregulated by the process of tumorigenesis. To test the function of PKR in breast cancer, we generated MCF7, T-47D and MDA-MB-231 breast cancer cell lines with significantly reduced PKR expression by siRNA knockdown. Importantly, while knockdown of PKR expression had no effect on cell proliferation under normal growth conditions, MCF7, T-47D or MDA-MB-231 cells with reduced PKR expression or treated with a small molecule PKR inhibitor were significantly less sensitive to doxorubicin or H2O2-induced toxicity compared to control cells. In addition, the rate of eIF2α phosphorylation following treatment with doxorubicin was delayed in breast cancer cell lines with decreased PKR expression. Significantly, treatment of breast cancer lines with reduced PKR expression with either interferon-α, which increases PKR expression, or salubrinal, which increases eIF2α phosphorylation, restored doxorubicin sensitivity to normal levels. Taken together these results indicate that increased PKR expression in primary breast cancer tissues may serve as a biomarker for response to doxorubicin-containing chemotherapy and that future therapeutic approaches to promote PKR expression/activation and eIF2α phosphorylation may be beneficial for the treatment of breast cancer.
Endospores of Bacillus subtilis HA101 were applied to a simulated Mars Exploration Rover (MER) wheel and exposed to Mars-normal UV irradiation for 1, 3, or 6 h. The experiment was designed to simulate a contaminated rover wheel sitting on its landing platform before rolling off onto the martian terrain, as was encountered during the Spirit and Opportunity missions. When exposed to 1 h of Mars UV, a reduction of 81% of viable endospores was observed compared to the non-UV irradiated controls. When exposed for 3 or 6 h, reductions of 94.6% and 96.6%, respectively, were observed compared to controls. In a second experiment, the contaminated rover wheel was rolled over a bed of heat-sterilized Mars analog soil; then the analog soil was exposed to full martian conditions of UV irradiation, low pressure (6.9 mbar), low temperature (-10°C), and an anaerobic CO(2) martian atmosphere for 24 h to determine whether endospores of B. subtilis on the contaminated rover wheel could be transferred to the surface of the analog soil and survive martian conditions. The experiment simulated conditions in which a rover wheel might come into contact with martian regolith immediately after landing, such as is designed for the upcoming Mars Science Laboratory (MSL) rover. The contaminated rover wheel transferred viable endospores of B. subtilis to the Mars analog soil, as demonstrated by 31.7% of samples showing positive growth. However, when contaminated soil samples were exposed to full martian conditions for 24 h, only 16.7% of samples exhibited positive growth-a 50% reduction in the number of soil samples positive for the transferred viable endospores.
Breast cancer is the second most frequently diagnosed cancer in U. S. women with >200,000 new cases estimated for 2010. Resistance to standard chemotherapy remains a significant clinical challenge. Thus, the identification of novel molecular biomarkers that predict response to treatment may improve therapeutic decision making and outcomes. Recently it has been reported that the level and activity of the dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and more aggressive primary tumor specimens. Previously, our laboratory has determined that PKR is a key regulator of cell proliferation, apoptosis and transformation. PKR is activated by diverse cellular stresses including growth factor withdrawal from hematopoietic cells, inflammatory cytokine treatment or viral infection to initiate apoptosis presumably as a result eIF2α phosphorylation and inhibition of protein synthesis. However, activated PKR may also promote apoptosis by inhibiting Bcl2 phosphorylation and regulate cell proliferation by mechanisms dependent on p53, STAT1 or NF-κB. In order to gain insight into how PKR signaling may affect breast cancer development and sensitivity to chemotherapy, we measured the level of PKR in primary breast cancer (and paired, normal adjacent tissue) by immunohistochemical staining of a breast tumor tissue microarray containing 50 cases. Increased PKR staining was observed in all tumor types tested (infiltrating ductal carcinoma, mucinous adenocarcinoma, medullary carcinoma, Paget's disease, carcinosarcoma, cystosarcoma phyllodes and benign fibroadenoma) compared to normal adjacent tissue. Furthermore, elevated PKR expression directly correlates with increased tumor grade indicating a more aggressive phenotype. To determine whether PKR may be required for tumor response to doxorubicin, we employed a siRNA strategy to knock-down PKR by >75% in several breast cell lines. Interestingly, basal PKR activity is dramatically higher in those cell lines derived from human mammary carcinoma (i.e. MCF7 and T-47D) compared to a “nontransformed” mammary epithelial cell line (MCF10A). Furthermore, timecourse and dose response studies with doxorubicin, a frequently used and effective chemotherapy, revealed that cells with reduced PKR are significantly less sensitive to the drug compared to control siRNA cells with higher PKR. Interestingly, phosphorylation of eIF2α is unaffected by reduced PKR expression in MCF7 cells even with the highest dose of doxorubicin. This indicates that the mechanism by which PKR promotes doxorubicin sensitivity may be independent of eIF2α phosphorylation and protein synthesis inhibition. Together, these results suggest that increased PKR expression may be a useful biomarker for more aggressive breast tumors and for predicting response to doxorubicin treatment. Based on this model, aggressive tumors that do not display elevated PKR are predicted to be resistant to doxorubicin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 203. doi:10.1158/1538-7445.AM2011-203
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