http://hdl.handle.net/10027/1133 Fri, 24 May 2013 07:07:52 GMT 2013-05-24T07:07:52Z http://hdl.handle.net/10027/8759 Specifically targeting ERK1 or ERK2 kills Melanoma cells Qin, Jianzhong; Xin, Hong; Nickoloff, Brian J. Background: Overcoming the notorious apoptotic resistance of melanoma cells remains a therapeutic challenge given dismal survival of patients with metastatic melanoma. However, recent clinical trials using a BRAF inhibitor revealed encouraging results for patients with advanced BRAF mutant bearing melanoma, but drug resistance accompanied by recovery of phospho-ERK (pERK) activity present challenges for this approach. While ERK1 and ERK2 are similar in amino acid composition and are frequently not distinguished in clinical reports, the possibility they regulate distinct biological functions in melanoma is largely unexplored. Methods: Rather than indirectly inhibiting pERK by targeting upstream kinases such as BRAF or MEK, we directly (and near completely) reduced ERK1 and ERK2 using short hairpin RNAs (shRNAs) to achieve sustained inhibition of pERK1 and/or pERK2. Results and discussion: Using A375 melanoma cells containing activating BRAFV600E mutation, silencing ERK1 or ERK2 revealed some differences in their biological roles, but also shared roles by reduced cell proliferation, colony formation in soft agar and induced apoptosis. By contrast, chemical mediated inhibition of mutant BRAF (PLX4032) or MEK (PD0325901) triggered less killing of melanoma cells, although they did inhibit proliferation. Death of melanoma cells by silencing ERK1 and/or ERK2 was caspase dependent and accompanied by increased levels of Bak, Bad and Bim, with reduction in p-Bad and detection of activated Bax levels and loss of mitochondrial membrane permeability. Rare treatment resistant clones accompanied silencing of either ERK1 and/or ERK2. Unexpectedly, directly targeting ERK levels also led to reduction in upstream levels of BRAF, CRAF and pMEK, thereby reinforcing the importance of silencing ERK as regards killing and bypassing drug resistance. Conclusions: Selectively knocking down ERK1 and/or ERK2 killed A375 melanoma cells and also increased the ability of PLX4032 to kill A375 cells. Thus, a new therapeutic window is open for future clinical trials in which agents targeting ERK1 and ERK2 should be considered in patients with melanoma. © 2012 Qin et al; BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sun, 01 Jan 2012 06:00:00 GMT http://hdl.handle.net/10027/8759 2012-01-01T06:00:00Z http://hdl.handle.net/10027/8606 Clonal immune responses of Mycobacterium-specific gammadelta T cells in tuberculous and non-tuberculous tissues during M. tuberculosis infection Huang, Dan; Chen, Crystal Y.; Zhang, Meihong; Qiu, Liyou; Shen, Yun; Du, George; Zhou, Keyuan; Wang, Richard; Chen, Zheng W. Background: We previously demonstrated that unvaccinated macaques infected with large-dose M.tuberculosis(Mtb) exhibited delays for pulmonary trafficking of Ag-specific alphabeta and gammadelta T effector cells, and developed severe lung tuberculosis(TB) and "secondary" Mtb infection in remote organs such as liver and kidney. Despite delays in lungs, local immunity in remote organs may accumulate since progressive immune activation after pulmonary Mtb infection may allow IFNgamma-producing gammadelta T cells to adequately develop and traffic to lately-infected remote organs. As initial efforts to test this hypothesis, we comparatively examined TCR repertoire/clonality, tissue trafficking and effector function of Vgamma2Vdelta2 T cells in lung with severe TB and in liver/kidney without apparent TB. Methodology/Principal Findings: We utilized conventional infection-immunity approaches in macaque TB model, and employed our decades-long expertise for TCR repertoire analyses. TCR repertoires in Vgamma2Vdelta2 T-cell subpopulation were broad during primary Mtb infection as most TCR clones found in lymphoid system, lung, kidney and liver were distinct. Polyclonally-expanded Vgamma2Vdelta2 T-cell clones from lymphoid tissues appeared to distribute and localize in lung TB granuloms at the endpoint after Mtb infection by aerosol. Interestingly, some TCR clones appeared to be more predominant than others in lymphocytes from liver or kidney without apparent TB lesions. TCR CDR3 spetratyping revealed such clonal dominance, and the clonal dominance of expanded Vgamma2Vdelta2 T cells in kidney/liver tissues was associated with undetectable or low-level TB burdens. Furthermore, Vgamma2Vdelta2 T cells from tissue compartments could mount effector function for producing anti-mycobacterium cytokine. Conclusion: We were the first to demonstrate clonal immune responses of mycobacterium-specific Vgamma2Vdelta2 T cells in the lymphoid system, heavily-infected lungs and lately subtly-infected kidneys or livers during primary Mtb infection. While clonally-expanded Vgamma2Vdelta2 T cells accumulated in lately-infected kidneys/livers without apparent TB lesions, TB burdens or lesions appeared to impact TCR repertoires and tissue trafficking patterns of activated Vgamma2Vdelta2 T cells. © 2012 Huang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. doi: 10.1371/journal.pone.0030631 Sun, 01 Jan 2012 06:00:00 GMT http://hdl.handle.net/10027/8606 2012-01-01T06:00:00Z http://hdl.handle.net/10027/8578 Regulation and Consequence of Serine Catabolism in Streptococcus pyogenes LaSarre, Breah; Federle, Michael J. The Gram-positive bacterium Streptococcus pyogenes (Group A Streptococcus, GAS), is found strictly in humans and is capable of causing a wide variety of infections. Here we demonstrate that serine catabolism in GAS is controlled by the transcriptional regulator, Spy49_0126c. We have designated this regulator SerR (serine catabolism regulator). Microarray and transcriptional reporter data show that SerR acts as a transcriptional repressor of multiple operons, including sloR and sdhBA. Purified recombinant SerR binds to promoters of both sloR and sdhB, demonstrating that this regulation is direct. Deletion of serR results in reduced culture yield of the mutant compared to wild-type when grown in defined medium unless additional serine is provided, suggesting that regulation of serine metabolism is 10 important for maximizing bacterial growth. Deletion of sloR or sdhB in the DserR mutant background restores growth to wild-type levels, suggesting that both operons have roles in serine catabolism. While reports have linked sloR function to streptolysin O expression, transport experiments with radiolabeled L-serine reveal that the sloR operon is required for rapid acquisition of serine, implicating a novel role for this operon in amino acid metabolism. Post print version of article may differ from published version. The definitive version is available through American Society for Microbiology at DOI: 10.1128/JB.01516-10 Fri, 01 Apr 2011 05:00:00 GMT http://hdl.handle.net/10027/8578 2011-04-01T05:00:00Z http://hdl.handle.net/10027/8568 Suppression of FOXM1 Sensitizes Human Cancer Cells to Cell Death Induced by DNA-Damage Halasi, Marianna; Gartel, Andrei L. Irradiation and DNA-damaging chemotherapeutic agents are commonly used in anticancer treatments. Following DNA damage FOXM1 protein levels are often elevated. In this study, we sought to investigate the potential role of FOXM1 in programmed cell death induced by DNA-damage. Human cancer cells after FOXM1 suppression were subjected to doxorubicin or c-irradiation treatment. Our findings indicate that FOXM1 downregulation by stable or transient knockdown using RNAi or by treatment with proteasome inhibitors that target FOXM1 strongly sensitized human cancer cells of different origin to DNA-damage-induced apoptosis. We showed that FOXM1 suppresses the activation of pro-apoptotic JNK and positively regulates anti-apoptotic Bcl-2, suggesting that JNK activation and Bcl-2 down-regulation could mediate sensitivity to DNA-damaging agent-induced apoptosis after targeting FOXM1. Since FOXM1 is widely expressed in human cancers, our data further support the fact that it is a valid target for combinatorial anticancer therapy. © 2012 Halasi, Gartel. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. doi:10.1371/journal.pone.0031761 Wed, 29 Feb 2012 06:00:00 GMT http://hdl.handle.net/10027/8568 2012-02-29T06:00:00Z