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Baskaran Rajasekaran, Ph.D.
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Associate Professor
W1251 BSTWR
200 Lothrop Street
Pittsburgh, Pennsylvania 15261
Phone: (412) 648-9023
Fax: (412) 648-9023
E-mail: bask@pitt.edu
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Research
Our lab is focused on understanding the role that several proto-oncogenes and tumor suppressors play in maintaining the fidelity of the genome. Specifically, our research examines the role of the c-Abl proto-oncogene, and the gene product mutated in the cancer-prone disorder Ataxia-Telangiectasia (termed ATM) in mediating cellular responses to genotoxic stress. During the course of his career, Dr. Rajasekaran has made several important findings in the area of DNA damage signaling. Such seminal advances include identification of RNA polymerase II as substrate for nuclear Abl kinase (a finding that eventually allowed elaboration of its role in genotoxic stress response), and the demonstration that ATM, despite possessing significant homology to the lipid kinase phosphatidylinositol 3-kinase, possesses protein kinase activity. Further, the identification of ATM phosphorylation site in c-Abl paved the way for identification of a repertoire of important substrates in ATM-mediated signaling cascades. His most recent findings highlight the involvement of the mismatch repair (MMR) system in ATM-mediated signaling. This work uncovered a molecular mechanism uniting a DNA repair mechanism with the triggering of damage-induced checkpoint and apoptotic pathways. Current research efforts are focused on understanding the role that the MMR system plays in the regulation of ATM and c-Abl activity in response to ionizing radiation and DNA alkylating agents. Other projects are examining the role that ATM performs in the BRCA1-medicated regulation of GADD45 in response to genotoxic stress.
All cells have intricately coupled sensing and signaling mechanisms that regulate the cellular outcome following exposure to genotoxic agents such as ionizing radiation (IR). In the IRinduced signaling pathway, specific protein events, such as ataxia-telangiectasia mutated protein (ATM) activation and histone H2AX phosphorylation (gamma-H2AX), are mechanistically well characterized. How these mechanisms can be altered, especially by clinically relevant agents, is not clear. Our recent study showed that hyperthermia, an effective radiosensitizer, can induce several steps associated with IR signaling in cells. Hyperthermia induces gamma-H2AX foci formation similar to foci formed in response to IR exposure, and heat-induced gamma-H2AX foci formation is dependent on ATM but independent of heat shock protein 70 expression. Hyperthermia also enhanced ATM kinase activity and increased cellular ATM autophosphorylation. The hyperthermia-induced increase in ATM phosphorylation was independent of Mre11 function. Similar to IR, hyperthermia also induced MDC1 foci formation; however, it did not induce all of the characteristic signals associated with irradiation because formation of 53BP1 and SMC1 foci was not observed in heated cells but occurred in irradiated cells. Additionally, induction of chromosomal DNA strand breaks was observed in IR-exposed but not in heated cells. These results indicate that hyperthermia activates signaling pathways that overlap with those activated by IR-induced DNA damage. Moreover, prior activation of ATM or other components of the IR-induced signaling pathway by heat may interfere with the normal IR-induced signaling required for chromosomal DNA double-strand break repair, thus resulting in increased cellular radiosensitivity.
Curcumin, a natural phenolic compound found in turmeric (Curcuma longa) exhibits anticancer properties, attributed to its antiproliferative and apoptosis-inducing activity. The ubiquitously expressed nonreceptor tyrosine kinase c-Abl regulates stress responses induced by oxidative agents such as ionizing radiation and H2O2. In this study, we show that c-Abl is an important component of the cell death response activated by curcumin and that Abl mediates this response partly through activation of c-Jun N-terminal kinase (JNK). Therefore, inhibition of Abl by STI571 [imatinib (Gleevec)] treatment or down-regulation of Abl expression through Abl-specific shorthairpin RNA (shRNA) diminished cell death induction and JNK activation. Highlighting the interdependent nature of the Abl and JNK signaling in the curcumin-induced cell death response, a JNK inhibitor [anthra(1,9-cd)pyrazol-6(2H)-one-1,9-pyrazoloanthrone (SP600125)] caused very little cell death inhibition in STI571-pretreated cells and in Abl shRNA-expressing cells. Moreover, treatment with Abl and JNK inhibitor alone or together caused similar levels of cell death inhibition. Although p53 induction in response to curcumin treatment is dependent on Abl, we found that Abl-->p53 signaling is not necessary for curcumin-induced cell death. Taken together, the results demonstrate the differential roles played by Abl-->p53 and Abl-->JNK signaling events in modulating the cell death response to curcumin.
About 45% of head and neck squamous cell carcinomas (HNSCC) are characterized by amplification of chromosomal band 11q13. This amplification occurs by a breakage-fusion-bridge (BFB) cycle mechanism. The first step in the BFB cycle involves breakage and loss of distal 11q, from FRA11F (11q14.2) to 11qter. Consequently, numerous genes, including three critical genes involved in the DNA damage response pathway, MRE11A, ATM, and H2AFX are lost in the step preceding 11q13 amplification. We hypothesized that this partial loss of genes on distal 11q may lead to a diminished DNA damage response in HNSCC. Characterization of HNSCC using fluorescence in situ hybridization (FISH) revealed concurrent partial loss of MRE11A, ATM, and H2AFX in all four cell lines with 11q13 amplification and in four of seven cell lines without 11q13 amplification. Quantitative microsatellite analysis and loss of heterozygosity studies confirmed the distal 11q loss. FISH evaluation of a small series of HNSCC, ovarian, and breast cancers confirmed the presence of 11q loss in at least 60% of these tumors. All cell lines with distal 11q loss exhibited a diminished DNA damage response, as measured by a decrease in the size and number of gamma-H2AX foci and increased chromosomal instability following treatment with ionizing radiation. In conclusion, loss of distal 11q results in a defective DNA damage response in HNSCC. Distal 11q loss was also unexpectedly associated with reduced sensitivity to ionizing radiation. Although the literature attributes the poor prognosis in HNSCC to 11q13 gene amplification, our results suggest that distal 11q deletions may be an equally significant factor.
Because systematic studies of global gene expression following genotoxin assault provide clues to the mechanisms of maintenance of genome integrity we are developing new genetic and biochemical approaches, and DNA microarrays to systematically map the stress-response machinery controlling cell cycle arrest, apoptosis and DNA repair. Efforts are also underway to identify how ATM and ATR sense DNA damage caused by IR and UV irradiation. Models envisaging the DNA repair proteins as upstream regulators and downstream targets of ATM and ATR are currently being tested. A recent exciting line of evidence indicates that ATM-activated Abl plays a role in mediating response to growth factor and anti-oxidant signaling which is another line of study that is being followed.
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Selected Publications
- Li H, Baskaran R, Krisky DM, Bein K, Grandi P, Cohen JB, Glorioso JC. 2008. "Chk2 is required for HSV-1 ICP0-mediated G2/M arrest and enhancement of virus growth" Virology 375:13-23. | Abstract
- Parikh RA, White JS, Huang X, Schoppy DW, Baysal BE, Baskaran R, Bakkenist CJ, Saunders WS, Hsu LC, Romkes M, Gollin SM. 2007. "Loss of distal 11q is associated with DNA repair deficiency and reduced sensitivity to ionizing radiation in head and neck squamous cell carcinoma" Genes Chromosomes Cancer 46:761-75. | Abstract
- Hunt CR, Pandita RK, Laszlo A, Higashikubo R, Agarwal M, Kitamura T, Gupta A, Rief N, Horikoshi N, Baskaran R, Lee JH, Löbrich M, Paull TT, Roti Roti JL, Pandita TK. 2007. "Hyperthermia activates a subset of ataxia-telangiectasia mutated effectors independent of DNA strand breaks and heat shock protein 70 status" Cancer Res. 67:3010-7. | Abstract
- Kamath R, Jiang Z, Sun G, Yalowich JC, Baskaran R. 2007. "c-Abl kinase regulates curcumin-induced cell death through activation of c-Jun N-terminal kinase" Mol Pharmacol. 71:61-72. | Abstract
- Adamson AW, Beardsley DI, Kim WJ, Gao Y, Baskaran R, Brown KD. 2005. "Methylator-induced, mismatch repair-dependent G2 arrest is activated through Chk1 and Chk2" Mol Biol Cell. 16:1513-26. | Abstract
- Singh SV, Herman-Antosiewicz A, Singh AV, Lew KL, Srivastava SK, Kamath R, Brown KD, Zhang L, Baskaran R. 2005. "Sulforaphane-induced G2/M phase cell cycle arrest involves checkpoint kinase 2-mediated phosphorylation of cell division cycle 25C" J Biol Chem. 2004 Jun 11;279(24):25813-22. | Abstract
- Shangary S, Lerner EC, Zhan Q, Corey SJ, Smithgall TE, Baskaran R. 2003. "Lyn regulates the cell death response to ultraviolet radiation through c-Jun N terminal kinase-dependent Fas ligand activation" Exp Cell Res. 289:67-76. | Abstract
- Brown KD, Rathi A, Kamath R, Beardsley DI, Zhan Q, Mannino JL, Baskaran R. 2003. "The mismatch repair system is required for S-phase checkpoint activation" Nat Genet. 33:80-4. | Abstract
- Adamson AW, Kim WJ, Shangary S, Baskaran R, Brown KD. 2002. "ATM is activated in response to N-methyl-N'-nitro-N-nitrosoguanidine-induced DNA alkylation" J Biol Chem. 277:38222-9. | Abstract
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