Huichen Wang

Publications of Huichen Wang

• MiR-21 is continually elevated long-term in the brain after exposure to ionizing radiation.

  Authors: Yan Shi, Xiangming Zhang, Xiaobing Tang, Ping Wang, Huichen Wang, Ya Wang

  Radiation research. 01/2012; 177(1):124-8.

  Ionizing radiation stimulates miR-21 expression in different types of mammalian cells in culture. However, it remains unclear whether radiation could stimulate miR-21 expression in brain cells andIonizing radiation stimulates miR-21 expression in different types of mammalian cells in culture. However, it remains unclear whether radiation could stimulate miR-21 expression in brain cells and tissue and, if so, how long the upregulation of miR-21 would be maintained after exposure to different types of radiation. To answer these questions, we examined the miR-21 levels in irradiated mouse hippocampal cells and brain tissue from mice at different times up to 1 year after whole-body exposure to 0.5 Gy of X rays [low linear energy transfer (LET)] or (56)Fe ions (high LET). The results showed that radiation stimulated miR-21 expression in mouse hippocampal cells and upregulation of EGFR, which is similar to that in human hepatocytes, as we reported previously. Interestingly, the miR-21 levels gradually increased within 1 year after irradiation, although there was no significant difference in the miR-21 low- and high-LET irradiated mice. The high expression of miR-21 in the brain was also associated with high expression of EGFR in irradiated mice; thus our data strongly support that EGFR and miR-21 are in a positive regulatory loop, because it is known that radiation stimulates miR-21 through the EGFR/Stat3 pathway and miR-21 activates the EGFR pathway. Since the brain is relatively resistant to radiation-induced histomorphological changes, our findings may provide a new way to explore radiation-induced pathological changes in the brain by investigating miR-21 and its multiple targets.

• S-phase cells are more sensitive to high-linear energy transfer radiation.

  Authors: Hongyan Wang, Shuang Liu, Piyan Zhang, Shimeng Zhang, Mamta Naidu, Huichen Wang, Ya Wang

  International journal of radiation oncology, biology, physics. 08/2009; 74(4):1236-41.

  PURPOSE: S-phase cells are more resistant to low-linear energy transfer (LET) ionizing radiation (IR) than nonsynchronized and G(1)-phase cells, because both nonhomologous end-joining (NHEJ) andPURPOSE: S-phase cells are more resistant to low-linear energy transfer (LET) ionizing radiation (IR) than nonsynchronized and G(1)-phase cells, because both nonhomologous end-joining (NHEJ) and homologous recombination repair can repair DNA double-strand breaks (DSBs) in the S phase. Although it was reported 3 decades ago that S-phase cells did not show more resistance to high-LET IR than cells in other phases, the mechanism remains unclear. We therefore attempted to study the phenotypes and elucidate the mechanism involved. METHODS AND MATERIALS: Wild-type and NHEJ-deficient cell lines were synchronized using the double-thymidine approach. A clonogenic assay was used to detect the sensitivity of nonsynchronized, synchronized S-phase, and G(2)-phase cells to high- and low-LET IR. The amounts of Ku bound to DSBs in the high- and low-LET-irradiated cells were also examined. RESULTS: S-phase wild-type cells (but not NHEJ-deficient cells) were more sensitive to high-LET IR than nonsynchronized and G(2)-phase cells. In addition, S-phase wild-type cells showed less efficient Ku protein binding to DSBs than nonsynchronized and G(2)-phase cells in response to high-LET IR, although all cells at all phases showed similarly efficient levels of Ku protein binding to DSBs in response to low-LET IR. CONCLUSIONS: S-phase cells are more sensitive to high-LET IR than nonsynchronized and G(2)-phase cells, because of the following mechanism: it is more difficult for Ku protein to bind to high-LET IR-induced DNA DSBs in S-phase cells than in cells at other phases, which results in less efficient NHEJ.

• Histone H1 functions as a stimulatory factor in backup pathways of NHEJ.

  Authors: Bustanur Rosidi, Minli Wang, Wenqi Wu, Aparna Sharma, Huichen Wang, George Iliakis

  Nucleic acids research. 04/2008; 36(5):1610-23.

  DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by ionizing radiation (IR) are predominantly removed by two pathways of non-homologous end-joining (NHEJ) termed D-NHEJ andDNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by ionizing radiation (IR) are predominantly removed by two pathways of non-homologous end-joining (NHEJ) termed D-NHEJ and B-NHEJ. While D-NHEJ depends on the activities of the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4/XLF, B-NHEJ utilizes, at least partly, DNA ligase III/XRCC1 and PARP-1. Using in vitro end-joining assays and protein fractionation protocols similar to those previously applied for the characterization of DNA ligase III as an end-joining factor, we identify here histone H1 as an additional putative NHEJ factor. H1 strongly enhances DNA-end joining and shifts the product spectrum from circles to multimers. While H1 enhances the DNA-end-joining activities of both DNA Ligase IV and DNA Ligase III, the effect on ligase III is significantly stronger. Histone H1 also enhances the activity of PARP-1. Since histone H1 has been shown to counteract D-NHEJ, these observations and the known functions of the protein identify it as a putative alignment factor operating preferentially within B-NHEJ.

• Evidence for the involvement of Puralpha in response to DNA replication stress.

  Authors: Huichen Wang, Meijuan Wang, Krzysztof Reiss, Nune Darbinian-Sarkissian, Edward M Johnson, George Iliakis, Shohreh Amini, Kamel Khalili, Jay Rappaport

  Cancer biology & therapy. 05/2007; 6(4):596-602.

  Puralpha is a sequence-specific nucleic acid binding protein that is involved in multiple cellular functions including regulation of transcription, initiation of DNA replication, cell cyclePuralpha is a sequence-specific nucleic acid binding protein that is involved in multiple cellular functions including regulation of transcription, initiation of DNA replication, cell cycle progression, and neuronal cell differentiation. Its potential role in DNA repair has not been explored. We have now analyzed the role of Puralpha in the cellular response to replication-associated DNA repair of double-strand breaks (DSBs) using Puralpha knockout mouse embryo fibroblasts (MEFs). We found that Puralpha negative cells are hypersensitive to the DNA replication inhibitor, hydroxyurea (HU), and that HU induces excessive DSBs, which delayed the resumption of cell cycle progression after HU treatment. Reintroduction of Pura into Pura null cells reduced the accumulation of DSBs and enhanced DNA end joining. These results suggest a role for Puralpha as a caretaker protein that is involved in the repair of DSBs induced by stalled replication forks.

• Plasmid-based assays for DNA end-joining in vitro.

  Authors: George Iliakis, Bustanur Rosidi, Minli Wang, Huichen Wang

  Methods in molecular biology (Clifton, N.J.). 02/2006; 314:123-31.

  Double-strand breaks (DSBs) disrupt DNA integrity and cause genomic instability and cancer, mutations, or cell death. Among the pathways utilized by cells of higher eukaryotes to repair this lesion,Double-strand breaks (DSBs) disrupt DNA integrity and cause genomic instability and cancer, mutations, or cell death. Among the pathways utilized by cells of higher eukaryotes to repair this lesion, nonhomologous end-joining (NHEJ) is the most dominant. The biochemical characterization of NHEJ has significantly benefited from in vitro plasmid end-joining assays that can complement and extend information obtained from genetic studies. There is evidence that several factors involved in DNA-PK-dependent NHEJ remain to be identified. In addition, under certain circumstances, cells utilize backup pathways of NHEJ that depend on unknown factors to remove DSBs. Characterization of these putative factors will benefit from plasmid-based assays of DNA end-joining. Here, we describe a protocol for in vitro end-joining using plasmid DNA as substrate. The required procedures include: (1) preparation of HeLa-cell nuclear extract; (2) preparation of plasmid substrate DNA; (3) assembly of in vitro DNA repair reactions; and (4) product analysis by gel electrophoresis. The assay is powerful and easy to perform, but one should be aware that it represents an oversimplification, as it does not consider the in vivo organization of DNA into chromatin.

• PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways.

  Authors: Minli Wang, Weizhong Wu, Wenqi Wu, Bustanur Rosidi, Lihua Zhang, Huichen Wang, George Iliakis

  Nucleic acids research. 02/2006; 34(21):6170-82.

  Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to formPoly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer.

• DNA ligase III as a candidate component of backup pathways of nonhomologous end joining.

  Authors: Huichen Wang, Bustanur Rosidi, Ronel Perrault, Minli Wang, Lihua Zhang, Frank Windhofer, George Iliakis

  Cancer research. 06/2005; 65(10):4020-30.

  Biochemical and genetic studies support the view that the majority of DNA double-strand breaks induced in the genome of higher eukaryotes by ionizing radiation are removed by two pathways ofBiochemical and genetic studies support the view that the majority of DNA double-strand breaks induced in the genome of higher eukaryotes by ionizing radiation are removed by two pathways of nonhomologous end joining (NHEJ) termed D-NHEJ and B-NHEJ. Whereas D-NHEJ depends on the activities of the DNA-dependent protein kinase and DNA ligase IV/XRCC4, components of B-NHEJ have not been identified. Using extract fractionation, we show that the majority of DNA end joining activity in extracts of HeLa cells derives from DNA ligase III. DNA ligase III fractionates through two columns with the maximum in DNA end joining activity and its depletion from the extract causes loss of activity that can be recovered by the addition of purified enzyme. The same fractionation protocols provide evidence for an additional factor strongly enhancing DNA end joining and shifting the product spectrum from circles to multimers. An in vivo plasmid assay shows that DNA ligase IV-deficient mouse embryo fibroblasts retain significant DNA end joining activity that can be reduced by up to 80% by knocking down DNA ligase III using RNA interference. These in vivo and in vitro observations identify DNA ligase III as a candidate component for B-NHEJ and point to additional factors contributing to NHEJ efficiency.

• Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA-PK in human cells exposed to ionizing radiation and treated with kinase inhibitors.

  Authors: Hongyan Wang, Minli Wang, Huichen Wang, Wilfried Böcker, George Iliakis

  Journal of cellular physiology. 03/2005; 202(2):492-502.

  In eukaryotic cells, DNA double strand breaks (DSBs) cause the prompt phosphorylation of serine 139 at the carboxy terminus of histone H2AX to generate gamma-H2AX, detectable by Western blotting orIn eukaryotic cells, DNA double strand breaks (DSBs) cause the prompt phosphorylation of serine 139 at the carboxy terminus of histone H2AX to generate gamma-H2AX, detectable by Western blotting or immunofluorescence. The consensus sequence at the phosphorylation site implicates the phosphatidylinositol 3-like family of protein kinases in H2AX phosphorylation. It remains open whether ATM (ataxia telangiectasia mutated) is the major H2AX kinase, or whether other members of the family, such as DNA-PK (DNA dependent protein kinase) or ATR (ATM and Rad3 related), contribute in a functionally complementary manner. To address this question, we measured global H2AX phosphorylation in cell lysates and foci formation in individual cells of either wild type or mutant (ATM or DNA-PK) genetic background. Normal global phosphorylation kinetics is observed after irradiation in cells defective either in ATM or DNA-PK alone, suggesting a complementary contribution to H2AX phosphorylation. This is further supported by the observation that initial H2AX phosphorylation is delayed when both kinases are inhibited by wortmannin, as well as when ATM is inhibited by caffeine in DNA-PK deficient cells. However, robust residual global phosphorylation is detectable under all conditions of genetic or chemical inhibition suggesting the function of additional kinases, such as ATR. Treatment with wortmannin, caffeine, or UCN-01 produces a strong DNA-PK dependent late global hyperphosphorylation of H2AX, uncoupled from DNA DSB rejoining and compatible with an inhibition of late steps in DNA DSB processing. Evaluation of gamma-H2AX foci formation confirms the major conclusions made on the basis of global H2AX phosphorylation, but also points to differences particularly several hours after exposure to IR. The results in aggregate implicate DNA-PK, ATM and possibly other kinases in H2AX phosphorylation. The functional significance and the mechanisms of coordination in space and time of these multiple inputs require further investigation.

• ATR affecting cell radiosensitivity is dependent on homologous recombination repair but independent of nonhomologous end joining.

  Authors: Hongyan Wang, Huichen Wang, Simon N Powell, George Iliakis, Ya Wang

  Cancer research. 11/2004; 64(19):7139-43.

  ATR is one of the most important checkpoint proteins in mammalian cells responding to DNA damage. Cells defective in normal ATR activity are sensitive to ionizing radiation (IR). The mechanism byATR is one of the most important checkpoint proteins in mammalian cells responding to DNA damage. Cells defective in normal ATR activity are sensitive to ionizing radiation (IR). The mechanism by which ATR protects the cells from IR-induced killing remains unclear. DNA double-strand breaks (DSBs) induced by IR are critical lesions for cell survival. Two major DNA DSB repair pathways exist in mammalian cells: homologous recombination repair (HRR) and nonhomologous end joining (NHEJ). We show that the doxycycline (dox)-induced ATR kinase dead (ATRkd) cells have the similar inductions and rejoining rates of DNA DSBs compared with cells without dox induction, although the dox-induced ATRkd cells are more sensitive to IR and have the deficient S and G(2) checkpoints. We also show that the dox-induced ATRkd cells have a lower HRR efficiency compared with the cells without dox induction. These results indicate that the effects of ATR on cell radiosensitivity are independent of NHEJ but are linked to HRR that may be affected by the deficient S and G(2) checkpoints.

• Backup pathways of NHEJ are suppressed by DNA-PK.

  Authors: Ronel Perrault, Huichen Wang, Minli Wang, Bustanur Rosidi, George Iliakis

  Journal of cellular biochemistry. 08/2004; 92(4):781-94.

  In cells of higher eukaryotes double strand breaks (DSBs) induced in the DNA after exposure to ionizing radiation (IR) are rapidly rejoined by a pathway of non-homologous end joining (NHEJ) thatIn cells of higher eukaryotes double strand breaks (DSBs) induced in the DNA after exposure to ionizing radiation (IR) are rapidly rejoined by a pathway of non-homologous end joining (NHEJ) that requires DNA dependent protein kinase (DNA-PK) and is therefore termed here D-NHEJ. When this pathway is chemically or genetically inactivated, cells still remove the majority of DSBs using an alternative, backup pathway operating independently of the RAD52 epistasis group of genes and with an order of magnitude slower kinetics (B-NHEJ). Here, we investigate the role of DNA-PK in the functional coordination of D-NHEJ and B-NHEJ using as a model end joining by cell extracts of restriction endonuclease linearized plasmid DNA. Although DNA end joining is inhibited by wortmannin, an inhibitor of DNA-PK, the degree of inhibition depends on the ratio between DNA ends and DNA-PK, suggesting that binding of inactive DNA-PK to DNA ends not only blocks processing by D-NHEJ, but also prevents the function of B-NHEJ. Residual end joining under conditions of incomplete inhibition, or in cells lacking DNA-PK, is attributed to the function of B-NHEJ operating on DNA ends free of DNA-PK. Thus, DNA-PK suppresses alternative pathways of end joining by efficiently binding DNA ends and shunting them to D-NHEJ.

• Caffeine inhibits homology-directed repair of I-SceI-induced DNA double-strand breaks.

  Authors: Huichen Wang, Wilfried Boecker, Hongyan Wang, Xiang Wang, Jun Guan, Larry H Thompson, Jac A Nickoloff, George Iliakis

  Oncogene. 02/2004; 23(3):824-34.

  We recently reported that two Chinese hamster mutants deficient in the RAD51 paralogs XRCC2 and XRCC3 show reduced radiosensitization after treatment with caffeine, thus implicating homology-directedWe recently reported that two Chinese hamster mutants deficient in the RAD51 paralogs XRCC2 and XRCC3 show reduced radiosensitization after treatment with caffeine, thus implicating homology-directed repair (HDR) of DNA double-strand breaks (DSBs) in the mechanism of caffeine radiosensitization. Here, we investigate directly the effect of caffeine on HDR initiated by DSBs induced by a rare cutting endonuclease (I-SceI) into one of two direct DNA repeats. The results demonstrate a strong inhibition by caffeine of HDR in wild-type cells, and a substantial reduction of this effect in HDR-deficient XRCC3 mutant cells. Inhibition of HDR and cell radiosensitization to killing shows similar dependence on caffeine concentration suggesting a cause-effect relationship between these effects. UCN-01, a kinase inhibitor that effectively abrogates checkpoint activation in irradiated cells, has only a small effect on HDR, indicating that similar to radiosensitization, inhibition of checkpoint signaling is not sufficient for HDR inhibition. Recombination events occurring during treatment with caffeine are characterized by rearrangements reminiscent to those previously reported for the XRCC3 mutant, and immunofluorescence microscopy demonstrates significantly reduced formation of IR-specific RAD51 foci after caffeine treatment. In summary, our results identify inhibition of HDR as a significant contributor to caffeine radiosensitization.

• DNA damage checkpoint control in cells exposed to ionizing radiation.

  Authors: George Iliakis, Ya Wang, Jun Guan, Huichen Wang

  Oncogene. 10/2003; 22(37):5834-47.

  Damage induced in the DNA after exposure of cells to ionizing radiation activates checkpoint pathways that inhibit progression of cells through the G1 and G2 phases and induce a transient delay inDamage induced in the DNA after exposure of cells to ionizing radiation activates checkpoint pathways that inhibit progression of cells through the G1 and G2 phases and induce a transient delay in the progression through S phase. Checkpoints together with repair and apoptosis are integrated in a circuitry that determines the ultimate response of a cell to DNA damage. Checkpoint activation typically requires sensors and mediators of DNA damage, signal transducers and effectors. Here, we review the current state of knowledge regarding mechanisms of checkpoint activation and proteins involved in the different steps of the process. Emphasis is placed on the role of ATM and ATR, as well on CHK1 and CHK2 kinases in checkpoint response. The roles of downstream effectors, such as P53 and the CDC25 family of proteins, are also described, and connections between repair and checkpoint activation are attempted. The role of checkpoints in genomic stability and the potential of improving the treatment of cancer by DNA damage inducing agents through checkpoint abrogation are also briefly outlined.

• Biochemical evidence for Ku-independent backup pathways of NHEJ.

  Authors: Huichen Wang, Ange Ronel Perrault, Yoshihiko Takeda, Wei Qin, Hongyan Wang, George Iliakis

  Nucleic acids research. 10/2003; 31(18):5377-88.

  Cells of higher eukaryotes process within minutes double strand breaks (DSBs) in their genome using a non-homologous end joining (NHEJ) apparatus that engages DNA-PKcs, Ku, DNA ligase IV, XRCC4 andCells of higher eukaryotes process within minutes double strand breaks (DSBs) in their genome using a non-homologous end joining (NHEJ) apparatus that engages DNA-PKcs, Ku, DNA ligase IV, XRCC4 and other as of yet unidentified factors. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DNA DSBs using an alternative pathway operating with an order of magnitude slower kinetics. This alternative pathway is active in mutants deficient in genes of the RAD52 epistasis group and frequently joins incorrect ends. We proposed, therefore, that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK-dependent (D-NHEJ) pathway, rather than homology directed repair of DSBs. The present study investigates the role of Ku in the coordination of these pathways using as a model end joining of restriction endonuclease linearized plasmid DNA in whole cell extracts. Efficient, error-free, end joining observed in such in vitro reactions is strongly inhibited by anti-Ku antibodies. The inhibition requires DNA-PKcs, despite the fact that Ku efficiently binds DNA ends in the presence of antibodies, or in the absence of DNA-PKcs. Strong inhibition of DNA end joining is also mediated by wortmannin, an inhibitor of DNA-PKcs, in the presence but not in the absence of Ku, and this inhibition can be rescued by pre-incubating the reaction with double stranded oligonucleotides. The results are compatible with a role of Ku in directing end joining to a DNA-PK dependent pathway, mediated by efficient end binding and productive interactions with DNA-PKcs. On the other hand, efficient end joining is observed in extracts of cells lacking DNA-PKcs, as well as in Ku-depleted extracts in line with the operation of alternative pathways. Extracts depleted of Ku and DNA-PKcs rejoin blunt ends, as well as homologous ends with 3' or 5' protruding single strands with similar efficiency, but addition of Ku suppresses joining of blunt ends and homologous ends with 3' overhangs. We propose that the affinity of Ku for DNA ends, particularly when cooperating with DNA-PKcs, suppresses B-NHEJ by quickly and efficiently binding DNA ends and directing them to D-NHEJ for rapid joining. A chromatin-based model of DNA DSB rejoining accommodating biochemical and genetic results is presented and deviations between in vitro and in vivo results discussed.

• Caffeine could not efficiently sensitize homologous recombination repair-deficient cells to ionizing radiation-induced killing.

  Authors: Huichen Wang, Xiang Wang, George Iliakis, Ya Wang

  Radiation research. 04/2003; 159(3):420-5.

  Caffeine inhibits ATM and ATR, two important checkpoint regulators, abolishes ionizing radiation-induced checkpoint response, and radiosensitizes cells. Radiation-induced DNA double-strand breaksCaffeine inhibits ATM and ATR, two important checkpoint regulators, abolishes ionizing radiation-induced checkpoint response, and radiosensitizes cells. Radiation-induced DNA double-strand breaks (DSBs) are repaired by two major processes, homologous recombination repair (HRR) and nonhomologous end joining (NHEJ). It remains unclear which repair process, HRR or NHEJ, is affected when the checkpoint responses are abolished by caffeine. In this study we observed the effect of caffeine on gene-targeted DT40 chicken lymphoblast cells. We show that caffeine efficiently abolishes S- and G(2)-phase checkpoint responses after irradiation in all cell lines tested and greatly radiosensitizes wild-type and ATM(-/-) cells, the partially checkpoint-deficient cells. However, caffeine has a much smaller radiosensitizing effect on RAD54(-/-) cells and has no effect on RAD51-deficient cells. RAD51 and RAD54 are the important factors for HRR. Our results indicate that the checkpoint responses abolished by caffeine (S and G(2)) mainly affect HRR, which results in cell radiosensitization.

• Caffeine-induced radiosensitization is independent of nonhomologous end joining of DNA double-strand breaks.

  Authors: Xiang Wang, Huichen Wang, George Iliakis, Ya Wang

  Radiation research. 04/2003; 159(3):426-32.

  After exposure to ionizing radiation, proliferating cells actively slow down progression through the cell cycle through the activation of checkpoints to provide time for repair. Two majorAfter exposure to ionizing radiation, proliferating cells actively slow down progression through the cell cycle through the activation of checkpoints to provide time for repair. Two major complementary DNA double-strand break (DSB) repair pathways exist in mammalian cells, homologous recombination repair (HRR) and nonhomologous end joining (NHEJ). The relationship between checkpoint activation and these two types of DNA DSB repair pathways is not clear. Caffeine, as a nonspecific inhibitor of ATM and ATR, abolishes multi-checkpoint responses and sensitizes cells to radiation-induced killing. However, it remains unknown which DNA repair process, NHEJ or HRR, or both, is affected by caffeine-abolished checkpoint responses. We report here that caffeine abolishes the radiation-induced G(2)-phase checkpoint and efficiently sensitizes both NHEJ-proficient and NHEJ-deficient mammalian cells to radiation-induced killing without affecting NHEJ. Our results indicate that caffeine-induced radiosensitization occurs by affecting an NHEJ-independent process, possibly HRR.

• hTERT associates with human telomeres and enhances genomic stability and DNA repair.

  Authors: Girdhar G Sharma, Arun Gupta, Huichen Wang, Harry Scherthan, Sonu Dhar, Varsha Gandhi, George Iliakis, Jerry W Shay, Charles S H Young, Tej K Pandita

  Oncogene. 02/2003; 22(1):131-46.

  Ectopic expression of telomerase in telomerase-silent cells is sufficient to overcome senescence and to extend cellular lifespan. We show here that the catalytic subunit of human telomerase (hTERT)Ectopic expression of telomerase in telomerase-silent cells is sufficient to overcome senescence and to extend cellular lifespan. We show here that the catalytic subunit of human telomerase (hTERT) crosslinks telomeres. This interaction is blocked by the telomere repeat binding factor 1, but not by a dominant negative form of this protein. It is also abolished by destruction of the RNA component of telomerase as well as by mutations in the hTERT protein. Ectopic expression of hTERT leads to transcriptional alterations of a subset of genes and changes in the interaction of the telomeres with the nuclear matrix. This is associated with reduction of spontaneous chromosome damage in G(1) cells, enhancement of the kinetics of DNA repair and an increase in NTP levels. The effect on DNA repair is likely indirect as TERT does not directly affect DNA end rejoining in vitro or meiotic recombination in vivo. The observed effects of hTERT occurred rapidly before any significant lengthening of telomeres was observed. Our findings establish an intimate relationship between hTERT-telomere interactions and alteration in transcription of a subset of genes that may lead to increased genomic stability and enhanced repair of genetic damage. These novel functions of telomerase are distinct from its known effect on telomere length and have potentially important biological consequences.

• Role of Puralpha in the modulation of homologous recombination-directed DNA repair by HIV-1 Tat.

  Authors: Huichen Wang, Martyn K White, Rafal Kaminski, Nune Darbinian, Shohreh Amini, Edward M Johnson, Kamel Khalili, Jay Rappaport

  Anticancer research. 28(3A):1441-7.

  The nucleic acid-binding protein Puralpha is involved at stalled DNA replication forks, in double-strand break (DSB) DNA repair and the cellular response to DNA replication stress. Puralpha interactsThe nucleic acid-binding protein Puralpha is involved at stalled DNA replication forks, in double-strand break (DSB) DNA repair and the cellular response to DNA replication stress. Puralpha interacts with HIV-1 Tat, which regulates homologous recombination-directed DNA repair (HRR). We investigated Rad51 and HRR regulation in mouse embryo fibroblasts (MEFs) from PURA -/- knockout mice that lack Puralpha. Rad51 was induced in PURA -/- MEFs but was repressed when Puralpha was ectopically expressed in these cells. Similarly Rad51 inversely correlated with the level of Puralpha in normal postnatal mouse brain. HIV-1 Tat stimulated HRR DNA repair of I-SceI induced DNA DSBs and the nuclear appearance of Rad51 foci. In contrast, Puralpha suppressed HRR DNA repair, Rad51 expression, and Rad51 foci formation. Tat stimulates the Rad51 promoter involving both Puralpha-dependent and Puralpha-independent mechanisms. Interaction between Puralpha and Tat may have opposing effects on Rad51 expression. The effects on HRR may contribute to HIV-1 associated pathogenesis.