Biomedical subjects

Benjamin P C Chen

Publications and source records attributed to Benjamin P C Chen.

14 recordsLinked to original sources

PubMed2006

Ataxia telangiectasia mutated (ATM) is essential for DNA-PKcs phosphorylations at the Thr-2609 cluster upon DNA double strand break.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is rapidly phosphorylated at the Thr-2609 cluster and Ser-2056 upon ionizing radiation (IR). Furthermore, DNA-PKcs phosphorylation at both regions is critical for its role in DNA double strand break (DSB) repair as well as cellular resistance to radiation. IR-induced DNA-PKcs phosphorylation at Thr-2609 and Ser-2056, however, exhibits distinct kinetics indicating that they are differentially regulated. Although DNA-PKcs autophosphorylates itself at Ser-2056 after IR, we have reported here that ATM mediates DNA-PKcs phosphorylation at Thr-2609 as well as at the adjacent (S/T)Q motifs within the Thr-2609 cluster. In addition, our data suggest that DNA-PKcs- and ATM-mediated DNA-PKcs phosphorylations are cooperative and required for the full activation of DNA-PKcs and the subsequent DSB repair. Elimination of DNA-PKcs phosphorylation at both regions severely compromises radioresistance and DSB repair. Finally, our result provides a possible mechanism for the direct involvement of ATM in non-homologous end joining-mediated DSB repair.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2006

ATR-dependent phosphorylation of DNA-dependent protein kinase catalytic subunit in response to UV-induced replication stress.

Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) upon ionizing radiation (IR) is essential for cellular radioresistance and nonhomologous-end-joining-mediated DNA double-strand break repair. In addition to IR induction, we have previously shown that DNA-PKcs phosphorylation is increased upon camptothecin treatment, which induces replication stress and replication-associated double-strand breaks. To clarify the involvement of DNA-PKcs in this process, we analyzed DNA-PKcs phosphorylation in response to UV irradiation, which causes replication stress and activates ATR (ATM-Rad3-related)/ATM (ataxia-telangiectasia mutated) kinases in a replication-dependent manner. Upon UV irradiation, we observed a rapid DNA-PKcs phosphorylation at T2609 and T2647, but not at S2056, distinct from that induced by IR. UV-induced DNA-PKcs phosphorylation occurs specifically only in replicating cells and is dependent on ATR kinase. Inhibition of ATR activity via caffeine, a dominant-negative kinase-dead mutant, or RNA interference led to the attenuation of UV-induced DNA-PKcs phosphorylation. Furthermore, DNA-PKcs associates with ATR in vivo and is phosphorylated by ATR in vitro, suggesting that DNA-PKcs could be the direct downstream target of ATR. Taken together, these results strongly suggest that DNA-PKcs is required for the cellular response to replication stress and might play an important role in the repair of stalled replication forks.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2006

Role of non-homologous end joining (NHEJ) in maintaining genomic integrity.

Of the various types of DNA damage that can occur within the mammalian cell, the DNA double strand break (DSB) is perhaps the most dangerous. DSBs are typically induced by intrinsic sources such as the by products of cellular metabolism or by extrinsic sources such as X-rays or gamma-rays and chemotherapeutic drugs. It is becoming increasing clear that an inability to respond properly to DSBs will lead to genomic instability and promote carcinogenesis. The mammalian cell, therefore, has in place several mechanisms that can respond rapidly to DSBs. In this review, we focus on the role of one such mechanism, the non-homologous end joining (NHEJ) pathway of DSB repair, in maintaining genome integrity and preventing carcinogenesis.

Animals↗

PubMed2006

DNA-PK phosphorylates histone H2AX during apoptotic DNA fragmentation in mammalian cells.

The phosphorylation of histone H2AX at serine 139 is one of the earliest responses of mammalian cells to ionizing radiation-induced DNA breaks. DNA breaks are also generated during the terminal stages of apoptosis when chromosomal DNA is cleaved into oligonucleosomal pieces. Apoptotic DNA fragmentation and the consequent chromatin condensation are important for efficient clearing of genomic DNA and nucleosomes and for protecting the organism from auto-immmunization and oncogenic transformation. In this study, we demonstrate that H2AX is phosphorylated during apoptotic DNA fragmentation in mouse, Chinese hamster ovary, and human cells. We have previously shown that ataxia telangiectasia mutated kinase (ATM) is primarily responsible for H2AX phosphorylation in murine cells in response to ionizing radiation. Interestingly, we find here that DNA-dependent protein kinase (DNA-PK) is solely responsible for H2AX phosphorylation during apoptosis while ATM is dispensable for the process. Moreover, the kinase activity of DNA-PKcs (catalytic subunit of DNA-PK) is specifically required for the induction of gammaH2AX. We further show that DNA-PKcs is robustly activated in apoptotic cells, as evidenced by autophosphorylation at serine 2056, before it is inactivated by cleavage. In contrast, ATM is degraded well before DNA fragmentation and gammaH2AX induction resulting in the predominance of DNA-PK during the later stages of apoptosis. Finally, we show that DNA-PKcs autophosphorylation and gammaH2AX induction occur only in apoptotic nuclei with characteristic chromatin condensation but not in non-apoptotic nuclei from the same culture establishing the most direct link between DNA fragmentation, DNA-PKcs activation, and H2AX phosphorylation. It is well established that DNA-PK is inactivated by cleavage late in apoptosis in order to forestall DNA repair. Our results demonstrate, for the first time, that DNA-PK is actually activated in late apoptotic cells and is able to initiate an early step in the DNA-damage response, namely H2AX phosphorylation, before it is inactivated by proteolysis.

Animals↗

PubMed2005

Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo.

Despite a clear link between ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using G0-G1 confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologically distinct subpool of p53Ser15 is ATM dependent and resistant to 26S-proteasomal degradation. p53Ser15 colocalizes and coimmunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear microbeam irradiation studies confirm p53Ser15 is recruited to sites of DNA damage containing gamma-H2AX, ATM(Ser1981), and DNA-PKcs(Thr2609) in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased gamma-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis.

Acid Anhydride Hydrolases↗

PubMed2005

Homologous recombination and nonhomologous end-joining repair pathways regulate fragile site stability.

Common fragile sites are specific loci that form gaps and constrictions on metaphase chromosomes exposed to replication stress, which slows DNA replication. These sites have a role in chromosomal rearrangements in tumors; however, the molecular mechanism of their expression is unclear. Here we show that replication stress leads to focus formation of Rad51 and phosphorylated DNA-PKcs, key components of the homologous recombination (HR) and nonhomologous end-joining (NHEJ), double-strand break (DSB) repair pathways, respectively. Down-regulation of Rad51, DNA-PKcs, or Ligase IV, an additional component of the NHEJ repair pathway, leads to a significant increase in fragile site expression under replication stress. Replication stress also results in focus formation of the DSB markers, MDC1 and gammaH2AX. These foci colocalized with those of Rad51 and phospho-DNA-PKcs. Furthermore, gammaH2AX and phospho-DNA-PKcs foci were localized at expressed fragile sites on metaphase chromosomes. These findings suggest that DSBs are formed at common fragile sites as a result of replication perturbation. The repair of these breaks by both HR and NHEJ pathways is essential for chromosomal stability at these sites.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Cell cycle dependence of DNA-dependent protein kinase phosphorylation in response to DNA double strand breaks.

DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PKcs subunits, is the key component of the non-homologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair. Although the kinase activity of DNA-PKcs is essential for NHEJ, thus far, no in vivo substrate has been conclusively identified except for an autophosphorylation site on DNA-PKcs itself (threonine 2609). Here we report the ionizing radiation (IR)-induced autophosphorylation of DNA-PKcs at a novel site, serine 2056, the phosphorylation of which is required for the repair of DSBs by NHEJ. Interestingly, IR-induced DNA-PKcs autophosphorylation is regulated in a cell cycle-dependent manner with attenuated phosphorylation in the S phase. In contrast, DNA replication-associated DSBs resulted in DNA-PKcs autophosphorylation and localization to DNA damage sites. These results indicate that although IR-induced DNA-PKcs phosphorylation is attenuated in the S phase, DNA-PKcs is preferentially activated by the physiologically relevant DNA replication-associated DSBs at the sites of DNA synthesis.

Amino Acid Sequence↗

PubMed2004

Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a).

Cellular senescence can be triggered by telomere shortening as well as a variety of stresses and signaling imbalances. We used multiparameter single-cell detection methods to investigate upstream signaling pathways and ensuing cell cycle checkpoint responses in human fibroblasts. Telomeric foci containing multiple DNA damage response factors were assembled in a subset of senescent cells and signaled through ATM to p53, upregulating p21 and causing G1 phase arrest. Inhibition of ATM expression or activity resulted in cell cycle reentry, indicating that stable arrest requires continuous signaling. ATR kinase appears to play a minor role in normal cells but in the absence of ATM elicited a delayed G2 phase arrest. These pathways do not affect expression of p16, which was upregulated in a telomere- and DNA damage-independent manner in a subset of cells. Distinct senescence programs can thus progress in parallel, resulting in mosaic cultures as well as individual cells responding to multiple signals.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2004

DNA-PKcs function regulated specifically by protein phosphatase 5.

Unrepaired DNA double-strand breaks can lead to apoptosis or tumorigenesis. In mammals double-strand breaks are repaired mainly by nonhomologous end-joining mediated by the DNA-PK complex. The core protein of this complex, DNA-PKcs, is a DNA-dependent serine/threonine kinase that phosphorylates protein targets as well as itself. Although the (auto)phosphorylation activity has been shown to be essential for repair of both random double-strand breaks and induced breaks at the immunoglobulin locus, the corresponding phosphatase has been elusive. In fact, to date, none of the putative phosphatases in DNA double-strand break repair has been identified. Here we show that protein phosphatase 5 interacts with DNA-PKcs and dephosphorylates with surprising specificity at least two functional sites. Cells with either hypo- or hyperphosphorylation of DNA-PKcs at these sites show increased radiation sensitivity.

Animals↗

PubMed2003

Signal transduction in matrix contraction and the migration of vascular smooth muscle cells in three-dimensional matrix.

The interaction of vascular smooth muscle cells (SMCs) and extracellular matrix plays important roles in vascular remodeling. We investigated the signaling pathways involved in SMC-induced matrix contraction and SMC migration in three-dimensional (3D) collagen matrix. Matrix contraction is inhibited by the disruption of actin filaments but not microtubules. Therefore, we investigated the roles of signaling pathways related to actin filaments in matrix contraction. SMC-induced matrix contraction was markedly blocked (-80%) by inhibiting the Rho-p160ROCK pathway and myosin light chain kinase, and was decreased to a lesser extent (30-40%) by a negative mutant of Rac and inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) or p38 mitogen-activated protein kinase (MAPK), but it was not affected by the inhibition of Ras and Cdc42-Wiskott-Aldrich syndrome protein (WASP) pathways. Inhibition of extracellular-signal-regulated kinase (ERK) decreased SMC-induced matrix contraction by only 15%. The migration speed and persistence of SMCs in the 3D matrix were decreased by the inhibition of p160ROCK, PI 3-kinase, p38 MAPK or WASP to different extents, and p160ROCK inhibitor had the strongest inhibitory effect. Our results suggest that the SMC-induced matrix contraction and the migration of SMCs in 3D matrix share some signaling pathways leading to force generation at cell-matrix adhesions and that various signaling pathways have different relative importance in the regulations of these processes in SMCs.

Actin Cytoskeleton↗

PubMed2003

Identification of transcription factor KLF8 as a downstream target of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression.

Focal adhesion kinase (FAK) is an important mediator of integrin signaling in the regulation of cell adhesion, migration, survival, and proliferation. Here we report the identification of the transcription factor KLF8 as a target of FAK in cell cycle regulation. KLF8 is induced by FAK and decreased by FAK dominant-negative mutant DeltaC14. Overexpression of KLF8 increases cell cycle progression, whereas inhibition of endogenous KLF8 by siRNA reduces it. Cyclin D1 promoter is identified as a target of KLF8, which is activated both directly by KLF8 binding to the GT box A and by an indirect mechanism through its repression of a potential inhibitory regulator of cyclin D1. Transcription activation of cyclin D1 by FAK requires both Ets family and KLF8 factors in a temporally differential manner. Together, our data provide further insights into molecular mechanism for FAK to regulate cell cycle progression.

3T3 Cells↗

PubMed2002

Improved significance test for DNA microarray data: temporal effects of shear stress on endothelial genes.

Statistical methods for identifying differentially expressed genes from microarray data are evolving. We developed a test for the statistical significance of differential expression as a function of time. When applied to microarray data obtained from endothelial cells exposed to shearing for different durations, the new multi-group test (G-test) identified three times as many genes as the one-way ANOVA at the same significance level. Using simulated data, we showed that this increase in sensitivity was achieved without sacrificing specificity. Several genes known to respond to shear stress by Northern blotting were identified by the G-test at P < or = 0.01 (but not by ANOVA), with similar temporal patterns. The validity and utility of the G-test were further supported by the examination of a few more example genes in relation to the present knowledge of their regulatory mechanisms. This new significance test may have broad application for the analysis of gene-expression studies and, in fact, to other biological studies in general.

Cells, Cultured↗

PubMed2002

Genomic analysis of smooth muscle cells in 3-dimensional collagen matrix.

The proliferation, differentiation, and protein synthesis of vascular smooth muscle cells (SMCs) play important roles in vascular remodeling. Here, we compared the genetic programming and signaling of SMCs in collagen matrix as a three-dimensional (3-D) environment and on a two-dimensional (2-D) surface. By using DNA microarrays with 9600 genes, we showed that 77 genes were expressed more than twofold and 22 genes were less than one-half in 3-D matrix, when compared with the 2-D condition. The higher expression level of cyclin-dependent kinase inhibitor 1 (p21) in 3-D matrix suggests that p21 may be responsible for the lower proliferation rate in 3-D matrix. The expression level of collagen I was higher in 3-D matrix, suggesting that SMCs in 3-D matrix have increased matrix synthesis. In addition, SMCs in 3-D matrix had less stress fibers and focal adhesions, and a lower level of tyrosine phosphorylation of focal adhesion kinase (FAK). Overexpression of FAK attenuated the expression of p21 and collagen I in 3-D matrix, suggesting that FAK functions as a molecular switch for cell cycle regulation and matrix synthesis. The information generated in this study helps to elucidate the molecular basis of the modulation of SMC phenotypes by the extracellular matrix.

Cell Culture Techniques↗

PubMed2002

Differential regulation of Rho GTPases by beta1 and beta3 integrins: the role of an extracellular domain of integrin in intracellular signaling.

Integrins mediate cell adhesion and signal transduction at focal adhesions. Here we investigate the roles of integrin beta subunits in the regulation of actin cytoskeletal structure and the activities of Rho and Rac. The overexpression of beta3 integrin in Chinese hamster ovary cells enhances Rho activity and stress fiber formation, whereas the overexpression of beta1 integrin increases Rac activity and lamellipodia formation. The overexpression of a mutant beta1-3-1 integrin, in which the extracellular I-domain-like sequence of beta1 integrin has been replaced with the corresponding sequence of beta3 integrin, also enhances Rho activity and the formation of stress fibers. Our results demonstrate that beta1 and beta3 integrins differentially regulate the activities of Rho family GTPases and that the extracellular domains of integrin beta subunits play a critical role in transducing the extracellular ligand-binding information into specific intracellular signaling events.

Actins↗