Benjamin P C Chen

Telomere Shortening Triggers Senescence of Human Cells through a Pathway Involving ATM, p53, and p21CIP1, but Not p16INK4a

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.

Analysis of ATF3, a Transcription Factor Induced by Physiological Stresses and Modulated by gadd153/Chop10

We demonstrate that ATF3, a member of the ATF/CREB family of transcription factors, is induced in a variety of stressed tissues: mechanically injured liver, toxin-injured liver, blood-deprived heart, and postseizure brain. We also demonstrate that an ATF3-interacting protein, gadd153/Chop10, forms a nonfunctional heterodimer with ATF3: the heterodimer, in contrast to the ATF3 homodimer, does not bind to the ATF/cyclic AMP response element consensus site and does not repress transcription. Interestingly, ATF3 and gadd153/Chop10 are expressed in inverse but overlapping manners during the livers response to carbon tetrachloride (CCl4): the level of gadd153/Chop10 mRNA is high in the normal liver and greatly decreases upon CCl4 treatment; the level of ATF3 mRNA, on the other hand, is low in the normal liver and greatly increases upon CCl4 treatment. We hypothesize that in nonstressed liver, gadd153/Chop10 inhibits the limited amount of ATF3 by forming an inactive heterodimer with it, whereas in CCl4-injured liver, the synthesis of gadd153/Chop10 is repressed, allowing the induced ATF3 to function.

Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends.

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.

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.

ATF3 Gene GENOMIC ORGANIZATION, PROMOTER, AND REGULATION

ATF3 gene, which encodes a member of the activating transcription factor/cAMP responsive element binding protein (ATF/CREB) family of transcription factors, is induced by many physiological stresses. As a step toward understanding the induction mechanisms, we isolated the human ATF3 gene and analyzed its genome organization and 5′-flanking region. We found that the human ATF3 mRNA is derived from four exons distributed over 15 kilobases. Sequence analysis of the 5′-flanking region revealed a consensus TATA box and a number of transcription factor binding sites including the AP-1, ATF/CRE, NF-κB, E2F, and Myc/Max binding sites. As another approach to understanding the mechanisms by which the ATF3 gene is induced by stress signals, we studied the regulation of the ATF3 gene in tissue culture cells by anisomycin, an approach that has been used to study the stress responses in tissue culture cells. We showed that anisomycin at a low concentration activates the ATF3 promoter and stabilizes the ATF3 mRNA. Significantly, co-transfection of DNAs expressing ATF2 and c-Jun activates the ATF3 promoter. A possible mechanism implicating the C-Jun NH-terminal kinase/stress-activated protein kinase (JNK/SAPK) stress-inducible signaling pathway in the induction of the ATF3 gene is discussed.

Distinct roles for the small GTPases Cdc42 and Rho in endothelial responses to shear stress

Shear stress, the tangential component of hemodynamic forces, plays an important role in endothelial remodeling. In this study, we investigated the role of Rho family GTPases Cdc42 and Rho in shear stress-induced signal transduction and cytoskeleton reorganization. Our results showed that shear stress induced the translocation of Cdc42 and Rho from cytosol to membrane. Although both Cdc42 and Rho were involved in the shear stress-induced transcription factor AP-1 acting on the 12-O-tetradecanoyl-13-phorbol-acetate-responsive element (TRE), only Cdc42 was sufficient to activate AP-1/TRE. Dominant-negative mutants of Cdc42 and Rho, as well as recombinant C3 exoenzyme, attenuated the shear stress activation of c-Jun NH2-terminal kinases (JNKs), suggesting that Cdc42 and Rho regulate the shear stress induction of AP-1/TRE activity through JNKs. Shear stress-induced cell alignment and stress fiber formation were inhibited by the dominant-negative mutants of Rho and p160ROCK, but not by the dominant-negative mutant of Cdc42, indicating that the Rho-p160ROCK pathway regulates the cytoskeletal reorganization in response to shear stress.

gadd153/Chop10, a potential target gene of the transcriptional repressor ATF3.

Recently, we demonstrated that the function of ATF3, a stress-inducible transcriptional repressor, is negatively regulated by a bZip protein, gadd153/Chop10. In this report, we present evidence that ATF3 can repress the expression of its own inhibitor, gadd153/Chop10. First, ATF3 represses a chloramphenicol acetyltransferase reporter gene driven by the gadd153/Chop10 promoter when assayed by a transfection assay in vivo and a transcription assay in vitro. Second, the gadd153/Chop10 promoter contains two functionally important binding sites for ATF3: an AP-1 site and a C/EBP-ATF composite site, a previously unidentified binding site for ATF3. The absence of either site reduces the ability of ATF3 to repress the promoter. Third, overexpression of ATF3 by transient transfection results in a reduction of the endogenous gadd153/Chop10 mRNA level. Fourth, as described previously, ATF3 is induced in the liver upon CCl4 treatment. Intriguingly, we show in this report that gadd153/Chop10 mRNA is not present in areas where ATF3 is induced. Taken together, these results strongly suggest that ATF3 represses the expression of gadd153/Chop10. The mutual negative regulation between ATF3 and gadd153/Chop10 is discussed.

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.

Somatic mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) abrogate EGFR-mediated radioprotection in non-small cell lung carcinoma

The epidermal growth factor receptor (EGFR) is an important determinant of radioresponse, whose elevated expression and activity frequently correlates with radioresistance in several cancers, including non-small cell lung carcinoma (NSCLC). We reported recently that NSCLC cell lines harboring somatic, activating mutations in the tyrosine kinase domain (TKD) of the EGFR exhibit significant delays in the repair of DNA double-strand breaks (DSB) and poor clonogenic survival in response to radiation. Here, we explore the mechanisms underlying mutant EGFR-associated radiosensitivity. In three representative NSCLC cell lines, we show that, unlike wild-type (WT) EGFR, receptors with common oncogenic TKD mutations, L858R or DeltaE746-E750, are defective in radiation-induced translocation to the nucleus and fail to bind the catalytic and regulatory subunits of the DNA-dependent protein kinase (DNA-PK), a key enzyme in the nonhomologous end-joining repair pathway. Moreover, despite the presence of WT EGFR, stable exogenous expression of either the L858R or the DeltaE746-E750 mutant forms of EGFR in human bronchial epithelial cells significantly delays repair of ionizing radiation (IR)-induced DSBs, blocks the resolution of frank or microhomologous DNA ends, and abrogates IR-induced nuclear EGFR translocation or binding to DNA-PK catalytic subunit. Our study has identified a subset of naturally occurring EGFR mutations that lack a critical radioprotective function of EGFR, providing valuable insights on how the EGFR mediates cell survival in response to radiation in NSCLC cell lines.