Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Cellular supply of dNTPs is essential in the DNA replication and repair processes. Here we investigated the regulation of thymidine kinase 1 (TK1) in response to DNA damage and found that genotoxic insults in tumor cells cause up-regulation and nuclear localization of TK1. During recovery from DNA damage, TK1 accumulates in p53-null cells due to a lack of mitotic proteolysis as these cells are arrested in the G₂ phase by checkpoint activation. We show that in p53-proficient cells, p21 expression in response to DNA damage prohibits G₁/S progression, resulting in a smaller G₂ fraction and less TK1 accumulation. Thus, the p53 status of tumor cells affects the level of TK1 after DNA damage through differential cell cycle control. Furthermore, it was shown that in HCT-116 p53^−/− cells, TK1 is dispensable for cell proliferation but crucial for dTTP supply during recovery from DNA damage, leading to better survival. Depletion of TK1 decreases the efficiency of DNA repair during recovery from DNA damage and generates more cell death. Altogether, our data suggest that more dTTP synthesis via TK1 take place after genotoxic insults in tumor cells, improving DNA repair during G₂ arrest.     

Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G₁ phase to G₂ and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G₁ cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression.     

Activation of the Prereplication Complex Is Blocked by Mimosine through Reactive Oxygen Species-activated Ataxia Telangiectasia Mutated (ATM) Protein without DNA Damage

Mimosine is an effective cell synchronization reagent used for arresting cells in late G₁ phase. However, the mechanism underlying mimosine-induced G₁ cell cycle arrest remains unclear. Using highly synchronous cell populations, we show here that mimosine blocks S phase entry through ATM activation. HeLa S3 cells are exposed to thymidine for 15 h, released for 9 h by washing out the thymidine, and subsequently treated with 1 mm mimosine for a further 15 h (thymidine → mimosine). In contrast to thymidine-induced S phase arrest, mimosine treatment synchronizes >90% of cells at the G₁-S phase boundary by inhibiting the transition of the prereplication complex to the preinitiation complex. Mimosine treatment activates ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint signaling without inducing DNA damage. Inhibition of ATM activity is found to induce mimosine-arrested cells to enter S phase. In addition, ATM activation by mimosine treatment is mediated by reactive oxygen species (ROS). These results suggest that, upon mimosine treatment, ATM blocks S phase entry in response to ROS, which prevents replication fork stalling-induced DNA damage.     

Non‐apoptotic function of caspases in a cellular model of hydrogen peroxide‐associated colitis

Oxidative stress, caused by reactive oxygen species (ROS), is a major contributor to inflammatory bowel disease (IBD)‐associated neoplasia. We mimicked ROS exposure of the epithelium in IBD using non‐tumour human colonic epithelial cells (HCEC) and hydrogen peroxide (H₂O₂). A population of HCEC survived H₂O₂‐induced oxidative stress via JNK‐dependent cell cycle arrests. Caspases, p21^WAF1 and γ‐H2AX were identified as JNK‐regulated proteins. Up‐regulation of caspases was linked to cell survival and not, as expected, to apoptosis. Inhibition using the pan‐caspase inhibitor Z‐VAD‐FMK caused up‐regulation of γ‐H2AX, a DNA‐damage sensor, indicating its negative regulation via caspases. Cell cycle analysis revealed an accumulation of HCEC in the G₁‐phase as first response to oxidative stress and increased S‐phase population and then apoptosis as second response following caspase inhibition. Thus, caspases execute a non‐apoptotic function by promoting cells through G₁‐ and S‐phase by overriding the G₁/S‐ and intra‐S checkpoints despite DNA‐damage. This led to the accumulation of cells in the G₂/M‐phase and decreased apoptosis. Caspases mediate survival of oxidatively damaged HCEC via γ‐H2AX suppression, although its direct proteolytic inactivation was excluded. Conversely, we found that oxidative stress led to caspase‐dependent proteolytic degradation of the DNA‐damage checkpoint protein ATM that is upstream of γ‐H2AX. As a consequence, undetected DNA‐damage and increased proliferation were found in repeatedly H₂O₂‐exposed HCEC. Such features have been associated with neoplastic transformation and appear here to be mediated by a non‐apoptotic function of caspases. Overexpression of upstream p‐JNK in active ulcerative colitis also suggests a potential importance of this pathway in vivo.     

DNA damage-induced S phase arrest in human breast cancer depends on Chk1, but G2 arrest can occur independently of Chk1, Chk2 or MAPKAPK2

Checkpoint kinases Chk1 and Chk2 are two key components in the DNA damage-activated checkpoint signaling pathways. To distinguish the roles of Chk1 and Chk2 in S and G₂ checkpoints after DNA damage, derivatives of the human breast cancer cell line MDA-MB-231 were established that express short hairpin RNAs to selectively suppress Chk1 or Chk2 expression. DNA damage was induced with the topoisomerase I inhibitor SN38 which arrests cells in S or G₂ phase depending on concentration. Depletion of Chk1 resulted in loss of S phase arrest upon incubation with SN38, but the cells still arrested in G₂. Suppression of Chk2 had no impact on cell cycle arrest, while cells concurrently suppressed for both Chk1 and Chk2 still arrested primarily in G₂ suggesting the presence of an alternate checkpoint regulator. One critical target for Chk1 is Cdc25A which is phosphorylated and degraded to prevent cell cycle progression. Cells arrested in G₂ in the absence of Chk1/Chk2 still showed regulation of Cdc25A consistent with the action of an alternate kinase. One candidate for an alternate checkpoint kinase is MAPKAPK2 (MK2), yet this kinase was minimally activated by DNA damage and its inhibition did not facilitate either S or G₂ progression. Furthermore, we were unable to substantiate the recent observation that the Chk1 inhibitor UCN-01 inhibits MK2. These results show that Chk1, but neither Chk2 nor MK2, is an important regulator of S phase arrest, and suggest that an additional kinase can contribute to the G2 arrest.     

Centromere fragmentation is a common mitotic defect of S and G2 checkpoint override

DNA damaging agents, including those used in the clinic, activate cell cycle checkpoints, which blocks entry into mitosis. Given that checkpoint override results in cell death via mitotic catastrophe, inhibitors of the DNA damage checkpoint are actively being pursued as chemosensitization agents. Here we explored the effects of gemcitabine in combination with Chk1 inhibitors in a panel of pancreatic cancer cell lines and found variable abilities to override the S phase checkpoint. In cells that were able to enter mitosis, the chromatin was extensively fragmented, as assessed by metaphase spreads and Comet assay. Notably, electron microscopy and high-resolution light microscopy showed that the kinetochores and centromeres appeared to be detached from the chromatin mass, in a manner reminiscent of mitosis with unreplicated genomes (MUGs). Cell lines that were unable to override the S phase checkpoint were able to override a G₂ arrest induced by the alkylator MMS or the topoisomerase II inhibitors doxorubicin or etoposide. Interestingly, checkpoint override from the topoisomerase II inhibitors generated fragmented kinetochores (MUGs) due to unreplicated centromeres. Our studies show that kinetochore and centromere fragmentation is a defining feature of checkpoint override and suggests that loss of cell viability is due in part to acentric genomes. Furthermore, given the greater efficacy of forcing cells into premature mitosis from topoisomerase II-mediated arrest as compared with gemcitabine-mediated arrest, topoisomerase II inhibitors maybe more suitable when used in combination with checkpoint inhibitors.     

Differential effects of caffeine on DNA damage and replication cell cycle checkpoints in the fission yeast Schizosaccharomyces pombe

Caffeine potentiates the lethal effects of ultraviolet and ionising radiation on wild-type Schizosaccharomyces pombe cells. In previous studies this was attributed to the inhibition by caffeine of a novel DNA repair pathway in S. pombe that was absent in the budding yeast Saccharomyces cerevisiae. Studies with radiation-sensitive S. pombe mutants suggested that this caffeine-sensitive pathway could repair ultraviolet radiation damage in the absence of nucleotide excision repair. The alternative pathway was thought to be recombinational and to operate in the G₂ phase of the cell cycle. However, in this study we show that cells held in G₁ of the cell cycle can remove ultraviolet-induced lesions in the absence of nucleotide excision repair. We also show that recombination-defective mutants, and those now known to define the alternative repair pathway, still exhibit the caffeine effect. Our observations suggest that the basis of the caffeine effect is not due to direct inhibition of recombinational repair. The mutants originally thought to be involved in a caffeine-sensitive recombinational repair process are now known to be defective in arresting the cell cycle in S and/or G₂ following DNA damage or incomplete replication. The gene products may also have an additional role in a DNA repair or damage tolerance pathway. The effect of caffeine could, therefore, be due to interference with DNA damage checkpoints, or inhibition of the DNA damage repair/tolerance pathway. Using a combination of flow cytometric analysis, mitotic index analysis and fluorescence microscopy we show that caffeine interferes with intra-S phase and G₂ DNA damage checkpoints, overcoming cell cycle delays associated with damaged DNA. In contrast, caffeine has no effect on the DNA replication S phase checkpoint in reponse to inhibition of DNA synthesis by hydroxyurea. Received: 16 June 1998 / Accepted: 13 July 1998     

The Protein Kinase C�� Catalytic Fragment Is Critical for Maintenance of the G2/M DNA Damage Checkpoint

Protein kinase Cδ (PKCδ) is an essential component of the intrinsic apoptotic program. Following DNA damage, such as exposure to UV radiation, PKCδ is cleaved in a caspase-dependent manner, generating a constitutively active catalytic fragment (PKCδ-cat), which is necessary and sufficient for keratinocyte apoptosis. We found that in addition to inducing apoptosis, expression of PKCδ-cat caused a pronounced G₂/M cell cycle arrest in both primary human keratinocytes and immortalized HaCaT cells. Consistent with a G₂/M arrest, PKCδ-cat induced phosphorylation of Cdk1 (Tyr¹⁵), a critical event in the G₂/M checkpoint. Treatment with the ATM/ATR inhibitor caffeine was unable to prevent PKCδ-cat-induced G₂/M arrest, suggesting that PKCδ-cat is functioning downstream of ATM/ATR in the G₂/M checkpoint. To better understand the role of PKCδ and PKCδ-cat in the cell cycle response to DNA damage, we exposed wild-type and PKCδ null mouse embryonic fibroblasts (MEFs) to UV radiation. Wild-type MEFs underwent a pronounced G₂/M arrest, Cdk1 phosphorylation, and induction of apoptosis following UV exposure, whereas PKCδ null MEFs were resistant to these effects. Expression of PKCδ-green fluorescent protein, but not caspase-resistant or kinase-inactive PKCδ, was able to restore G₂/M checkpoint integrity in PKCδ null MEFs. The function of PKCδ in the DNA damage-induced G₂/M cell cycle checkpoint may be a critical component of its tumor suppressor function.     

Phospho-Bcl-xL(Ser62) plays a key role at DNA damage-induced G2 checkpoint

Accumulating evidence suggests that Bcl-xL, an anti-apoptotic member of the Bcl-2 family, also functions in cell cycle progression and cell cycle checkpoints. Analysis of a series of phosphorylation site mutants reveals that cells expressing Bcl-xL(Ser62Ala) mutant are less stable at the G₂ checkpoint and enter mitosis more rapidly than cells expressing wild-type Bcl-xL or Bcl-xL phosphorylation site mutants, including Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala and Thr115Ala. Analysis of the dynamic phosphorylation and location of phospho-Bcl-xL(Ser62) in unperturbed, synchronized cells and during DNA damage-induced G₂ arrest discloses that a pool of phospho-Bcl-xL(Ser62) accumulates into nucleolar structures in etoposide-exposed cells during G₂ arrest. In a series of in vitro kinase assays, pharmacological inhibitors and specific siRNAs experiments, we found that Polo kinase 1 and MAPK9/JNK2 are major protein kinases involved in Bcl-xL(Ser62) phosphorylation and accumulation into nucleolar structures during the G₂ checkpoint. In nucleoli, phospho-Bcl-xL(Ser62) binds to and co-localizes with Cdk1(cdc2), the key cyclin-dependent kinase required for entry into mitosis. These data indicate that during G₂ checkpoint, phospho-Bcl-xL(Ser62) stabilizes G₂ arrest by timely trapping of Cdk1(cdc2) in nucleolar structures to slow mitotic entry. It also highlights that DNA damage affects the dynamic composition of the nucleolus, which now emerges as a piece of the DNA damage response.     

Outcome of treatment of human HeLa cervical cancer cells with roscovitine strongly depends on the dosage and cell cycle status prior to the treatment

Exposure of asynchronously growing human HeLa cervical carcinoma cells to roscovitine (ROSC), a selective cyclin‐dependent kinases (CDKs) inhibitor, arrests their progression at the transition between G₂/M and/or induces apoptosis. The outcome depends on the ROSC concentration. At higher dose ROSC represses HPV‐encoded E7 oncoprotein and initiates caspase‐dependent apoptosis. Inhibition of the site‐specific phosphorylation of survivin and Bad, occurring at high‐dose ROSC treatment, precedes the onset of apoptosis and seems to be a prerequisite for cell death. Considering the fact that in HeLa cells the G₁/S restriction checkpoint is abolished by E7, we addressed the question whether the inhibition of CDKs by pharmacological inhibitors in synchronized cells would be able to block the cell‐cycle in G₁ phase. For this purpose, we attempted to synchronize cells by serum withdrawal or by blocking of the mitotic apparatus using nocodazole. Unlike human MCF‐7 cells, HeLa cells do not undergo G₁ block after serum starvation, but respond with a slight increase of the ratio of G₁ population. Exposure of G₁‐enriched HeLa cells to ROSC after re‐feeding does not block their cell‐cycle progression at G₁‐phase, but increases the ratio of S‐ and G₂‐phase, thereby mimicking the effect on asynchronously growing cells. A quite different impact is observed after treatment of HeLa cells released from mitotic block. ROSC prevents their cell cycle progression and cells transiently accumulate in G₁‐phase. These results show that inhibition of CDKs by ROSC in cells lacking the G₁/S restriction checkpoint has different outcomes depending on the cell‐cycle status prior to the onset of treatment. J. Cell. Biochem. 106: 937–955, 2009. © 2009 Wiley‐Liss, Inc.     

Dovitinib induces mitotic defects and activates the G2 DNA damage checkpoint

Dovitinib (TKI258; formerly CHIR‐258) is an orally bioavailable inhibitor of multiple receptor tyrosine kinases. Interestingly, Dovitinib triggered a G₂/M arrest in cancer cell lines from diverse origins including HeLa, nasopharyngeal carcinoma, and hepatocellular carcinoma. Single‐cell analysis revealed that Dovitinib promoted a delay in mitotic exit in a subset of cells, causing the cells to undergo mitotic slippage. Higher concentrations of Dovitinib induced a G₂ arrest similar to the G₂ DNA damage checkpoint. In support of this, DNA damage was triggered by Dovitinib as revealed by γ‐H2AX and comet assays. The mitotic kinase CDK1 was found to be inactivated by phosphorylation in the presence of Dovitinib. Furthermore, the G₂ arrest could be overcome by abrogation of the G₂ DNA damage checkpoint using small molecule inhibitors of CHK1 and WEE1. Finally, Dovitinib‐mediated G₂ cell cycle arrest and subsequent cell death could be promoted after DNA damage repair was disrupted by inhibitors of poly(ADP‐ribose) polymerases. These results are consistent with the recent finding that Dovitinib can also target topoisomerases. Collectively, these results suggest additional directions for use of Dovitinib, in particular with agents that target the DNA damage checkpoint.     

Modulation of G(2) arrest enhances cell death induced by the antitumor 1-nitroacridine derivative,Nitracrine

Nitracrine (Ledakrin) is an antitumor drug which is activated by cellular enzymes and binds covalently to DNA. Previous studies have shown that covalent binding and crosslinking of DNA is associated with the cytotoxic and antitumor activities of this compound. In this study, cell cycle perturbations, effects on DNA synthesis and the cell death process initiated by Nitracrine were studied in murine leukemia L1210 cells. We show that exposure of L1210 cells to Nitracrine at the IC₉₉ concentration delayed progression through the S phase and transiently arrested cells in G₂/M as found by flow cytometry. Higher drug concentration (2 × IC₉₉) inhibited cell cycle progression in the S phase and induced rapid cell death. Both studied concentrations of the drug produced different effects on DNA synthesis as determined by bromodeoxyuridine incorporation, with a delay in the S phase progression at EC₉₉ concentration and irreversible arrest in early S phase at the higher dose (2 × IC₉₉). At both concentrations of Nitracrine cell death occurred preferentially in the S phase as revealed by the TUNEL assay. When cells treated with the drug for 4 hours were post-incubated in the presence of 1 mM caffeine this led to rapid cell death and suppression of the G₂ arrest. This was associated with a about 10-fold increase in the cytotoxicity of Nitracrine. Similar effects were observed for another DNA crosslinking agent, cis-platinum, and to a lesser extent, for DNA topoisomerase I inhibitor, camptothecin. Together, our studies show that suppression of G₂ arrest induced by Nitracrine greatly enhances its cytotoxicity toward L1210 cells.     

BRCA1 Is Required for Maintenance of Phospho-Chk1 and G2/M Arrest during DNA Cross-Link Repair in DT40 Cells

The Fanconi anemia DNA repair pathway is pivotal for the efficient repair of DNA interstrand cross-links. Here, we show that FA-defective (Fancc⁻) DT40 cells arrest in G₂ phase following cross-link damage and trigger apoptosis. Strikingly, cell death was reduced in Fancc⁻ cells by additional deletion of the BRCA1 tumor suppressor, resulting in elevated clonogenic survival. Increased resistance to cross-link damage was not due to loss of toxic BRCA1-mediated homologous recombination but rather through the loss of a G₂ checkpoint. This proapoptotic role also required the BRCA1-A complex member ABRAXAS (FAM175A). Finally, we show that BRCA1 promotes G₂ arrest and cell death by prolonging phosphorylation of Chk1 on serine 345 after DNA damage to sustain arrest. Our data imply that DNA-induced cross-link death in cells defective in the FA pathway is dependent on the ability of BRCA1 to prolong cell cycle arrest in G₂ phase.     

TGF-beta1-induced cardiac myofibroblasts are nonproliferating functional cells carrying DNA damages

TGF-β₁ induces differentiation and total inhibition of cardiac MyoFb cell division and DNA synthesis. These effects of TGF-β₁ are irreversible. Inhibition of MyoFb proliferation is accompanied with the expression of Smad1, Mad1, p15Ink4B and total inhibition of telomerase activity. Surprisingly, TGF-β₁-activated MyoFbs are growth-arrested not only at G1-phase but also at S-phase of the cell cycle. Staining with TUNEL indicates that these cells carry DNA damages. However, the absolute majority of MyoFbs are non-apoptotic cells as established with two apoptosis-specific methods, flow cytometry and caspase-dependent cleavage of cytokeratin 18. Expression in MyoFbs of proliferative cell nuclear antigen even in the absence of serum confirms that these MyoFbs perform repair of DNA damages. These results suggest that TGF-β₁-activated MyoFbs can be growth-arrested by two checkpoints, the G1/S checkpoint, which prevents cells from entering S-phase and the intra-S checkpoint, which is activated by encountering DNA damage during the S phase or by unrepaired damage that escapes the G1/S checkpoint. Despite carrying of the DNA damages TGF-β₁-activated MyoFbs are highly functional cells producing lysyl oxidase and contracting the collagen matrix.     

Arrest of the cell cycle reduces susceptibility of target cells to perforin-mediated lysis

Cytotoxic T lymphocytes secrete a pore-forming cytolysin, perforin, that damages membranes of target cells. They also ligate Fas receptors on target cells and provoke apoptotic death. A20 (B lymphoma) and P815 (mastocytoma) cell lines were examined for their susceptibility to perforin-mediated lysis and to Fas-induced apoptosis after blockade of the cell cycle at the G₁/S interface. Cells were arrested at the G₁/S interface by inhibition of DNA synthesis with thymidine or aphidicolin. Subsequently, the treated cells were incubated either with CTL cytotoxic granules or the Fas-specific monoclonal antibody Jo-2. We show that arrest of the cell cycle at the G₁/S interface markedly reduced the susceptibility of target cells to perforin-mediated lysis. In contrast, growth arrest with thymidine or aphidicolin increased susceptibility of A20 and P815 cells to Fas-mediated apoptosis. Susceptibility to lysis by intact CTLs was not affected significantly by blockade of target cells with aphidicolin or thymidine. When cells surviving exposure to perforin-containing granules were isolated on Ficoll density gradients and cell-cycle profiles were examined by flow cytometry, the ratio of G₁ to G₂cells increased among the survivors exposed to granules in contrast to controls incubated with buffer alone. The data suggest that cells in G₁ phase of the cell cycle are less susceptible to the perforin pathway than cells in G₂and S phases but are more susceptible to the Fas pathway. J. Cell. Biochem. 69:425–435, 1998. © 1998 Wiley-Liss, Inc.     

Phosphorylation of cyclin D1 regulated by ATM or ATR controls cell cycle progression

Cyclin D1 is required at high levels for passage through G₁ phase but must be reduced to low levels during S phase to avoid the inhibition of DNA synthesis. This suppression requires the phosphorylation of Thr286, which is induced directly by DNA synthesis. Because the checkpoint kinase ATR is activated by normal replication as well as by DNA damage, its potential role in regulating cyclin D1 phosphorylation was tested. We found that ATR, activated by either UV irradiation or the topoisomerase IIβ binding protein 1 activator, promoted cyclin D1 phosphorylation. Small interfering RNA against ATR inhibited UV-induced Thr286 phosphorylation, together with that seen in normally cycling cells, indicating that ATR regulates cyclin D1 phosphorylation in normal as well as stressed cells. Following double-stranded DNA (dsDNA) breakage, the related checkpoint kinase ATM was also able to promote the phosphorylation of cyclin D1 Thr286. The relationship between these checkpoint kinases and cyclin D1 was extended when we found that normal cell cycle blockage in G₁ phase observed following dsDNA damage was efficiently overcome when exogenous cyclin D1 was expressed within the cells. These results indicate that checkpoint kinases play a critical role in regulating cell cycle progression in normal and stressed cells by directing the phosphorylation of cyclin D1.