Induction Of DNA Damage Response Research Articles

The HINT1 tumor suppressor regulates both gamma-H2AX and ATM in response to DNA damage.

Hint1 is a haploinsufficient tumor suppressor gene and the underlying molecular mechanisms for its tumor suppressor function are unknown. In this study we demonstrate that HINT1 participates in ionizing radiation (IR)–induced DNA damage responses. In response to IR, HINT1 is recruited to IR-induced foci (IRIF) and associates with γ-H2AX and ATM. HINT1 deficiency does not affect the formation of γ-H2AX foci; however, it impairs the removal of γ-H2AX foci after DNA damage and this is associated with impaired acetylation of γ-H2AX. HINT1 deficiency also impairs acetylation of ATM and activation of ATM and its downstream effectors, and retards DNA repair, in response to IR. HINT1-deficient cells exhibit resistance to IR-induced apoptosis and several types of chromosomal abnormalities. Our findings suggest that the tumor suppressor function of HINT1 is caused by, at least in part, its normal role in enhancing cellular responses to DNA damage by regulating the functions of both γ-H2AX and ATM.

Research on Hutchinson-Gilford Progeria Syndrome

IN this issue of the Journal, I have included a summary of a workshop held in November 2007 on the topic of Hutchinson-Gilford Progeria Syndrome (HGPS) (1). This syndrome was first described over 120 years ago by Hutchinson (2), and although the phenotype does include some aging-like changes, biogerontologists have questioned whether it is a viable model for studying accelerated aging (3). The question has justifiably risen again with the recent identification of LMNA as the gene responsible for this sporadic autosomal dominant syndrome (4). It is now clear that the syndrome results from the accumulation of a metabolite formed during processing of the mutated pre-lamin A protein. This metabolite has been named progerin, and it induces nuclear blebbing in HGPS cells grown in culture (5), suggesting that serious disruption of DNA metabolism may be occurring in these cells. At least several outcomes are possible, including induction of cell senescence, induction of apoptosis, dysregulated gene expression, dysregulation of differentiation, and so forth. Whatever the critical defect may be, Kudlow and colleagues (6) have argued that progeroid syndromes, including HGPS, ‘‘might have partial mechanistic overlap with normal aging and therefore might provide uniquely informative opportunities to formulate and test hypotheses regarding the biology of aging and age-dependent disease.’’ Warner and Sierra (7) also have suggested such possible overlap. Scaffidi and Misteli (8) have recently shown that small amounts of progerin are produced in normal cells by occasional use of the aberrant splice site used in HGPS cells, but it is not known whether these small amounts actually have any impact on longevity. However, progerin does accumulate in the skin with age in a subset of dermal fibroblasts, suggesting that this could be a possible biomarker of cellular aging (1,9). Kudlow and colleagues (6) suggest three general approaches to identify such putative mechanistic links between normal aging and accelerated aging models, whether human or murine. Because many mouse mutations that induce progeroid phenotypes share disruption of DNA metabolism as a common feature, they suggest that it would be useful to know what roles lamin A plays in the various DNA transactions important to the proper function of a mammalian cell, such as replication, transcription, repair, and recombination. Progerin has in fact been reported to interfere with mitosis (10) as well as induce DNA damage responses (11). Secondly, they suggest that persistent DNA damage signaling, cell death, and cellular changes may contribute to the pathology of HGPS, and that it is important to determine whether and why these changes are selectively targeted to specific cell lineages and tissues. Finally, a third approach is to look for single nucleotide polymorphisms (SNPs) in the gene for lamin A that associate with longevity; Francis Collins reported at the workshop that four such SNPs have been found in older people (1). Besides the interesting question of whether HGPS is or is not a valid model of accelerated human aging, the most important translational question is the development of a cure—or at the very least an effective treatment. Numerous studies have reported that farnesyltransferase inhibitors reverse the morphological defects observed in HGPS fibroblasts (12). This provides the basis for the clinical trial currently underway under the direction of Mark Kieran at Children’s Hospital in Boston. The primary endpoint is weight gain because failure to grow, starting at about age 2 years, represents a consistent and predictable characteristic of the syndrome. Secondary biomarkers such as alopecia, short stature, subcutaneous fat, bone integrity, and a variety of other abnormalities will also be followed. However, the ideal solution would be a total cure rather than an ongoing treatment. In trying to understand the underlying cause of HGPS, it may be instructive to ask what causes and ‘‘times’’ the onset of growth failure and the subsequent low weight and height gain, and why do mesenchymally derived tissues seem to be most vulnerable (13). Sharpless and DePinho (14) have reviewed mounting evidence that accumulation of damage to cellular macromolecules such as DNA could be one cause of cellular attrition with aging. Continued cellular attrition by apoptosis would result in forced regeneration of tissue cells in response to homeostatic demands, possibly resulting ultimately in an inability of stem cell pools to respond to these demands (15). Such a process could explain an onset of growth failure, at about 2 years of age, such as observed in HGPS. In addition to DNA damage accumulation in HGPS cells, the aberrant morphology of HGPS nuclei might also trigger the cellular attrition reported by Bridger and Kill (16). HGPS stem cells may also be subject to apoptosis, which would compound the attrition problem. Scaffidi and Misteli (17) have recently reported that progerin ‘‘interferes with the

Constitutive overexpression of CDC25A in primary human mammary epithelial cells results in both defective DNA damage response and chromosomal breaks at fragile sites

CDC25A phosphatase, an essential component of the cell cycle machinery, is also a key player in integrating the specific signals of checkpoint control in response to DNA damage. There are several lines of evidence that indicate a role for CDC25A in cancer development, consistent with the fact that its overexpression is detected in human cancers. In particular we previously reported that CDC25A is overexpressed also in early breast carcinoma. Recent data suggest that oncogene activation during early stages of tumor development causes DNA replication stress resulting in the induction of DNA damage response (DDR) and that the selection of cells defecting in their DDR could lead to malignant progression. To address how CDC25A overexpression contributes to breast cancer development we established a cell model in which CDC25A was constitutively overexpressed in hTERT-immortalized primary human mammary epithelial cells. At the earliest passages following CDC25A transduction we observed DDR signs associated with unscheduled DNA replication origins. In the latest passages DDR was significantly impaired and, even after ionizing radiation exposition, cells failed to induce G1 and G2 checkpoints; moreover DNA replication stress conditions, such as aphidicolin treatment, highlighted increased fragile site breakages and destabilized chromosomes just in these latest passages cells. Our data suggest that CDC25A overexpression, pushing the cell through the cell cycle transitions, induces DDR alterations that might enhance genomic instability.

Induction of Cellular Senescence and DNA Damage Response by Sodium Selenite

Cells respond to DNA damage by induction of apoptosis, DNA damage checkpoint, and senescence, all of which have been proposed as barriers of tumorigenesis. Although selenium chemoprevention by apoptosis has been well studied, it is not clear whether and how other tumorigenesis barriers participate in selenium chemoprevention. To address these issues, we treated MRC-5 normal lung fibroblasts with 0–5 μM sodium selenite in a series of time course studies. We employed a number of senescence markers, including the measurement of senescence-associated expression of β-galactosidase (SA-β-gal). At day 7, there are clear features of stress-induced senescence as evidenced by SA-β-gal staining in cells treated with selenite at 1 and 2 μM. At a concentration of 5 μM, a significant portion of cells die. In response to clastogens, ATM is rapidly activated, which in turn initiates a cascade of DNA damage response including histone H2AX phosphorylation at Ser-139 (also known as γH2AX). By using antibodies against the phosphorylated form of the proteins, the results showed that there is a selenium dose-dependent increase in the level of γH2AX at 3 h and 6 h. Taken together, the results suggest a novel mechanism of selenium chemoprevention by which selenium induces DNA damage response and senescence to inhibit cancer cell proliferation.

Loss of cyclin D1 in concert with deregulated estrogen receptor α expression induces DNA damage response activation and interrupts mammary gland morphogenesis

We have previously shown that increased and deregulated estrogen receptor alpha expression in the mammary gland leads to the development of proliferative disease and cancer. To address the importance of cyclin D1 in ERalpha-mediated mammary tumorigenesis, we crossed ERalpha-overexpressing mice with cyclin D1 knockout mice. Mammary gland morphogenesis was completely interrupted in the ERalpha-overexpressing cyclin D1-deficient triple transgenic mice. In addition to a highly significant reduction in mammary epithelial cell proliferation, cyclin E was upregulated resulting in DNA damage checkpoint activation and apoptosis. This imbalance between proliferative and apoptotic rates in conjunction with remarkable structural defects and cellular disorganization in the terminal end buds interrupted ductal morphogenesis. Interestingly, the structure of the mammary fat pad was fundamentally altered by the consequences of overexpressing ERalpha in the epithelial cells in the absence of cyclin D1 illustrating how alterations in the epithelial compartment can impact surrounding stromal composition. Transplantation of embryonic ERalpha-overexpressing and cyclin D1-deficient mammary epithelium into the cleared fat pad of wild-type mice did not rescue the aberrant mammary gland phenotype indicating that it was intrinsic to the mammary epithelial cells. In conclusion, although cyclin D1 is not essential for proliferation of normal mammary epithelial cells, ERalpha-overexpressing cells are absolutely dependent on cyclin D1 for proliferation. This differential requirement for cyclin D1 in normal vs abnormal mammary epithelial cells supports the application of cyclin D1 inhibitors as therapeutic interventions in ERalpha-overexpressing breast cancers.

Photochemically enhanced delivery of a cell‐penetrating peptide nucleic acid conjugate targeting human telomerase reverse transcriptase: effects on telomere status and proliferative potential of human prostate cancer cells

Peptide nucleic acids (PNAs) are DNA mimics that have been demonstrated to be efficient antisense/antigene tools in cell-free systems. However, their potential as in vivo regulators of gene expression has been hampered by their poor uptake by living cells, and strategies need to be developed for their intracellular delivery. This study has aimed to demonstrate the possibility (i) of efficiently delivering a PNA, which targets mRNA of the catalytic component of human telomerase reverse transcriptase (hTERT), into DU145 prostate cancer cells through a combined approach based on conjugation of the PNA to Tat internalizing peptide (hTERT-PNA-Tat) and subsequent photochemical internalization, and (ii) to interfere with telomerase function. Treated cells were analysed for telomerase activity, hTERT expression, growth rate, ability to undergo apoptosis and telomere status. After exposure to light, DU145 cells treated with hTERT-PNA-Tat and the photosensitiser TPPS2a showed dose-dependent inhibition of telomerase activity, which was accompanied by marked reduction of hTERT protein expression. A dose-dependent decline in DU145 cell population growth and induction of caspase-dependent apoptosis were also observed from 48 h after treatment. Such an antiproliferative effect was associated with the presence of telomeric dysfunction, as revealed by cytogenetic analysis, in the absence of telomere shrinkage, and with induction of DNA damage response as suggested by the increased expression of gamma-H2AX. Our results (i) indicate photochemical internalization as an efficient approach for intracellular delivery of chimaeric PNAs, and (ii) corroborate earlier evidence suggesting pro-survival and anti-apoptotic roles of hTERT, which are independent of its ability to maintain telomere length.

Exonuclease-1 Deletion Impairs DNA Damage Signaling and Prolongs Lifespan of Telomere-Dysfunctional Mice

Exonuclease-1 (EXO1) mediates checkpoint induction in response to telomere dysfunction in yeast, but it is unknown whether EXO1 has similar functions in mammalian cells. Here we show that deletion of the nuclease domain of Exo1 reduces accumulation of DNA damage and DNA damage signal induction in telomere-dysfunctional mice. Exo1 deletion improved organ maintenance and lifespan of telomere-dysfunctional mice but did not increase chromosomal instability or cancer formation. Deletion of Exo1 also ameliorated the induction of DNA damage checkpoints in response to gamma-irradiation and conferred cellular resistance to 6-thioguanine-induced DNA damage. Exo1 deletion impaired upstream induction of DNA damage responses by reducing ssDNA formation and the recruitment of Replication Protein A (RPA) and ATR at DNA breaks. Together, these studies provide evidence that EXO1 contributes to DNA damage signal induction in mammalian cells, and deletion of Exo1 can prolong survival in the context of telomere dysfunction.

Viral Oncogene–Induced DNA Damage Response Is Activated in Kaposi Sarcoma Tumorigenesis

Kaposi sarcoma is a tumor consisting of Kaposi sarcoma herpesvirus (KSHV)–infected tumor cells that express endothelial cell (EC) markers and viral genes like v-cyclin, vFLIP, and LANA. Despite a strong link between KSHV infection and certain neoplasms, de novo virus infection of human primary cells does not readily lead to cellular transformation. We have studied the consequences of expression of v-cyclin in primary and immortalized human dermal microvascular ECs. We show that v-cyclin, which is a homolog of cellular D-type cyclins, induces replicative stress in ECs, which leads to senescence and activation of the DNA damage response. We find that antiproliferative checkpoints are activated upon KSHV infection of ECs, and in early-stage but not late-stage lesions of clinical Kaposi sarcoma specimens. These are some of the first results suggesting that DNA damage checkpoint response also functions as an anticancer barrier in virally induced cancers.

Protection of DNA Ends by Telomeric 3′ G-Tail Sequences

The extreme ends of eukaryotic chromosomes contain 3' extensions in the form of single-stranded G-rich repeats, referred to as telomeric 3' G-tails or overhangs. Increasing evidence has suggested that telomeric 3' G-tails can adopt a G-quadruplex conformation both in vitro and in vivo. However, the role of G-quadruplexes on the structure and function of telomeric 3' G-tails remains unclear. In the current study, we showed that the human telomeric 3' G-tail sequence protected the duplex DNA ends in cis from being recognized as double strand breaks. This protection is dependent on the G-quadruplex conformation of the 3' G-tail sequence. These results suggest that the ability of telomeric 3' G-tails to adopt the endprotecting G-quadruplex conformation may be one of the reasons for the existence of the evolutionarily conserved G-stretch motifs in telomeric DNA sequences.

Inactivation of the Myc−Induced DNA Damage Response Promotes Lymphomagenesis and Treatment Resistance In Vivo.

Activation of the DNA damage response (DDR) was recently claimed to serve as a critical anti−cancer barrier in early human tumorigenesis (Bartkova−J et al., and Gorgoulis−VG et al., Nature, 2005). As a consequence, deregulation of the DDR machinery may be required for tumor manifestation.Myc activation is an oncogenic event frequently detected in malignant lymphomas. It is known that constitutive Myc expression provokes apoptosis via the ARF/p53 pathway as a cellular failsafe mechanism to counteract malignant transformation, but the potential role of Myc signaling to the DDR machinery, mediated via the Atm (ataxia telangiectasia mutated) kinase, as another putative tumor suppressive restraint remains still elusive.Utilizing a tamoxifen−regulatable Myc system as well as RNA interference in vitro, we found in cell−based models that acute induction of Myc, associated with an increased production of reactive oxygen species (ROS) and marks of DNA strand breaks, leads to p53 activation which is mainly mediated by Atm and its upstream regulator PP5.To further examine the role of Atm during Myc−driven tumorigenesis in vivo, we intercrossed Atm knockout mice to Eμ−myc transgenic mice that develop B−cell lymphomas. Lymphomas formed significantly faster in the absence of Atm, mostly due to a significant apoptotic defect in Atm−/− lymphomas compared to Atm+/+ derived (referred to as ‘control') lymphomas.In accordance with our in vitro findings, we detected significantly elevated levels of ROS−induced oxidative DNA damage and γ−H2AX foci in Myc−driven lymphomas. Importantly, constitutive expression of Myc selected against components of the Atm−governed DDR in a subset of control lymphomas. Like Atm deficiency, these lesions predetermined a poor response in subsequent cytotoxicity assays in vitro and anticancer therapy in vivo, since DNA damaging treatments also produce apoptosis via PP5/Atm.The role for ROS as inducers of DNA damage during the execution of Myc−induced apoptosis was further substantiated by the finding that Eμ−myc transgenic mice continuously exposed to the ROS scavenger N−acetylcysteine (starting prenatally) developed lymphomas with significantly reduced levels of spontaneous apoptosis and with less marks of DNA damage (i.e. γ−H2AX foci), but a retained ability to respond to anticancer therapy in vitro and in vivo.Hence, Atm eliminates pre−neoplastic lesions by converting oncogenic signaling into apoptosis, while selection for an attenuated Atm response permits lymphoma formation, and, in turn, has profound implications for reduced efficacy of DNA damaging therapies.

ATR, PML, and CHK2 Play a Role in Arsenic Trioxide-induced Apoptosis

Arsenic trioxide (ATO) is a potent anti-leukemic chemotherapeutic agent for acute promyelocytic leukemia (APL) that results from a t (15, 17) chromosomal translocation that produces PML-RARalpha, a fusion protein between a tumor suppressor PML and the retinoic acid receptor RARalpha. APL patients are initially treated with retinoic acid, but most develop resistance and relapse. In contrast, ATO induces prolonged remissions even in the relapsed cases. However, the molecular mechanisms by which ATO kills the leukemic cells are not fully understood. We find that ATO induces apoptosis, at least in part, by activating proapoptotic kinase Chk2. ATO does this by stimulating ATR (ataxia telangiectasia mutated and Rad3-related) kinase, a Chk2-activating kinase. In conjunction, ATO degrades PML-RARalpha, resulting in the restoration of PML, which is required for autophosphorylation and full activation of Chk2. As a result, the p53-dependent apoptosis pathway is activated. Based on this, we propose that a pathway composed of ATR, PML, Chk2, and p53 plays a role in ATO-mediated apoptosis, a notion that is consistent with the observation that Chk2 is genetically intact and mutations in the p53 gene are extremely rare in APL.

Caenorhabditis elegans에서 분리한 자외선 유도유전자 (UV100과 UV150)의 발현 및 특성에 관한 연구

본 연구는 DNA 상해유도기작을 규명하기 위하여 꼬마선충 (Caenorhabditis elegans) 으로 부터 subtraction hybridization 방법을 이용하여 자외선 유도 유전자인 UV100과 UV150을 분리하고 그 유전자 구조와 발현양상을 조사하였다. 분리한 유전자의 발현양상을 Northern hybridization 방법으로 살펴본 결과 자외선 조사 후에 최대 2배 이상의 발현 증가를 나타내었다. 이 결과 이미 밝혀진 다른 UV-inducible 유전자와 유사하게 UV100과 UV150 유전자는 자외선에 의해서만 발현이 증가됨을 알 수 있었다. 또한 분리한 유전자의 기능을 알기 위하여 RNAi 실험을 한 결과 분리한 자외선 유도유전자는 발생단계에 따라 다양한 DNA 회복기작을 나타냄을 알 수 있었다. The present study intends to characterize the DNA damage-inducible responses in Caenorhabditis elegans. To study UV-inducible responses in C. elegans, two UV-inducible cDNA clones were isolated from C. elegans by using subtration hybridization method. To investigate the expression of isolated genes, UV100 and UV150, the cellular levels of the transcript were determined by Northern blot analysis after UV-irradiation. The transcripts of isolated gene increased rapidly and reached maximum accumulation after UV-irradiation. Compared to the message levels of control, the levels of maximal increase were approximately 2 folds to UV-irradiation. These results implied that the effects of damaging agents are complex and different regulatory pathways exist for the induction of these genes. To study the function of UV100 and UV150 gene in response to UV irradiation, we carried out a RNAi experiment and investigated the UV sensivity. This result indicated that UV100 gene involved in stage-specific repair pathway or regulated by development.

효모 Schizosaccharomyces pombe에서 자외선 유도유전자 UVI-155의 분리 및 특성 연구

본 연구는 DNA상해유도기작을 규명하기 위하여 하등 진핵생물인 분열형 효모 Schizosaccharomyces pombe로부터 subtraction hybridization방법을 이용하여 자외선 유도 유전자인 UVI-155을 분리하고 그 유전자 구조와 발현양상을 조사하였다. UVI-155 유전자의 발현양상을 Northern hybridization 방법으로 살펴본 결과 자외선(ultraviolet-light) 조사 1시간 후에 최대의 발현 증가를 나타내었다. 반면 알킬화제 인 MMS (methyl methanesulfonate) 처리에 의해서도 발현이 증가되었다. 이 결과 다른 UV-inducible 유전자와는 다르게 UVI-l55 유전자는 UV와 MMS 등의 DNA 상해에 모두 발현이 증가됨을 알 수 있었다. 또 한 유전자의 기능을 알기 위하여 null-mutant세포 주를 제조하여 그 특성을 살펴본 결과 이 유전자는 세포의 성장에 필수적인 유전자임을 알 수 있었다. The present study intends to characterize the DNA damage-inducible responses in yeast. The fission yeast, Schizosaccharomyces pombe was used in this study as a model system for higher eukaryotes. To study UV-inducible responses in S. pombe, five UV-inducible cDNA clones were isolated from S. pombe by using subtration hybridization method. To investigate the expression of isolated genes, UVI-155, the cellular levels of the transcripts were determined by Northern blot analysis after UV-irradiation. The transcripts of isolated gene (UVI-155) increased rapidly and reached maximum accumulation after UV-irradiation. Compared to the message levels of control, the levels of maximal increase were approximately 5 fold to UV-irradiation. In order to investigation whether the increase of UVI-l55 trascripts was a specific results of UV-irradiation, UVI-155 transcript levels were examined after treating the cells to mthylmethane sulfonate (MMS). The transcripts of UVI-155 were not induced by treatment of <TEX>$0.25\%$</TEX> MMS. These results implied that the effects of damaging agents are complex and different regulatory pathways exist for the induction of these genes. To characterize the UVI-155 gene, gene deletion experiments were analyzed. The deleted strain was not well grown. This result indicated that the UVI-155 gene is essential for cell viability.

Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses

Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20 J/m 2 UVC (LD50 = 5.6 J/m 2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21 CIP1/WAF1, and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6 h and from the non-transcribed strand was 73% complete in 24 h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.

RAD6B overexpression confers chemoresistance: RAD6 expression during cell cycle and its redistribution to chromatin during DNA damage-induced response.

The HR6A and HR6B genes, homologs of the yeast RAD6 gene, encode ubiquitin conjugating enzymes that are required for postreplication repair (PRR) of DNA and damage-induced mutagenesis. We show here that consistent with its role as a PRR protein, HR6 protein (referred as RAD6) expression is cell cycle regulated, with maximal levels expressed in late S/G2 phases of the cell cycle. Exposure of MCF10A cells to adriamycin (ADR) causes enhancement in the levels of RAD6B mRNA and protein. Inclusion of actinomycin D abolishes both basal and ADR-induced RAD6B transcription indicating that ADR-induced effects on RAD6B transcription result from an increase in transcriptional activity rather than from regulation of RAD6B mRNA stability. The increase in RAD6 protein expression observed in ADR-treated cells is dependent upon transcription and de novo protein synthesis, as addition of actinomycin D and cycloheximide eliminated the induction effects. Using in vivo crosslinking experiments, we demonstrate that only a small proportion of RAD6 is associated with chromatin in untreated MCF10A cells. However, treatment with ADR or cisplatin is accompanied by a significant increase and redistribution of RAD6 to DNA, and RAD6, RAD18, PCNA, phosphohistone H3, as well as p53 proteins are all found in the DNA fractions. These findings suggest that although RAD6 protein is present in the nucleus, its recruitment to the chromatin appears to be modulated by DNA damage. Whereas MCF10A cells engineered to overexpress ectopic RAD6B are significantly more resistant to ADR and cisplatin as compared to empty vector-transfected cells, MCF10A cells stably transfected with antisense RAD6B display hypersensitivity to these damage-inducing drugs. Analysis of PRR capacities in cisplatin-treated MCF10A cells stably transfected with empty vector, RAD6B or antisense RAD6B showed that whereas RAD6B-overexpressing and vector control MCF10A cells possessed the ability to convert newly synthesized DNA to higher molecular weight species, MCF10A cells depleted of RAD6B are PRR-compromised. Although no human diseases have been linked to mutations in the PRR pathway genes, these data suggest that RAD6 may play an essential role in DNA damage tolerance and recovery via modulation of PRR, and that imbalances in the levels of RAD6 could lead to changes in drug sensitivity and damage-induced mutagenesis.

Death and more: DNA damage response pathways in the nematode C. elegans.

Genotoxic stress is a threat to our cells' genome integrity. Failure to repair DNA lesions properly after the induction of cell proliferation arrest can lead to mutations or large-scale genomic instability. Because such changes may have tumorigenic potential, damaged cells are often eliminated via apoptosis. Loss of this apoptotic response is actually one of the hallmarks of cancer. Towards the effort to elucidate the DNA damage-induced signaling steps leading to these biological events, an easily accessible model system is required, where the acquired knowledge can reveal the mechanisms underlying more complex organisms. Accumulating evidence coming from studies in Caenorhabditis elegans point to its usefulness as such. In the worm's germline, DNA damage can induce both cell cycle arrest and apoptosis, two responses that are spatially separated. The latter is a tightly controlled process that is genetically indistinguishable from developmental programmed cell death. Upstream of the central death machinery, components of the DNA damage signaling cascade lie and act either as sensors of the lesion or as transducers of the initial signal detected. This review summarizes the findings of several studies that specify the elements of the DNA damage-induced responses, as components of the cell cycle control machinery, the repairing process or the apoptotic outcome. The validity of C. elegans as a tool to further dissect the complex signaling network of these responses and the high potential for it to reveal important links to cancer and other genetic abnormalities are addressed.

Supramolecular complex formation between Rad6 and proteins of the p53 pathway during DNA damage-induced response.

The HR6A and -B genes, homologues of the yeast Rad6 gene, encode ubiquitin-conjugating enzymes that are required for postreplication repair of DNA and damage-induced mutagenesis. Using surface plasmon resonance, we show here that HR6 protein (referred as Rad6) physically interacts with p53. Analysis of proteins coimmunoprecipitated with Rad6 antibody from metabolically labeled normal MCF10A human breast epithelial cells not only confirmed Rad6-p53 interactions in vivo but also demonstrated for the first time that exposure of MCF10A cells to cisplatin or adriamycin (ADR) induces recruitment of p14ARF into Rad6-p53 complexes. Further analysis of ADR-induced p53 response showed that stable Rad6-p53-p14ARF complex formation is associated with a parallel increase and decrease in monoubiquitinated and polyubiquitinated p53, respectively, and arrest in G(2)/M phase of the cell cycle. Interestingly, the ADR-induced suppression of p53 polyubiquitination correlated with a corresponding decline in intact Hdm2 protein levels. Treatment of MCF10A cells with MG132, a 26S proteasome inhibitor, effectively stabilized monoubiquitinated p53 and rescued ADR-induced downregulation of Hdm2. These data suggest that ADR-induced degradation of Hdm2 occurs via the ubiquitin-proteasome pathway. Rad6 is present in both the cytoplasmic and nuclear compartments of normal MCF10A cells, although in response to DNA damage it is predominantly found in the nucleus colocalizing with ubiquitinated p53, whereas Hdm2 is undetectable. Consistent with in vivo data, results from in vitro ubiquitination assays show that Rad6 mediates addition of one (mono-) to two (multimono-) ubiquitin molecules on p53 and that inclusion of Mdm2 is essential for its polyubiquitination. The data presented in the present study suggest that Rad6-p53-p14ARF complex formation and p53 ubiquitin modification are important damage-induced responses that perhaps determine the fidelity of DNA postreplication repair.

Enhanced S phase delay and inhibition of replication of an undamaged shuttle vector in UVC-irradiated xeroderma pigmentosum variant.

Xeroderma pigmentosum variant (XP-V) cells are defective in bypass replication of UVC-induced thymine dimers in DNA because they lack a novel DNA polymerase (polymerase eta). In this study the effects of UVC on S phase cells were compared in fibroblasts derived from normal donors (IDH4) and XP-V patients (CTag) and immortalized by expression of the SV40 large T antigen. These transformed fibroblasts did not activate the G(1) checkpoint or inhibit replicon initiation when damaged by UVC or gamma-rays. The transformed XP-V cells (CTag) retained the increased sensitivity to UVC-induced inhibition of DNA strand growth previously observed with their diploid counterpart. Cell cycle progression analyses showed that CTag cells displayed a stronger S phase delay than transformed fibroblasts from normal individuals (IDH4) after treatment with only 2 J/m(2) UVC. Low doses of UVC also caused a lag in CTag cell proliferation. The extent of replication of an episomal DNA (pSV011), not previously exposed to radiation, was measured after the host cells were irradiated with 1-3 J/m(2) UVC. Replication of pSV011 was barely affected in irradiated IDH4 cells. Plasmid replication was inhibited by 50% in irradiated CTag cells and this inhibition could not be accounted for by increased killing of host cells by UVC. These results suggest that even in transformed cells UVC induces DNA damage responses that are reflected in transient cell cycle arrest, delay in proliferation and inhibition of episomal DNA replication. These responses are enhanced in CTag cells, presumably because of their bypass replication defect. The accumulation of replication complexes blocked at thymine dimers and extended single-stranded regions in chromosomal DNA might sequester replication factors that are needed for plasmid and chromosomal replication. Alternatively, aberrant replication structures might activate a signal transduction pathway that down-regulates DNA synthesis.

Abnormal DNA Damage-inducible Protein in Cells from Sjögren's Syndrome Patients

Antinuclear antibodies are commonly found in patients with Sjögren's syndrome. It has been suggested that the development of antinuclear antibodies depends on the activation of the spliceosome and other transcription-related subcellular particles, some of which have recently been shown also to function in DNA-modifying processes, such as DNA repair and V(D)J recombination. These observations add weight to a previously proposed model for the aetiology of Sjögren's syndrome. This includes the abnormal processing of the T-cell receptor and immunoglobulin genes. To test this hypothesis further, the present study on DNA-modifying proteins in Sjögren's syndrome was initiated. Gel-shift experiments using protein extracted from UV-treated Sjögren cells provided evidence of high molecular weight DNA-binding protein in six out of 12 Sjögren patients studied (but not among seven healthy controls). Some Sjögren sera displayed antibodies to protein extracts from cells treated with psoralen plus UVA radiation. These results indicate an abnormal DNA damage-inducible response in Sjögren's syndrome. It may therefore be concluded that alterations in nuclear protein may play a role in the aetiology of Sjögren's syndrome.

DNA damage inducible-gene expression following platinum treatment in human ovarian carcinoma cell lines.

DNA damage-inducible genes, such as gadd153, gadd45, p21 and c-jun, have previously been shown to be induced by the chemotherapeutic agent cisplatin. One of these genes, gadd153, has previously been reported to be differentially expressed in cisplatin-resistant cell lines and, therefore, to be a potential prognostic indicator for tumor response to cisplatin-based chemotherapy. It is not currently known whether such damage-inducible genes are turned on by the DNA damage itself (e.g. by the formation of Pt-DNA adducts) or by the downstream biological consequences of that damage. It is also not known whether the increased expression of these DNA-damage-inducible genes is related to immediate protective responses such as DNA repair or to more delayed responses such as cell cycle arrest or apoptosis. These experiments were initiated to characterize more fully the nature of the DNA damage-inducible response to cisplatin treatment and to determine whether any of these genes might be useful prognostic indicators of tumor response to cisplatin chemotherapy. The dose-response and time-course for the induction of the DNA damage-inducible genes gadd153, gadd45, p21 and c-jun were examined by Northern analysis in the human ovarian carcinoma cell line 2008 and its resistant subclone C13* following treatment with platinum anticancer agents. The extent of gene expression was correlated with cytotoxicity determined by growth inhibition assay, Pt-DNA adducts determined by atomic absorption spectrometry and inhibition of DNA synthesis determined by 3H-thymidine incorporation. All four genes were induced maximally in both sensitive and resistant cell lines at lethal cisplatin doses (> or = ID90). Induction was maximal between 24 and 48 h following exposure to the drug for all genes except c-jun which was induced by 6 h. At 24 h following cisplatin treatment the overall levels of gadd153 were less in the resistant C13* cell line than in the parental 2008 cell line, while those of gadd45 were greater in C13* than in 2008. Maximal expression of p21 and c-jun was not significantly different in the two cell lines. The dose-response of these genes correlated with the cytotoxicity of cisplatin and the inhibition of DNA synthesis by cisplatin, rather than to the actual levels of Pt-DNA adducts. The more cytotoxic platinum analog, ormaplatin, also induced gadd153 and its induction was also based on cytotoxicity. These results suggest that the regulation of gadd153 and gadd45 expression occurs thorough separate pathways in the 2008 and C13* cell lines. The DNA damage-inducible gene response for all four damage-inducible genes tested appeared to be more directly correlated with downstream biologic effects of cisplatin damage than with actual Pt-DNA adduct levels. The time-course and dose-response for induction of these genes was more consistent with delayed responses such as apoptosis rather than more immediate responses such as DNA repair. Finally, these results strengthen previous suggestions that the expression of gadd153, and possibly other DNA damage-inducible genes, may be useful indicators of tumor response to cisplatin-based chemotherapy.