Measuring gene-specific nucleotide excision repair in human cells using quantitative amplification of long targets from nanogram quantities of DNA

We have been developing a rapid and convenient assay for the measurement of DNA damage and repair in specific genes using quantitative polymerase chain reaction (QPCR) methodology. Since the sensitivity of this assay is limited to the size of the DNA amplification fragment, conditions have been found for the quantitative generation of PCR fragments from human genomic DNA in the range of 6-24 kb in length. These fragments include: (1) a 16.2 kb product from the mitochondrial genome; (2) 6.2, 10.4 kb, and 15.4 kb products from the hprt gene, and (3) 13.5, 17.7, 24.2 kb products from the human beta-globin gene cluster. Exposure of SV40 transformed human fibroblasts to increasing fluences of ultraviolet light (UV) resulted in the linear production of photoproducts with 10 J/m(2) of UVC producing 0.085 and 0.079 lesions/kb in the hprt gene and the beta-globin gene cluster, respectively. Kinetic analysis of repair following 10 J/m(2) of UVC exposure indicated that the time necessary for the removal of 50% of the photoproducts, in the hprt gene and beta-globin gene cluster was 7.8 and 24.2 h, respectively. Studies using lymphoblastoid cell lines show very little repair in XPA cells in both the hprt gene and beta-globin locus. Preferential repair in the hprt gene was detected in XPC cells. Cisplatin lesions were also detected using this method and showed slower rates of repair than UV-induced photoproducts. These data indicate that the use of long targets in the gene-specific QPCR assay allows the measurement of biologically relevant lesion frequencies in 5-30 ng of genomic DNA. This assay will be useful for the measurement of human exposure to genotoxic agents and the determination of human repair capacity.     

CITED2 silencing sensitizes cancer cells to cisplatin by inhibiting p53 trans-activation and chromatin relaxation on the ERCC1 DNA repair gene

In this study, we show that silencing of CITED2 using small-hairpin RNA (shCITED2) induced DNA damage and reduction of ERCC1 gene expression in HEK293, HeLa and H1299 cells, even in the absence of cisplatin. In contrast, ectopic expression of ERCC1 significantly reduced intrinsic and induced DNA damage levels, and rescued the effects of CITED2 silencing on cell viability. The effects of CITED2 silencing on DNA repair and cell death were associated with p53 activity. Furthermore, CITED2 silencing caused severe elimination of the p300 protein and markers of relaxed chromatin (acetylated H3 and H4, i.e. H3K9Ac and H3K14Ac) in HEK293 cells. Chromatin immunoprecipitation assays further revealed that DNA damage induced binding of p53 along with H3K9Ac or H3K14Ac at the ERCC1 promoter, an effect which was almost entirely abrogated by silencing of CITED2 or p300. Moreover, lentivirus-based CITED2 silencing sensitized HeLa cell line-derived tumor xenografts to cisplatin in immune-deficient mice. These results demonstrate that CITED2/p300 can be recruited by p53 at the promoter of the repair gene ERCC1 in response to cisplatin-induced DNA damage. The CITED2/p300/p53/ERCC1 pathway is thus involved in the cell response to cisplatin and represents a potential target for cancer therapy.     

Analysis of DNA damage and repair in murine leukemia L1210 cells using a quantitative polymerase chain reaction assay.

The polymerase chain reaction (PCR) represents an alternative to the current methods for investigating DNA damage and repair in specific genomic segments. In theory, any DNA lesion which blocks Taq polymerase can be measured by this assay. We used quantitative PCR (QPCR) to determine the lesion frequencies produced by cisplatin and ultraviolet light (UV) in a 2.3 kilobase (kb) segment of mitochondrial DNA and a 2.6 kb segment of the DHFR gene in mouse leukemia L1210 cells. The frequency of UV-induced lesions increased linearly with dose, and was 0.58 lesions/10 kb/10 J/m2 in the mitochondrial DNA, and 0.37 lesions/10 kb/10 J/m2 in the DHFR gene. With cisplatin, the lesion frequency also increased linearly with dose, and was 0.17 lesions/10 kb/10 microM in the DHFR gene, and 0.07 lesions/10 kb/10 microM in mitochondrial DNA. This result is contrary to that of Murata et al., 1990 (1), in which mitochondrial DNA received greater cisplatin damage than did nuclear DNA. Using PCR to measure the repair of UV-induced lesions in the DHFR gene segment, we observed that less than 10% of the lesions were removed by 4 h, but over 70% of the lesions were removed by 8 h. Repair of 43% of UV-induced lesions in mitochondrial DNA was also observed during a 24 h period.     

淋巴细胞凋亡与p53蛋白表达关系的研究

培养B95-8细胞,分离EB病毒,转染外周血和扁桃体淋巴细胞,建立永生化的LCLs和TLCL细胞株; 带有wt P₅₃基因的LCLs在DNA损伤剂——顺铂处理前未检出p53蛋白,经顺铂处理后,LCLs随作用时间延长细胞存活率明显下降、p53蛋白水平升高、DNA电泳显出梯状带;含mt P₅₃基因的淋巴瘤细胞在顺铂处理前可检出高浓度的p53蛋白,经顺铂处理后,细胞存活率与p53蛋白并无明显改变.这些结果表明：顺铂引起细胞DNA损伤、激活wt p53蛋白的表达、继而wt p53蛋白又促进了DNA损伤细胞凋亡.     

Repair of mitomycin C cross-linked DNA in mammalian cells measured by a host cell reactivation assay

DNA repair capacity in a cell could be detected by a host-cell reactivation assay (HCR). Since relation between DNA repair and genetic susceptibility to cancer remains unclear, it is necessary to identify DNA repair defects in human cancer cells. To assess DNA repair for breast cancer susceptibility, we developed a modified HCR assay using a plasmid containing a firefly luciferase gene damaged by mitomycin C (MMC), which forms interstrand cross-link (ICL) adducts. In particular, interstrand cross-link is thought to induce strand breaks being repaired by homologous recombination. The MMC-ICLs were verified by electrophoresis. Damaged plasmids were transfected into apparently normal human lymphocytes and NER-deficient XP cell lines and the DNA repair capacity of the cells were measured by quantifying the activity of the firefly luciferase. MMC lesion was repaired as much as UV adducts in normal lymphocytes and the XPC cells. However, the XPA cells have a lower repair capacity for MMC lesion than the XPC cell, indicating that the XPA protein may be involved in initial damage recognition of MMC-ICL adducts. Since several repair pathways including NER and recombination participate in MMC-ICL removal, this host cell reactivation assay using MMC-ICLs can be used in exploring DNA repair defects in human cancer cells.     

p53-degradation by HPV-16 E6 preferentially affects the removal of cyclobutane pyrimidine dimers from non-transcribed strand and sensitizes mammary epithelial cells to UV-irradiation

Nucleotide excision repair (NER), the most versatile and ubiquitous mechanism for DNA repair, operates to remove many types of DNA base lesions. We have studied the role of p53 function in modulating the repair of DNA damage following UV irradiation in normal and p53-compromised human mammary epithelial cells (HMEC). The effect of UV-induced DNA damage on cellular cytotoxicity and apoptosis was determined in conjunction with global, gene- and strand-specific repair. Cytotoxicity studies, using clonogenic survival and MTT assays, showed that HPV-16 E6-expressing HMEC were more UV sensitive than p53-WT cell lines. High apoptotic index obtained with p53-compromised cells was in conformity to both the low clonogenic survival and the low cellular viability. No discernible differences in the formation of initial UV-induced cyclobutane pyrimidine dimers (CPD) were observed in the cell lines of varying p53 functional status. However, the extent and the rate of damage removal from genome overall were highest for p53-WT cells. Further examination of strand-specific repair in the p53 gene revealed that the removal of CPD in the non-transcribed strand (NTS) was slower in p53-compromised cells compared to the normal p53-WT cell lines. These results suggest that loss of p53 function, in the absence of other genetic alterations, decreased both overall amount of CPD repaired and their removal rate from the genome. Additionally, normal function of p53 is required for the repair of the NTS, but not of the transcribed strand (TS) in genomic DNA in human epithelial cells. Thus, failure of quantitative removal of CPD by global genomic repair (GGR), due to loss of p53 function, causes the enhanced UV sensitivity and increased damage-induced apoptosis via a p53-independent pathway. Nevertheless, recovery of cells from UV damage requires normal p53 function and efficient GGR.     

Role and regulation of p53 during an ultraviolet radiation-induced G1 cell cycle arrest.

p53 can play a key role in response to DNA damage by activating a G1 cell cycle arrest. However, the importance of p53 in the cell cycle response to UV radiation is unclear. In this study, we used normal and repair-deficient cells to examine the role and regulation of p53 in response to UV radiation. A dose-dependent G1 arrest was observed in normal and repair-deficient cells exposed to UV. Expression of HPV16-E6, or a dominant-negative p53 mutant that inactivates wildtype p53, caused cells to become resistant to this UV-induced G1 arrest. However, a G1 to S-phase delay was still observed after UV treatment of cells in which p53 was inactivated. These results indicate that UV can inhibit G1 to S-phase progression through p53-dependent and independent mechanisms. Cells deficient in the repair of UV-induced DNA damage were more susceptible to a G1 arrest after UV treatment than cells with normal repair capacity. Moreover, no G1 arrest was observed in cells that had completed DNA repair prior to monitoring their movement from G1 into S-phase. Finally, p53 was stabilized under conditions of a UV-induced G1 arrest and unstable when cells had completed DNA repair and progressed from G1 into S-phase. These results suggest that unrepaired DNA damage is the signal for the stabilization of p53, and a subsequent G1 phase cell cycle arrest in UV-irradiated cells.     

In vivo formation and repair of cyclobutane pyrimidine dimers and 6-4 photoproducts measured at the gene and nucleotide level in Escherichia coli

In vivo formation and repair of the major UV-induced DNA photoproducts, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4 PPs), have been examined at the gene and nucleotide level in Escherichia coli. Each type of DNA photoproduct has individually been studied using photoreactivation and two newly developed assays; the multiplex QPCR assay for damage detection at the gene level and the reiterative primer extension (PE) assay for damage detection at the nucleotide level. In the E. coli lacI and lacZ genes, CPDs and 6-4 PPs form in a 2:1 ratio, respectively, during UV irradiation. Repair of 6-4 PPs is more efficient than repair of CPDs since, on the average, 42% of 6-4 PPs are repaired in both genes in the first 40 min following 200 J/m² UV irradiation, while 1% of CPDs are repaired. The location, relative frequency of formation, and efficiency of repair of each type of photoproduct was examined in the first 52 codons of the E. coli lacI gene at the nucleotide level. Hotspots of formation were found for each type of lesion. Most photoproducts are at sites where both CPDs and 6-4 PPs are formed. Allowing 40 min of recovery following 200 J/m² shows that in vivo repair of 6-4 PPs is about fourfold more efficient than the repair of CPDs. Comparison of the lesion-specific photoproduct distribution of the lacI gene with a UV-induced mutation spectrum from wild-type cells shows that most mutational hotspots are correlated with sites of a majority of CPD formation. However, 6-4 PPs are also formed at some of these sites with relatively high frequency. This information, taken together with the observation that 6-4 PPs are repaired faster than CPDs, suggest that the cause of mutagenic hotspots in wild-type E. coli is inefficient repair of CPDs.     

Role of the p53 homologue from Drosophila melanogaster in the maintenance of histone H3 acetylation and response to UV-light irradiation

It has been demonstrated that the human tumor suppressor p53 has an important role in modulating histone modifications after UV light irradiation. In this work we explored if the p53 Drosophila homologue has a similar role. Taking advantage of the existence of polytene chromosomes in the salivary glands of third instar larvae, we analyzed K9 and K14 H3 acetylation patterns in situ after UV irradiation of wild-type and Dmp53 null flies. As in human cells, after UV damage there is an increase in H3 acetylation in wild-type organisms. In Dmp53 mutant flies, this response is significantly affected at the K9 position. These results are similar to those found in human p53 mutant tumor cells with one interesting difference, only the basal H3 acetylation of K14 is reduced in Dmp53 mutant flies, while the basal H3-K9 acetylation is not affected. This work shows, that the presence of Dmp53 is necessary to maintain normal H3-K14 acetylation levels in Drosophila chromatin and that the function of p53 to maintaining histone modifications, is conserved in Drosophila and humans.     

Activation of DNA damage response pathways in human mesenchymal stem cells exposed to cisplatin or γ-irradiation

DNA damaging agents are widely used in treatment of hematogical malignancies and solid tumors. While effects on hematopoietic stem cells have been characterized, less is known about the DNA damage response in human mesenchymal stem cells (hMSCs) in the bone marrow stroma, progenitors of osteoblasts, chondrocytes and adipocytes. To elucidate the response of undifferentiated hMSCs to γ-irradiation and cisplatin, key DNA damage responses have been characterised in hMSCs from normal adult donors. Cisplatin and γ-irradiation activated the DNA damage response in hMSCs, including induction of p53 and p21, and activation of PI3 kinase-related protein kinase (PIKK)-dependent phosphorylation of histone H2AX on serine 139, and replication protein A2 on serine4/serine8. Chemical inhibition of ATM or DNA-PK reduced DNA damage-induced phosphorylation of H2AX, indicating a role for both PIKKs in the response of hMSCs to DNA damage. Consistent with repair of DNA strand breaks, γ-H2AX staining decreased by 24 hours following gamma-irradiation. γ-irradiation arrested hMSCs in the G₁ phase of the cell cycle, while cisplatin induced S-phase arrest, mediated in part by the ATR/Chk1 checkpoint pathway. In hMSCs isolated from a chronic lymphocytic leukemia (CLL) patient, p53 and p21 were induced by cisplatin and γ-irradiation, while RPA2 was phosphorylated on serine4/8 in particular following cisplatin. Compared to peripheral blood lymphocytes or the leukemia cell line K562, both normal hMSCs and CLL-derived hMSCs were more resistant to cisplatin and γ-irradiation. These results provide insights into key pathways mediating the response of bone marrow-derived hMSCs to DNA damaging agents used in cancer treatment.     

X-ray irradiation altered chemosensitivity of a p53-null non-small cell lung cancer cell line

Radiotherapy is an effective approach to treating many types of cancer. Recent progress in radiotherapy technology, such as intensity-modulated radiation therapy (IMRT) and three-dimensional (3D) radiotherapy, allow precise energy transfer to the tumor, which has improved local control rates. However, the emergence of tolerant cells during or after radiotherapy remains problematic. In the present study, we first established a cell population from H1299, the p53-null non-small cell lung cancer cell line, by 10 Gy irradiation using 6 MV X-rays. The radio- and chemosensitivity of this cell population (referred to as H1299-IR) was determined using colony formation analyses and MTS assays. Compared with the parental cell line, the radiosensitivity of H1299-IR was apparently the same. H1299 and H1299-IR were both more radio tolerant than the A549 cell line. However, H1299-IR became significantly more sensitive to cisplatin, an antitumor agent. After exposure to 25 mug/ml cisplatin for 2 h, parental cells steadily grew during the MTS assay, whereas the sensitivity of H1299-IR cells doubled both at 24 and 48 h. Microarray analysis of over 30,000 H1299-IR genes (Agilent Technology) revealed that 12 and 15 genes were up- (> 2.0) and down- (< 2.0) regulated, respectively. Rad51d (homologous recombination repair protein) gene was down-regulated 2.8-fold, whereas matrix metalloproteinase 1 (collagenase-1) gene was up-regulated 4.4-fold. These results indicated that some p53-null non-small cell lung cancers could be successfully treated when X-ray radiotherapy was administered with subsequent or concurrent cisplatin chemotherapy.     

ATR-Chk2 signaling in p53 activation and DNA damage response during cisplatin-induced apoptosis

Cisplatin is one of the most effective anti-cancer drugs; however, the use of cisplatin is limited by its toxicity in normal tissues, particularly injury of the kidneys. The mechanisms underlying the therapeutic effects of cisplatin in cancers and side effects in normal tissues are largely unclear. Recent work has suggested a role for p53 in cisplatin-induced renal cell apoptosis and kidney injury; however, the signaling pathway leading to p53 activation and renal apoptosis is unknown. Here we demonstrate an early DNA damage response during cisplatin treatment of renal cells and tissues. Importantly, in the DNA damage response, we demonstrate a critical role for ATR, but not ATM (ataxia telangiectasia mutated) or DNA-PK (DNA-dependent protein kinase), in cisplatin-induced p53 activation and apoptosis. We show that ATR is specifically activated during cisplatin treatment and co-localizes with H2AX, forming nuclear foci at the site of DNA damage. Blockade of ATR with a dominant-negative mutant inhibits cisplatin-induced p53 activation and renal cell apoptosis. Consistently, cisplatin-induced p53 activation and apoptosis are suppressed in ATR-deficient fibroblasts. Downstream of ATR, both Chk1 and Chk2 are phosphorylated during cisplatin treatment in an ATR-dependent manner. Interestingly, following phosphorylation, Chk1 is degraded via the proteosomal pathway, whereas Chk2 is activated. Inhibition of Chk2 by a dominant-negative mutant or gene deficiency attenuates cisplatin-induced p53 activation and apoptosis. In vivo in C57BL/6 mice, ATR and Chk2 are activated in renal tissues following cisplatin treatment. Together, the results suggest an important role for the DNA damage response mediated by ATR-Chk2 in p53 activation and renal cell apoptosis during cisplatin nephrotoxicity.