Segregational fidelity of chromosomes in human thyroid tumour cells

Using fluorescence in situ hybridisation (FISH) we have analysed the segregational fidelity of all the human chromosomes during mitotic cell division. The losses and gains of chromosomes were analysed in human polyploid cell lines derived from a well-differentiated papillary thyroid cancer. These thyroid cells can be cultured for more than 300 population doublings. For the purpose of our study the polyploid nature of the cells may act as a protective buffer against the cell-lethal effects of the loss of individual chromosomes. To evaluate the role of the p53 gene product in maintaining the fidelity of chromosome segregation we compared the frequencies of chromosome loss and gain in cultures with wild-type p53 activity (K1E7neo3) and cultures transfected with plasmids expressing a mutant p53 product (K1E7scx6). Cultures were analysed for the presence of both structurally normal and rearranged chromosomes at both early and late passages. Cell cultures with defective p53 activity showed progressive chromosome loss from a median chromosome number of 87–97 to 75–86. Cell growth in cultures with wild-type p53 activity showed the loss of chromosomes 6, 7, and 8 and the gain of 17 and 20. Cultures expressing mutant p53 activity showed the loss of chromosomes 2, 5, 14 and 17 and the gain of 4 and 22. The combination of defective p53 and growth resulted in further destabilisation with the additional losses of chromosomes 3, 11, 15, 16 and 21. Chromosomes 1, 9, 10, 12, 13, 18, 19, X and Y segregated stably under all the culture conditions as did the structurally rearranged marker chromosomes. The study has demonstrated variation in the fidelity of mitotic chromosome segregation and the influence of p53 gene activity upon the segregation of individual human chromosomes. Received: 7 August 1998; in revised form: 28 August 1998 / Accepted: 29 August 1998     

Noncomplementary DNA double-strand-break rejoining in bacterial and human cells.

We examined the rejoining of noncomplementary restriction enzyme-produced DNA double-strand breaks in Escherichia coli and in cultured human cells. The enzymes used in this study, ClaI, BamHI and SalI, produce double-strand breaks with 5 protruding single strands. The joining of a ClaI-produced DNA end to a BamHI-produced end or to a SalI-produced end was examined at the DNA sequence level. End rejoining in E.coli was studied by transforming cultures with linear plasmid DNA that was gel purified from restriction digests, and end rejoining in cultured human cells was studied by introducing enzymes into the cells by electroporation. The human cells used contain an Epstein-Barr virus (EBV)-based shuttle vector, pHAZE, that was recovered and introduced into E.coli for further analysis. The major products of DNA end-joining processes observed in linear plasmid-transformed E.coli and in the human cells exposed to restriction enzymes were identical. Furthermore, the deletions observed in both systems and in the spontaneous mutant plasmids in untreated human cells had a common underlying feature: short stretches of directly repeated DNA at the junction sites.     

A repair competition assay to assess recognition by human nucleotide excision repair.

We developed a competition assay to compare, in a quantitative manner, the ability of human nucleotide excision repair (NER) to recognise structurally different forms of DNA damage. This assay uses a NER substrate consisting of M13 double-stranded DNA with a single and uniquely located acetylaminofluorene (AAF) adduct, and measures the efficiency by which multiply damaged plasmid DNA competes for excision repair of the site-directed modification. To validate this assay, we tested competitor DNA containing defined numbers of either AAF adducts or UV radiation products. In both cases, repair of the site-directed NER substrate was inhibited in a damage-specific and dose-dependent manner. We then exploited this competition assay to determine the susceptibility of bulky adozelesin-DNA adducts to human NER.     

Calmodulin antagonists and cAMP inhibit ionizing-radiation-enhancement of double-strand-break repair in human cells

Ionizing radiation (IR) enhances double-strand-break (DSB)-repair fidelity in plasmids processed in normal lymphoblasts but not in lymphoblasts from ataxia telangiectasia (A-T) patients. Putatively, signal-transduction pathways mediate this DNA-repair induction. Because IR inhibition of DNA synthesis is defective in A-T cells and is mediated by a calmodulin (caM)-dependent pathway, we evaluated the involvement of caM-dependent pathways in DSB-repair induction. Human lymphoblasts were gamma-irradiated with or without treatment with caM antagonists and the cells' abilities to repair shuttle pZ189 carrying a single DSB (linDNA) were assessed. In untreated controls, IR enhanced DSB-rejoining fidelity if transfection occurred promptly but diminished fidelity if transfection was delayed. Treatment with two caM antagonists, W-7 and W-13, prior to irradiation blocked this IR-enhancement of DSB-rejoining fidelity. Vinpocetine, a caM-dependent phosphodiesterase inhibitor, and 8-bromo-cAMP also inhibited IR enhancement of repair fidelity, but caM-dependent protein kinase II inhibitor KN62 had no effect. Other protein kinase inhibitors, staurosporine and genistein, also did not inhibit IR enhancement of DSB repair fidelity. However, staurosporine blocked the twofold reduction in DSB-repair fidelity seen if linDNA transfection was delayed 2 h after irradiation. These findings point to the involvement of caM/cAMP-dependent pathway(s) in mediating IR-enhancement of DSB-rejoining fidelity in mammalian cells.     

Incorporation of large heterologies into heteroduplex DNA during double-strand-break repair in mouse cells

In this study, the formation and repair of large (>1 kb) insertion/deletion (I/D) heterologies during double-strand-break repair (DSBR) was investigated using a gene-targeting assay that permits efficient recovery of sequence insertion events at the haploid chromosomal immunoglobulin (Ig) mu-locus in mouse hybridoma cells. The results revealed that (i) large I/D heterologies were generated on one or both sides of the DSB and, in some cases, formed symmetrically in both homology regions; (ii) large I/D heterologies did not negatively affect the gene targeting frequency; and (iii) prior to DNA replication, the large I/D heterologies were rectified.     

Development of a novel assay for human tyrosyl DNA phosphodiesterase 2

Tyrosyl DNA phosphodiesterase 2 (TDP2), a newly discovered enzyme that cleaves 5′-phosphotyrosyl bonds, is a potential target for chemotherapy. TDP2 possesses both 3′- and 5′-tyrosyl-DNA phosphodiesterase activity, which is generally measured in a gel-based assay using 3′- and 5′-phosphotyrosyl linkage at the 3′ and 5′ ends of an oligonucleotide. To understand the enzymatic mechanism of this novel enzyme, the gel-based assay is useful, but this technique is cumbersome for TDP2 inhibitor screening. For this reason, we have designed a novel assay using p-nitrophenyl-thymidine-5′-phosphate (T5PNP) as a substrate. This assay can be used in continuous colorimetric assays in a 96-well format. We compared the salt and pH effect on product formation with the colorimetric and gel-based assays and showed that they behave similarly. Steady-state kinetic studies showed that the 5′ activity of TDP2 is 1000-fold more efficient than T5PNP. Tyrosyl DNA phosphodiesterase 1 (TDP1) and human AP-endonuclease 1 (APE1) could not hydrolyze T5PNP. Sodium orthovanadate, a known inhibitor of TDP2, inhibits product formation from T5PNP by TDP2 (IC₅₀ = 40 mM). Our results suggest that this novel assay system with this new TDP2 substrate can be used for inhibitor screening in a high-throughput manner.     

Mechanisms of double-strand-break repair during gene targeting in mammalian cells

In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism.     

Coordination of DNA ends during double-strand-break repair in bacteriophage T4

The extensive chromosome replication (ECR) model of double-strand-break repair (DSBR) proposes that each end of a double-strand break (DSB) is repaired independently by initiating extensive semiconservative DNA replication after strand invasion into homologous template DNA. In contrast, several other DSBR models propose that the two ends of a break are repaired in a coordinated manner using a single repair template with only limited DNA synthesis. We have developed plasmid and chromosomal recombinational repair assays to assess coordination of the broken ends during DSBR in bacteriophage T4. Results from the plasmid assay demonstrate that the two ends of a DSB can be repaired independently using homologous regions on two different plasmids and that extensive replication is triggered in the process. These findings are consistent with the ECR model of DSBR. However, results from the chromosomal assay imply that the two ends of a DSB utilize the same homologous repair template even when many potential templates are present, suggesting coordination of the broken ends during chromosomal repair. This result is consistent with several coordinated models of DSBR, including a modified version of the ECR model.     

Assessment of DNA damage and repair in human peripheral blood mononuclear cells using a novel DNA unwinding technique.

A newly developed, fast and sensitive microplate assay (Fast Micromethod) was used for the assessment of gamma-radiation-induced DNA damage in peripheral blood mononuclear cells (PBMC) from healthy donors of various ages and from cancer patients undergoing radiotherapy. This assay detects the presence of DNA single-strand breaks and alkali-labile sites by monitoring the rate of DNA unwinding under alkaline conditions using the fluorescent dye PicoGreen, which preferentially binds to double-stranded DNA at high pH (>12.0); it requires only minimal amounts of material (approximately 3 x 10(3) cells/well) and can be performed within 3 hrs. or less. EDTA blood samples were collected from patients not undergoing chemotherapy prior and immediately after irradiation, or were collected from healthy donors and irradiated ex vivo. The results revealed that the amount of DNA strand breaks in PBMC, induced by application of a single dose to patients in the course of radiotherapy treatment, markedly varied between different individuals. To examine the effect of age on DNA damage, the basal levels of DNA damage in PBMC from a total of 30 healthy donors were determined: 10 were 20 to 30 years of age, 10 were 40 to 60 years of age and 10 were >70 years of age. It was found that the mean basal level of DNA damage from donors in the >70-year age group was significantly higher (by 97%) than that of the 20- to 30-year age group and 27% higher than that of the 40- to 60-year age group. Measurements of the level of induced DNA damage in PBMC isolated from blood after 2 Gy irradiation with 60Co gamma-rays revealed no significant differences between donors aged 20-30 and 40-60. However, there was a strong increase (by 2.3- to 2.9-fold) in radiosensitivity in the age group >70. The microplate assay described may be used as a pretherapeutic sensitivity test for the assessment of the individual radiosensitivity of patients prior to radiation therapy.     

DNA damage and DNA repair in cultured human cells exposed to chromate

DNA damage and DNA repair have been observed in cultured human skin fibroblasts exposed to potassium chromate but not to a chromic glycine complex. DNA repair synthesis (unscheduled incorporation of [3H]thymidine (TdR)) was measured in cells during or following exposure to chromate and was significant for chromate concentrations above 10(-6) M. Maximal DNA repair was observed at about 10(-4) M chromate. DNA repair capacity was found to be saturated at this concentration. Chromate was stable for at least 8 h in culture medium and produced approximately a linear increase in repair with duration of exposure. DNA damage as determined by alkaline sucrose gradient sedimentation was detected after treatment for 1.5 h with 5 . 10(-4) M chromate. Exposure to 10(-7) M chromate solution for 7 days inhibited colony formation while acute (1 h) treatment was toxic at 5 . 10(-6) M. The chromic glycine complex was toxic above 10(-3) M for a 1-week exposure but was not observably toxic after a 1-h treatment. These results indicate that chromate and not chromic compounds may be the carcinogenic form for man. The nature of the ultimate carcinogen is discussed. These findings illustrate the utility of the DNA repair technique to study the effects on human cells of inorganic carcinogens and mutagens.     

Influence of double-strand-break repair pathways on radiosensitivity throughout the cell cycle in CHO cells

Unrepaired DNA double-strand breaks (DSBs) produced by ionizing radiation (IR) are a major determinant of cell killing. To determine the contribution of DNA repair pathways to the well-established cell cycle variation in IR sensitivity, we compared the radiosensitivity of wild-type CHO cells to mutant lines defective in nonhomologous end joining (NHEJ), homologous recombination repair (HRR), and the Fanconi anemia pathway. Cells were irradiated with IR doses that killed approximately 90% of each asynchronous population, separated into synchronous fractions by centrifugal elutriation, and assayed for survival (colony formation). Wild-type cells had lowest resistance in early G1 and highest resistance in S phase, followed by declining resistance as cells move into G2/M. In contrast, HR-defective cells (xrcc3 mutation) were most resistant in early G1 and became progressively less resistant in S and G2/M, indicating that the S-phase resistance in wild-type cells requires HRR. Cells defective in NHEJ (dna-pk(cs) mutation) were exquisitely sensitive in early G1, most resistant in S phase, and then somewhat less resistant in G2/M. Fancg mutant cells had almost normal IR sensitivity and normal cell cycle dependence, suggesting that Fancg contributes modestly to survival and in a manner that is independent of cell cycle position.     

Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells

The eukaryotic sliding DNA clamp, proliferating cell nuclear antigen (PCNA), is essential for DNA replication and repair synthesis. In order to load the ring-shaped, homotrimeric PCNA onto the DNA double helix, the ATPase activity of the replication factor C (RFC) clamp loader complex is required. Although the recruitment of PCNA by RFC to DNA replication sites has well been documented, our understanding of its recruitment during DNA repair synthesis is limited. In this study, we analyzed the accumulation of endogenous and fluorescent-tagged proteins for DNA repair synthesis at the sites of DNA damage produced locally by UVA-laser micro-irradiation in HeLa cells. Accumulation kinetics and in vitro pull-down assays of the large subunit of RFC (RFC140) revealed that there are two distinct modes of recruitment of RFC to DNA damage, a simultaneous accumulation of RFC140 and PCNA caused by interaction between PCNA and the extreme N-terminus of RFC140 and a much faster accumulation of RFC140 than PCNA at the damaged site. Furthermore, RFC140 knock-down experiments showed that PCNA can accumulate at DNA damage independently of RFC. These results suggest that immediate accumulation of RFC and PCNA at DNA damage is only partly interdependent.     

Human WIPI-1 puncta-formation: a novel assay to assess mammalian autophagy

Autophagy depends on the activity of phosphoinositide-3 kinase class III to generate PI(3)P. We identified the human WIPI protein family of PI(3)P-binding factors and showed that WIPI-1 (Atg18) is linked to autophagy in human cells. Induction of autophagy by rapamycin, gleevec, thapsigargin and amino acid deprivation led to an accumulation of WIPI-1 at LC3-positive membrane structures (WIPI-1 puncta-formation), suggested to represent autophagosomal isolation membranes. WIPI-1 puncta-formation is inhibited by wortmannin and LY294002, and PI(3)P-binding-deficient WIPI-1 is puncta-formation-incompetent. Quantification of WIPI-1 puncta should be suitable to assay mammalian autophagy.     

DNA repair and repair fidelity in metastatic variants of the B16 murine melanoma

We have examined the concept of genomic instability in relation to the metastatic progression of low (F1) and high metastasis (BL6, ML8) clones of the B16 mouse melanoma, by using a mutation assay, and DNA strand break repair and repair fidelity assays. The frequency of induced ouabain resistant colonies between the variant cell lines was consistent with the difference between their metastatic properties. Survival data for X-irradiation and bleomycin were similar among the 3 cell lines. When X-rays or bleomycin were used to induce strand breakage, no difference was detectable in either the rate or extent of DNA repair using the techniques of alkaline unwinding and alkaline elution for total strand breaks, and neutral elution for double strand breaks. DNA repair fidelity was measured using the PMH16 plasmid. A Kpn I restriction site was used to introduce a break within the gpt gene of the plasmid, prior to transfection. We found that ～ 100% and ～ 65% of the highly metastatic ML8 and BL6 clones, respectively, religated the gene with the required fidelity, compared with only ～ 25% of the low metastasis F1 clones. In summary, the metastatic variants show similar sensitivities to X-irradiation and bleomycin, but a differential response to EMS. This difference is not reflected in any subsequent DNA strand break religation, but the variants do differ in their fidelity of repair. However, although the fidelity of DNA religation is related to metastatic potential, it is not consistent with the mutation frequency data. © 1993 Wiley-Liss, Inc.     

BCR/ABL modifies the kinetics and fidelity of DNA double-strand breaks repair in hematopoietic cells

The oncogenic BCR/ABL tyrosine kinase facilitates the repair of DNA double-strand breaks (DSBs). We find that after gamma-irradiation BCR/ABL-positive leukemia cells accumulate more DSBs in comparison to normal cells. These lesions are efficiently repaired in a time-dependent fashion by BCR/ABL-stimulated non-homologous end-joining (NHEJ) followed by homologous recombination repair (HRR) mechanisms. However, mutations and large deletions were detected in HRR and NHEJ products, respectively, in BCR/ABL-positive leukemia cells. We propose that unfaithful repair of DSBs may contribute to genomic instability in the Philadelphia chromosome-positive leukemias.