An abnormal profile of DNA replication intermediates in Bloom's syndrome

Bloom's syndrome (BS) cells display a characteristic genomic instability, notably an elevated frequency of sister-chromatid exchange. Replicating DNA in cultured BS cells was labeled with [3H]thymidine using several time schedules. Separation of DNA in agarose gels showed high molecular weight DNA and three classes of DNA replication intermediates: 20-kilobase DNA, 10-kilobase DNA, and Okazaki fragments. In contrast newly replicated DNA from normal cells showed no 20-kilobase DNA replication intermediates. Certain BS cells, exceptional in that their characteristic genomic instability has for unknown reasons been corrected, also differed from normal cells in having the 20-kilobase intermediate, but they differed from both normal cells and the other (the uncorrected) BS cells in lacking the 10-kilobase DNA replication intermediates.     

Age-related radical-induced DNA damage is linked to prostate cancer

We measured concentrations and ratios of mutagenic (8-OH) lesions to putatively nonmutagenic formamidopyrimidine (Fapy) lesions of adenine (Ade) and guanine (Gua) to elucidate radical (.OH)-induced changes in DNA of normal, normal from cancer, and cancer tissues of the prostate. The relationship between the lesions was expressed using the mathematical model log(10)[(8-OH-Ade + 8-OH-Gua)/(FapyAde + FapyGua)]. Logistic regression analysis of the log ratios for DNA of normal and cancer tissues discriminated between the two tissue groups with high sensitivity and specificity. Correlation analysis of log ratios for normal prostates revealed a highly significant increase in the proportion of mutagenic base lesions with age. Data from correlation analysis of the log ratios for normal tissues from cancer were consistent with an age-dependent, dose-response relationship. The slopes for both correlations intersected at approximately 61 years, an age when prostate cancer incidence is known to rise sharply. The age-related increase in the proportion of.OH-induced mutagenic base lesions is likely a significant factor in prostate cancer development.     

Monoclonal antibodies detect conformationl abnormality of uracil DNA glycosylase in Bloom's syndrome cells

The immunoreactivity of normal human and Bloom's syndrome uracilDNA glycosylase was examined using a series of three anti-humanplacenta uracil DNA glycosylase monoclonal antibodies. Immunoreactivitywas determined by three separate and independent criteria: enzyme-linkedimmuno-sorbent assay (ELISA), enzyme inhibition studies andimmunoblot analysis. As defined by each criteria, normal humanuracil DNA glycosylase was immunoreactive with each antibody(37.04.12, 40.10.09 and 42.08.07). In contrast, each glycosylasepurified from two separate non-transformed Bloom's syndromecell strains was not reactive with antibody 40.10.09. First,no ELISA reactivity was observed with each glycosylase protein.Second, catalysis by each Bloom's syndrome glycosylase was notinhibited by antibody 40.10.09. However, each Bloom's syndromeenzyme was immunoreactive with antibodies 37.04.12 and 42.08.07.No immunoreactive glycosylase species was observed during theinduction of the Bloom's syndrome enzyme during cell proliferation.However, immunoreactivity of the denatured Bloom's syndromeenzyme with 40.10.09 antibody was observed by immunoblot analysis.These results suggest that Bloom's syndrome uracil DNA glycosylaseis characterized by a structural alteration in the native glycosylaseprotein secondary to the primary antigenic site recognized bythe 40.10.09 antibody. This altered antigeniclty may providean immunological marker for the identification of this humangenetic syndrome.     

DNA replication arrest and tolerance to DNA methylation damage

Inhibition of DNA replication by different DNA damaging agentshas been investigated in HeLaMR cells and amethylation damage-tolerantvariant HeLa5Al. In synchronous HeLaMR and HeLa5A1 cells exposedto N-ethyl-N-nitrosourea or ionizing radiation in mid-G₁ phase,DNA synthesis was inhibited in the following S phase. N-methyl-N-nitrosourea-inducedreplication inhibition in HeLaMR cells was delayed until thesecond S phase after treatment In contrast, N-methyl-N-nitrosoureatreatment of HeLa5Al cells affected neither the timing nor theextent of the first or second S phases. Both radiation and chemicaltreatment inhibited replication of an episomal plasmid and ofgenomic DNA in unison. Inhibition was observed at levels ofDNA damage that did not directly damage the plasmid molecules.Thus, DNA replication inhibition occurs immediately after ionizingradiation or ethylation damage, but methylation damage requiresprocessing through one cell cycle to generate an inhibitorysignal. The inhibitory signal appears to act in trans on undamagedDNA. Although methylationtolerantcells are responsive to inhibitionafter     

Two types of DNA ligase I activity in lymphoblastoid cells from patients with Bloom's syndrome

DNA ligases I and II were separated by hydroxylapatite (HA) column chromatography in cell-free extracts of lymphoblastoid cell lines (LCLs) derived from two unrelated patients with Bloom's syndrome (BS) and two healthy individuals. The specific activity of ligase I from the crude extract was consistently lower in GM3403, a BS LCL from an Ashkenazi Jewish patient, than in normal control LCLs. By contrast, the level of ligase I activity in BSL-2KA, another BS LCL derived from a Japanese patient, was equivalent to those in normal LCLs, although GM3403 and BSL-2KA shared the feature of exceedingly high frequency of spontaneous sister-chromatid exchange. The levels of total ligase activity in crude extracts without the separation into the two forms, however, were approximately two-fold higher for the two BS LCLs than for the normal LCLs. Partial purification by chromatography on a DEAE-cellulose 23 column and a phosphocellulose column did not affect the superiority of the two BS LCLs over the normal LCLs in the specific activity of the total ligases. Nonetheless, subsequent application to an HA column again resulted in much less elevation of the specific activity of ligase I for GM3403 than for BSL-2KA and control LCLs. The levels of ligase II activity, accounting for 4-13% of total ligase activity, were similar among the LCLs examined. Irrespective of the extent of purification, essentially no difference in the heat lability of DNA ligase I was detected among the four LCLs. These findings suggest that there may exist among BS LCLs at least two types of subtle abnormality of DNA ligase I itself and/or a putative substance modulating the enzyme function.     

Increased levels of 5-fluorouracil-induced DNA lesions in Bloom's syndrome

In Bloom's syndrome (BS) the regulation of uracil-DNA glycosylase, an enzyme involved in the repair of DNA containing 5-FU, is altered. 5-FU induces higher levels of DNA fragmentation in BS cells than in non-BS cells. The increase in DNA fragmentation is connected to the cytotoxic mechanism where 5-FU is incorporated into DNA. When 5-FU induces DNA fragmentation by a mechanism not involving the incorporation of drug into DNA, the levels of DNA fragmentation in BS and non-BS cells remain similar.     

Bloom's syndrome protein response to ultraviolet-C radiation and hydroxyurea-mediated DNA synthesis inhibition

Bloom's syndrome (BS) arises through mutations in both copies of the BLM gene that encodes a RecQ 3'-5' DNA helicase. BS patients are predisposed to developing all the cancers that affect the general population, and BS cells exhibit marked genetic instability. We showed recently that BLM protein contributes to the cellular response to ionizing radiation by acting as downstream ATM kinase effector. We now show that following UVC treatment, BLM-deficient cells exhibit a reduction in the number of replicative cells, a partial escape from the G2/M cell cycle checkpoint, and have an altered p21 response. Surprisingly, we found that hydroxyurea-treated BLM-deficient cells exhibit an intact S phase arrest, proper recovery from the S phase arrest, and intact p53 and p21 responses. We also show that the level of BLM falls sharply in response to UVC radiation. This UVC-induced reduction in BLM does not require a functional ATM gene and does not result from a subcellular compartment change. Finally, we demonstrate that exposure to UVC and hydroxyurea treatment both induce BLM phosphorylation via an ATM-independent pathway. These results are discussed in the light of their potential physiological significance with regard to the role of BLM in the cellular pathways activated by UVC radiation or HU-mediated inhibition of DNA synthesis.     

DNA repair and the molecular mechanisms of Bloom's syndrome

This critical review considers recent work on alterations in DNA repair capacity in Bloom's syndrome as a molecular mechanism for this human disorder. Four main types of DNA repair deficiencies are discussed. These include perturbations in the temporal regulation of DNA repair pathways during the cell cycle, failure to enhance DNA repair pathways during cell proliferation, reduced levels of DNA ligase in Bloom's syndrome cells, and the identification of mutant repair enzyme proteins. These deficiencies are considered in relation to the cellular characteristics of Bloom's syndrome, including delays in DNA replication, hypermutability, and increased incidence of chromosomal aberrations (spontaneously occurring or observed after exposure to environmental agents). The relationship between DNA repair deficiencies and the genetic basis of Bloom's syndrome is described. Previous evidence suggested an autosomal recessive mode of inheritance for Bloom's syndrome. A discussion is presented as to the molecular mechanism through which an alteration in a single gene could result in multiple DNA repair defects.     

Enhanced deoxyribonuclease activity in human transformed cells and in Bloom's syndrome cells

Human hereditary diseases such as xeroderma pigmentosum, Fanconi's anemia, ataxia telangiectasia, and Bloom's syndrome are characterized by a proneness for developing cancer associated with abnormalities in the processing of DNA damage. The molecular defects responsible for predisposing human tissues to cancer are still not well understood, despite the fact that a considerable amount of work has already been done on this problem. In this paper, we show that in human tumor cell lines, in cells transformed by DNA tumor viruses, and in cells derived from certain cancer-prone disorders, the level of activity of a 42-kDa deoxyribonuclease is many times higher than in diploid untransformed control cells. This suggests that this activity is linked to, or may play a role in, malignant transformation.     

Regulation of hypoxanthine DNA glycosylase in normal human and Bloom's syndrome fibroblasts

The regulation of the base excision repair enzyme hypoxanthine DNA glycosylase was examined in normal human skin fibroblasts (NHS) and fibroblasts from a patient with Bloom's syndrome. Using randomly proliferating cells and those synchronized at specific intervals in the cell cycle, enzyme levels were shown to become elevated severalfold in a proliferation-associated manner. In NHS synchronized in G0 by serum deprivation or in G1 by isoleucine deprivation, maximal enzyme levels were reached prior to maximal rates of DNA synthesis. In Bloom's syndrome cells synchronized in this manner, these two activities were coincident. Cells synchronized at the G1-S border by hydroxyurea exhibit an initial wave of DNA synthesis upon removal of the drug. The cells then undergo another DNA synthetic cycle climaxing 18-21 h after release. Maximal hypoxanthine glycosylase activity of hydroxyurea-synchronized Bloom's cells was observed during the second round of DNA synthesis. However, in NHS the peak of enzyme activity was observed as early as 9 h prior to the second round of DNA synthesis. To determine if hypoxanthine glycosylase could be induced in the absence of DNA synthesis, serum-synchronized NHS were released in the presence of hydroxyurea. The results showed that inhibition of DNA synthesis did not diminish glycosylase induction which demonstrated that DNA replication was not required for glycosylase induction.     

Tolerance to methylnitrosourea-induced DNA damage is associated with 6-thioguanine resistance in CHO cells

Clones (13 and B) of O6-methylguanine-DNA-methyl-transferase-proficient (MT+) CHO cells showing different levels of resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) but similar MT activity, were found to be sensitive to methyl methanesulphonate and resistant to N-methyl-N-nitrosourea (MNU). A 2.8-fold increase in resistance to MNU-induced cytotoxicity was observed in clone 13 and a 16-fold increase in clone B. A slight increase in survival (1.5-fold) after N-ethyl-N-nitrosourea treatment was observed in clone B. These data indicate that the resistant phenotype is specific for agents that preferentially methylate O atoms in DNA. The survival of MNNG- and MNU-resistant clones as well as of the parental CHO cell line was analysed after exposure to purine analogues substituted in different positions, 8-azaguanine (8-AG), 8-azaadenine (8-AA) and 6-thioguanine (6-TG). A 6-fold increase in resistance to 6-TG was found in clone B, although the hypoxanthine guanine phosphoribosyltransferase gene is functional in these cells. The same cytotoxicity was found in all the lines after treatment with 8-AG and 8-AA. These data are in agreement with the previous observation that clone 13 and clone B belong to two different classes of resistance, clone 13 resistance being explained by MT levels. The finding that clone B is cross-resistant to 6-TG is discussed in the light of a mechanism of tolerance to modifications at specific positions of guanine.     

DNA damage is able to induce senescence in tumor cells in vitro and in vivo

Often the use of cytotoxic drugs in cancer therapy results in stable disease rather than regression of the tumor, and this is typically seen as a failure of treatment. We now show that DNA damage is able to induce senescence in tumor cells expressing wild-type p53. We also show that cytotoxics are capable of inducing senescence in tumor tissue in vivo. Our results suggest that p53 and p21 play a central role in the onset of senescence, whereas p16(INK4a) function may be involved in maintaining senescence. Thus, like apoptosis, senescence appears to be a p53-induced cellular response to DNA damage and an important factor in determining treatment outcome.     

DNA replication in Syrian hamster cells transiently exposed to hydroxyurea

DNA rereplication has been observed in mammalian cells transiently treated during S phase with hydroxyurea, an inhibitor of DNA synthesis. Recently, evidence has been presented which does not support this proposal. We have performed a series of similar but more defined studies with the tumorigenic Syrian hamster cell line BP6T to further investigate the possibility of DNA rereplication after transient inhibition during S phase. Synchronized BP6T cells (greater than 75%) were given a 6-h exposure to 1 mM hydroxyurea after having progressed 2 h into S phase. The DNA species synthesized up to 24 h after removal of the inhibitor have been identified by bromodeoxyuridine pulse labeling and density gradient centrifugation studies. The results indicate that no DNA rereplication had occurred in the S phase of the same cell cycle as the transient hydroxyurea treatment. DNA replicated early in the S phase in which treatment was given was not replicated again until the next cell cycle. Our observations reveal that DNA rereplication does not occur in tumorigenic Syrian hamster cells transiently insulted with hydroxyurea during S phase. Thus, these findings imply that DNA synthesis is stringently controlled in these transformed cells.     

Cell cycle regulation of the endogenous wild type Bloom's syndrome DNA helicase

Bloom's syndrome (BS) is a rare human autosomal recessive disorder characterized by an increased risk to develop cancer of all types. BS cells are characterized by a generalized genetic instability including a high level of sister chromatid exchanges. BS arises through mutations in both alleles of the BLM gene which encodes a 3' - 5' DNA helicase identified as a member of the RecQ family. We developed polyclonal antibodies specific for the NH2- and COOH-terminal region of BLM. Using these antibodies, we analysed BLM expression during the cell cycle and showed that the BLM protein accumulates to high levels in S phase, persists in G2/M and sharply declines in G1, strongly suggestive of degradation during mitosis. The BLM protein is subject to post-translational modifications in mitosis, as revealed by slow migrating forms of BLM found in both demecolcine-treated cells and in mitotic cells isolated from non-treated asynchronous populations. Phosphatase treatment indicated that phosphorylation events were solely responsible for the appearance of the retarded moieties, a possible signal for subsequent degradation. Together, these results are consistent with a role of BLM in a replicative (S phase) and/or post-replicative (G2 phase) process. Oncogene (2000).     

Increased error-prone non homologous DNA end-joining--a proposed mechanism of chromosomal instability in Bloom's syndrome

BS is an inherited cancer predisposition disorder caused by inactivation of the RecQ family helicase, BLM. One of the defining features of cells from BS individuals is chromosomal instability, characterized by elevated sister chromatid exchanges (SCEs), as well as chromosomal breaks, deletions, and rearrangements. Although the basis for chromosomal instability is poorly understood, there is evidence that chromosomal abnormalities can arise through an alteration in the efficiency or fidelity of DNA double strand break (DSB) repair. Here, we show that BS cells demonstrate aberrant DSB repair mediated by the non-homologous end-joining (NHEJ) pathway for DNA repair, one of the two main pathways for the repair of DSBs in mammalian cells. Through a comparison of BS cell lines, and a derivative in which the BS phenotype has been reverted by expression of the BLM cDNA, we show that BS cells display aberrant end-joining of DSBs. Importantly, DNA end-joining in BS cells is highly error-prone and frequently results in DNA ligation at distant sites of microhomology, creating large DNA deletions. This aberrant repair is dependent upon the presence of the Ku70/86 heterodimer, a key component in the NHEJ pathway. We propose that aberrant NHEJ is a candidate mechanism for the generation of chromosomal instability in BS.     

Oxidative DNA damage in bone marrow cells of patients with low-risk myelodysplastic syndrome

Bone marrow aspirates of 19 patients with low-risk myelodysplastic syndromes (MDS) and 14 control subjects were collected in order to assess the level of oxidative DNA damage. Glycophorin A positive and negative cells separated by miniMACS magnetic cell sorting were subjected to single cell gel electrophoresis (comet assay) combined with enzymes of base excision repair (endonuclease III and formamido-pyrimidine-glycosylase) that specifically recognize oxidized nucleotides. Compared to controls, MDS patients exhibited a significant increase of oxidative damage to DNA which could contribute to genomic instability and disease progression.     

53BP1 is associated with replication protein A and is required for RPA2 hyperphosphorylation following DNA damage

p53-binding protein 1 (53BP1) acts as an 'adaptor/mediator' for transducing DNA damage signals, especially following detection of DNA double-strand breaks. In an effort to broaden our understanding of the protein network surrounding 53BP1, we isolated possible 53BP1 binding partners by co-immunoprecipitation, and identified them via tandem mass spectrometric analysis. The 53BP1-associated proteins included RPA1 and RPA2, two components of the replication protein A (RPA) complex. The presence of RPA components in the immunoprecipitates was confirmed by immunoblotting, and we found that the association between 53BP1 and RPA2 was disrupted following DNA damage induced by treatment with camptothecin, a topoisomerase I inhibitor. To investigate the functional meaning of the 53BP1 and RPA interaction, we established U2OS osteosarcoma cell lines stably expressing dominant-negative fragments of 53BP1. We found that camptothecin-induced RPA2 phosphorylation was inhibited in these cells, and also following 53BP1 knockdown by siRNA transfection. On the cellular level, camptothecin-induced apoptosis was augmented in the dominant-negative cell lines, resulting in increased chemosensitivity to this drug. Taken together, these results suggest that 53BP1 is involved in DNA damage-induced RPA2 hyperphosphorylation, and inhibition of 53BP1 function may sensitize cancer cells to camptothecin treatment.     

Differential expression of p53, p21waf1/cip1 and hdm2 dependent on DNA damage in Bloom's syndrome fibroblasts

The Bloom's syndrome gene, BLM, encodes a protein which bears homology to the RecQ helicases. It is believed to be involved in DNA replication and has been implicated in the maintenance of genomic stability. To investigate whether BLM was involved in cellular responses to DNA damage Bloom's syndrome fibroblasts were treated with either UV or ionizing radiation and the levels of p53 and two of its down stream effectors, p21waf1/cip1 and hdm2, were determined by western blot analysis. Following 20 J/m2 UVC-radiation we observed that the maximal accumulation of p21waf1/cip1 and hdm2 proteins preceded that of p53 in both a normal diploid fibroblast cell strain (GM0038) and in two Bloom's syndrome cell strains. Furthermore, the Bloom's syndrome cells demonstrated a delayed and prolonged accumulation of all three proteins and a delayed recovery of the protein levels back to pre-damage levels compared with the normal cell strain. Conversely, normal and Bloom's syndrome cell response following 2.5 Gy of ionizing radiation was quite similar for p21waf1/cip1 and hdm2, but differed significantly for p53. Maximum accumulation of p53 occurred within 2 h of damage and preceded that of p21waf1/cip1 and hdm2. These results suggest that the BLM protein may play a role in the detection of certain types of DNA damage and in the cellular response to that damage.      

Delayed DNA maturation, a possible cause of the elevated sister-chromatid exchange in Bloom's syndrome

Differences in behaviour between the 5-bromodeoxyuridine (BrdU)-substitutedtemplate strands in Bloom's syndrome (BS) and normal human fibroblastshave been investigated in order to elucidate the mechanism responsiblefor the elevated baseline sister-chromatid exchange (SCE) frequencyin BS. Alkaline sucrose gradient analysis of the normal andBrdU-substituted DNA strands showed the former to be of highermol. wt. and of mature size while the latter were of lower molecularsize, resulting from breaks introduced during the repair ofthe BrdU with no differences discernible between BS and normalcells. The rates of removal of BrdU were similar in BS and normalcells, which indicates that the increased SCE level in BS isnot due to different rates of repair of the BrdU. The maturationof newly synthesized DNA on a normal template is delayed inBS cells compared with normal cells although it is completeat 18 h, the time it is acting as a template for DNA synthesis.In the presence of a BrdU-substituted template the maturationalthough further delayed is complete in normal cells by 12 hbut in BS cells is not complete even by 30 h, when the newlysynthesized strand, due to cell cycle delay produced by theincorporation of BrdU, becomes a template in the next roundof DNA synthesis. It is suggested that a similar delay in maturationprobably occurs when a new strand containing BrdU is synthesizedon a normal template in BS cells. When these strands act asa template they will contain two types of breaks — thosedue to BrdU repair and those due to delayed maturation. Thelatter will be responsible for the elevated SCEs in BS cellsas the DNA replication forks move through them in a manner similarto that previously reported. The possible implications of differentialdelays in cell proliferation in BrdU, rates of BrdU removaland extent of DNA maturation in this syndrome are discussed.     

Mitoxantrone-induced DNA damage in leukemia cells is enhanced by treatment with high-dose arabinosylcytosine

Summary In a phase II study, patients with chronic myelogenous leukemia in blast crisis (CML-BC) were treated with intravenous (IV) mitoxantrone (5 mg/m²) per day given over 30 min x 5 days and high-dose arabinosylcytosine (ara-C) (3 g/m² IV q 12 h x 6).The effect of this treatment on DNA damage was studied in the leukemia cells of four patients using the alkaline elution technique modified to measure DNA in unlabeled human cells. A fluorescence assay using Hoechst 33258 dye was applied for the determination of eluted DNA.After a single infusion of mitoxantrone, neither frank nor protein-associated single-strand breaks (SSB) were observed.Even repeated treatment with mitoxantrone on 3 consecutive days did not induce significant SSB. However, after the combined sequential infusion of ara-C and mitoxantrone the DNA elution pattern changed, showing significant DNA damage.SSB remained apparent after 24 h and increased with subsequent doses of ara-C and mitoxantrone. Studies of other patients treated with ara-C alone did not reveal significant SSB (n=5).Following mitoxantrone infusion the median peak concentrations of intracellular ara-CTP (the triphosphate of ara-C) exceeded 900 M,a value greater than that observed in CML-BC patients receiving ara-C alone (230 M, n=15, P<0.02). The present study shows the applicability of the alkaline elution method for the assay of DNA damage in vivo. The enhanced DNA damage after combined treatment with mitoxantrone and high-dose ara-C suggests a synergistic drug effect.Abbreviations ara-C 1--D-arabinosylcytosine - ara-CTP the triphosphate of ara-C - SSB single-strand breaks - SSF strand scission factor - PK proteinase K - CML chronic myelogenous leukemia - BC blast crisis - AML acute myelogenous leukemia Supported by grants CA28596, CA32839, and CA23270 from the National Cancer Institute, Department of Health and Human Services