Bloom Syndrome Patients Research Articles

DNA topoisomerases and the DNA lesion in human genetic instability syndromes

Genetically determined chromosomal instability entails, among other sequelae, a condition of elevated cancer risk. Patients with the autosomal recessive disorder Fanconi's anemia (FA) often develop leukemias of the monocytic lineage together with pancytopenia, whereas the Bloom's syndrome (BS) mutation confers an early and elevated incidence of neoplasia of no particular type. Cultured cells from FA patients show spontaneously elevated rates of chromosome aberrations and a hypersensitivity to DNA cross-linking agents. Cytogenetic evaluation of cells from BS patients revealed elevated rates of sister chromatid exchanges, which were sensitive to the bromodeoxyuridine (BrdU) concentration used in the assay. Such a BrdU sensitivity was also found in cultured cells from healthy subjects exposed to the intracellular superoxide generator paraquat or to bleomycin. Skin fibroblasts from FA and BS patients show poor growth, which in the case of FA could be mitigated by lowering the oxygen concentration to 5%. Lymphoblastoid B-cell lines derived from peripheral blood samples from FA and BS patients show elevated numbers of cells arrested in the G2 phase of the cell cycle. This phenomenon could also be provoked by exposing cell lines from healthy subjects to compounds interfering with the function of DNA topoisomerase I (camptothecin) or II ( m-AMSA). To test for a putative deficiency of either DNA topoisomerase, B-cell cultures from FA and BS patients were compared with cell cultures from healthy subjects regarding their sensitivity towards camptothecin and m-AMSA. No difference in sensitivity to these agents was found in patient vs. control cell lines, thus ruling out a deficiency in DNA topoisomerase I or II as the prime defect in these conditions of elevated cancer risk. The similarity between the cell cycle kinetic patterns found in untreated FA cell lines and in normal cell lines exposed to camptothecin or m-AMSA suggest that the DNA lesion in FA, presumably being caused by an oxygen-related mechanism, may interfere with DNA topoisomerase function.

Immunological alteration of the Bloom's syndrome uracil DNA glycosylase in Epstein-Barr virus-transformed human lymphoblastoid cells

The immunological reactivity of the uracil DNA glycosylase was investigated in three Epstein-Barr virus-transformed human lymphoblastoid cell lines. Two were derived from normal human lymphocytes while the third was derived from a Bloom's syndrome patient. A panel of 3 anti-human placental uracil DNA glycosylase monoclonal antibodies (37.04.12, 40.10.09 and 42.08.07) was used. Immunological reactivity was determined in a double-blind enzyme-linked immunosorbent assay (ELISA); by inhibition of enzyme activity; and by immunoblot analysis. In the ELISA, the glycosylase from each lymphoblastoid cell line was recognized by glycosylase antibodies 37.04.12 and 42.18.07. In contrast, antibody 40.10.09 failed to recognize the glycosylase from the Bloom's syndrome cell line. Further analysis demonstrated that the 40.10.09 antibody was unable to inhibit catalysis by the Bloom's syndrome lymphoblast glycosylase. In contrast, the 40.10.09 antibody inhibited the activity of the two normal human lymphoblast enzymes. Denaturation of the Bloom's syndrome lymphoblast glycosylase rendered that protein immunoreactive with the 40.10.09 antibody. These results demonstrated that: (1) the immunological alteration in the Bloom's syndrome uracil DNA glycosylase was detected in hematopoietic cells; and (2) viral transformation did not affect the immunoreactivity of the enzyme from either normal human or Bloom's syndrome cells.

Frequency of variant erythrocytes at the glycophorin-A locus in two Bloom's syndrome patients

Blood type MN is determined by a glycoprotein termed glycophorin A (GPA) which exists on the surface of erythrocytes, and the difference between the M and N types is derived from the presence of 2 different amino acids in the amino-terminal portion (Furthmayer, 1978). Using a pair of fluorescence-labeled monoclonal antibodies specific to each GPA, somatic mutations in erythrocytes of MN heterozygotes at the GPA-M and -N alleles can be quantitatively determined using a flow sorter (Langlois et al., 1986). Our results for 2 Bloom's syndrome (BS) patients showed that variants either lost expression of one allele (simple gene inactivation or loss) or expressed only one allele at twice the normal level (most probably somatic recombination) occurring at a frequency of about 1–3 per 10 3 erythrocytes. The flow cytometric patterns of erythrocytes from the BS patients showed a typical smear of variants bearing intermediate levels of expression of one GPA allele, indicating that the real variant frequency is even greater than that measured. On the other hand, the parents heterozygous for the BS gene showed variant frequencies (1–8 × 10 −5) within the normal range. These data strongly support the hypothesis that the cancer proneness of BS patients is due to their increased frequency of spontaneous mutations and somatic recombination.

DNA ligase activity in human cell lines from normal donors and Bloom's syndrome patients.

DNA ligase activity was studied in several untransformed or virus-transformed human cell lines from normal donors and from Bloom's syndrome (BS) patients. This proneness genetic disease is characterized by several cytological abnormalities and cancer proneness and, recently, some transformed cell lines from these patients were described to present a reduced activity of DNA ligase I. Results presented in this work indicate that: (i) the total DNA ligase activity in crude extract from untransformed or transformed cell lines from several BS patients was significantly higher than in control cells; (ii) the partial purification of the enzyme after gel filtration on fast protein liquid chromatography of crude extracts from lymphoblastoid BS cells showed that the enzyme activity was eluted in a major 180 kDa form in which activity was higher than in control cells; (iii) the activity gel analysis of these enzyme fractions revealed that DNA ligase of human cells was correlated to a major 130 kDa polypeptide and, in BS cells, the extent of the activity of this band was equal or higher than that in control untransformed or transformed cells.

Concomitant reversion of the characteristic phenotypic properties of a cell line of Bloom's syndrome origin.

The fibroblast strain GM3498 and the lymphoblastoid line GM4408 have been established from the same Bloom's syndrome patient, as confirmed by DNA hybridization with hypervariable probes and isoenzyme analysis. Strain GM3498, but not GM4408, exhibits a high frequency of spontaneous sister chromatid exchange, hypersensitivity to ethyl methanesulphonate and an anomalously low level of DNA ligase I activity. We conclude that GM3498 is representative of Bloom's syndrome, whereas GM4408 is a revertant cell line. These data agree with a model in which a malfunctioning DNA ligase I is the primary cause of the syndrome.

Effects of cell fusion and deoxynucleosides on sister-chromatid exchanges in B-lymphoblastoid cell lines from 5 Bloom syndrome patients

The effect of cell fusion and deoxynucleosides (deoxyadenosine, dA; deoxyguanosine, dG; deoxycytidine, dC; thymidine, T) on sister-chromatid exchanges (SCEs) in Bloom syndrome (BS) was studied in two types of BrdU (bromodeoxyuridine)-sensitive and BrdU-resistant B-lymphoblastoid cell lines (LCLs) with respect to cellular proliferation in BrdU-labeled culture conditions. Cell fusion between BrdU-sensitive and BrdU-resistant BS B-LCLs did not exhibit complementation, although when any of the BS B-LCLs (retaining high SCE character) labeled with BrdU were fused with non-labeled normal cells, the hybrid cells had a normal level of SCE at the first mitosis after fusion. Deoxycytidine addition showed no effect on SCEs in normal cells but decreased SCEs in BS cells from the baseline level of 70 SCEs/cell to about 60 SCE/cell. Purine deoxyribonucleosides (dG and dA) caused a significant concentration-dependent increase in SCE frequency both in normal and BS cells. Although T caused a 2-fold increase in normal SCEs, it highly decreased BS SCE from 70 SCEs/cell to 35 SCEs/cell. FrdU did not greatly affect BS SCE in the presence of BrdU and T. These observations indicate strongly that BS cells may have a low thymidine pool compared with normal cells, which could account for a more efficient BrdU substitution in the DNA thus potentiating the template effect on SCE.

Effects of cell fusion and deoxynucleosides on sister-chromatid exchanges in B-lymphoblastoid cell lines from 5 Bloom syndrome patients

The effect of cell fusion and deoxynucleosides (deoxyadenosine, dA; deoxyguanosine, dG; deoxycytidine, dC; thymidine, T) on sister-chromatid exchanges (SCEs) in Bloom syndrome (BS) was studied in two types of BrdU (bromodeoxyuridine)-sensitive and BrdU-resistant B-lymphoblastoid cell lines (LCLs) with respect to cellular proliferation in BrdU-labeled culture conditions. Cell fusion between BrdU-sensitive and BrdU-resistant BS B-LCLs did not exhibit complementation, although when any of the BS B-LCLs (retaining high SCE character) labeled with BrdU were fused with non-labeled normal cells, the hybrid cells had a normal level of SCE at the first mitosis after fusion. Deoxycytidine addition showed no effect on SCEs in normal cells but decreased SCEs in BS cells from the baseline level of 70 SCEs/cell to about 60 SCE/cell. Purine deoxyribonucleosides (dG and dA) caused a significant concentration-dependent increase in SCE frequency both in normal and BS cells. Although T caused a 2-fold increase in normal SCEs, it highly decreased BS SCE from 70 SCEs/cell to 35 SCEs/cell. FrdU did not greatly affect BS SCE in the presence of BrdU and T. These observations indicate strongly that BS cells may have a low thymidine pool compared with normal cells, which could account for a more efficient BrdU substitution in the DNA thus potentiating the template effect on SCE.

Sister-chromatid exchanges induced by ultraviolet light in Bloom's syndrome fibroblasts

The relationships between the cytotoxic effect of ultraviolet light and the UV-induced sister-chromatid exchanges (SCEs) were compared among fibroblast cell strains from two unrelated Bloom's syndrome (BS) patients, one xeroderma pigmentosum (XP) patient belonging to complementation group A and two unrelated normal controls. The “net” induced SCEs as a function of UV fluence, obtained by subtracting spontaneous SCEs from observed SCEs, were much higher in both BS cells and XP group A cells than in normal cells. The relative efficiency of induced SCE, defined as the “net” induced SCEs as a function of surviving fraction after UV irradiation, was higher in BS cells than in normal and XP cells, and there was essentially no difference between XP and normal cells. These results imply that in addition to the extremely high frequency of spontaneous SCEs, the increased efficiency in UV induction of SCEs may reflect the intrinsic defect(s) in BS cells.

Radiation sensitivity of bloom's syndrome lymphocytes during S and G 2 phases

In order to study chromosome sensitivity of Bloom's syndrome (BS) cells in relation to the replication stage, γ-ray irradiation was performed immediately before adding bromodeoxyuridine (BrdU) to lymphocyte cultures of one BS patient and of one control. It was found that BS cells are much more sensitive to the irradiation than control cells at the end of S and at G 2 phases. The rate of induction of chromosome breaks is significantly increased and that of chromatid breaks and exchanges is also increased, though to a lesser degree. Our results also favor the existence of a cell subpopulation in BS characterized by a slow cycle, a high spontaneous chromosome aberration rate, and a high radiation sensitivity.

Background allelic variants in normal hemopoietic cells and Bloom's syndrome erythrocytes and the possible implication of somatic crossingover

The existence of rare cells with blood group A or B phenotype among the red cells of AB heterozygotes is a well-known phenomenon. However, its origin remains unclear due to methodological problems. A direct quantitation of non-B and non-A erythrocytes in A 1B donors revealed minor populations of only-A and only-B cells, respectively, both in a frequency of 10 −3. Null cells comprise, at the most, a fraction of about 5 × 10 5. In order to discriminate between somatic crossingover (SCO) and gene inactivation as the underlying mechanism, three individuals were selected who were double heterozygotes for the blood group (AB) and the linked locus of adenylate kinase (AK-2-1). Separation of cells with A or B phenotypes did not result in cosegregation of the AK isoenzymes. Thus, the variant blood group phenotypes represent the normal frequency of allelic silence, not the product of SCO. This sets a background variant level for the estimation of somatic recombination in blood cells from normal donors. Determination of variant phenotypes in a Bloom's syndrome patient, heterozygous for AB, gave a frequency six times higher than the value of normals. It is suggested that this elevated figure might indicate the frequency of SCO, which is known to be higher in Bloom's syndrome.

Value and significance of SCE in human leukemia and cancer.

The introduction and refinement of sister chromatid exchange (SCE) techniques generated much enthusiasm, interest, and promise for their application in human neoplasia. In particular, the demonstration of high SCE levels in the cells of Bloom syndrome (BS) patients (1,2), a condition with a very high susceptibility to the development of cancer (primarily lymphoma or leukemia), and the application of SCE as a sensitive and specific index of carcinogenic and/or mutagenic activity (3,4), generated much excitement for the application of SCE studies in human neoplasia. It was hoped that unique changes, akin to those at the chromosome level, would characterize certain malignant conditions and states (5–7). Thus, the general concept was developed that individuals or families with high risk of cancer might show SCE changes characteristic of these groups and that cells involved in certain neoplastic conditions would display unique SCE patterns different from those of normal cells and allow for differential delineation of some neoplastic processes.

Effect of poly(ADP-ribose)polymerase inhibitors on the frequency of sister-chromatid exchanges in Bloom syndrome cells

The effect of inhibitors of poly(ADP-ribose)polymerase, benzamide (Bam) and m-aminobenzamide (m-AB), on sister-chromatid exchanges (SCEs) and cell growth, was examined in lymphoblastoid cell lines from a normal adult (KS-64) and from a Bloom syndrome patient (BS 1–2). The presence of Bam and m-AB increased the levels of SCEs in KS-64 and BS 1–2 lymphoblastoid cells. Though the net increase was similar in the two types of cell, the relative increase was much lower in the BS 1–2 cells. Bam and m-AB increased the number of SCEs in BS 1–2 cells to levels of 95.4 ± 3.24 and 98.1 ± 3.23 per cell, respectively, as compared with the baseline level of 75.5 ± 2.16. On the other hand, when KS-64 cells were treated with Bam and m-AB, the number of SCEs increased to 27.1 ± 1.98 and 28.6 ± 2.71 per cell, respectively, compared with the baseline number of 6.7 ± 0.41 per cell. These inhibitors of poly(ADP-ribose)polymerase also inhibited cell growth at concentrations which induced SCEs in KS-64 as well as in BS 1–2 cells. No significant decrease in the poly(ADP-ribose)polymerase activity or in the amount of poly-(ADP-ribose) was detected in BS 1–2 cells as compared with KS-64 cells. The mechanism by which SCEs are increased in BS 1–2 cells is discussed.

Establishment of B-lymphoid cell lines retaining cytogenetic characteristics of Bloom syndrome

The present study describes the establishment of and chromosomal changes in B-lymphoid cell lines from cells of Bloom syndrome (BS) patients using Epstein-Barr virus (EBV). Even though PHA-stimulated BS lymphocytes from all five patients studied showed high levels of sister chromatid exchange (SCE), three EBV-transformed BS-B-lymphoid cell lines had normal levels of SCE and two yielded two types of cell populations, i.e., one with increased SCE and chromosome instability (including breaks and quadriradials) and another with normal levels of SCE and without structural aberrations. The karyotypic abnormalities, as observed in the BS lines have not been seen in the cells of any established normal B-lymphoid lines transformed by EBV and strongly suggest that the chromosome abnormalities in the BS-B-cell lines with abnormal karyotypes originated in vivo and not through an in vitro effect of EBV. Furthermore, in the EBV-transformed B-cell lines, we found quadriradial formation between sister chromosomes during endomitoses instead of between homologous chromosomes, strongly suggesting that quadriradial formation may be closely related to SCE. The coexistence in BS subjects of abnormal and normal populations of cells with respect to the number of SCE awaits explanation.

Transformation of Bloom syndrome T-lymphocytes by cocultivation with a lethally irradiated human T-cell line carrying type C virus particles.

The present study describes the establishment of T-cell lines from the peripheral blood of two Bloom syndrome (BS) patients and one healthy female by co-cultivation with a lethally irradiated human T-cell line (MT-2) carrying adult T-cell leukemia (ATL) virus (ATLV). The cell lines from normal and BS subjects exhibited cell surface markers compatible with T-cell origin, and BS T-cell lines retained the original cytogenetic characteristics of the syndrome. Even though phytohemagglutinin-stimulated BS lymphocytes from the two BS patients studied all showed high levels of sister chromatid exchange (SCE), one of the BS T-lines retained the high SCE level in 100% of the cells and the other BS T-line contained two populations, one with high SCE (70%) and the other with normal SCE (30%), at a relatively constant frequency over 6 months. Transformation of normal and BS cells by cocultivation with MT-2, which carries ATLV, did not cause karyotypic changes over 6 months. ATLV infection, chromosome instability, karyotypic abnormality and SCE in BS T-lymphocytes are also discussed.

Chromosome breakage factor in the plasma of two Bloom's syndrome patients.

Chromosome breakage factor in the plasma of two Bloom's syndrome patients.

Normalization by cell fusion of sister chromatid exchange in Bloom syndrome lymphocytes.

Fusion of fresh lymphocytes from a Bloom syndrome (BS) patient with those of normal subjects or a BS heterozygote resulted in complete normalization of the frequency of sister chromatid exchanges in the chromosomes of BS cells. This normalization took place by the first mitosis in hybrid cells. In contrast, cultivation of BS lymphocytes with those of normal subjects or the BS heterozygote had no effect on sister chromatid exchanges. The cell fusion experiments suggest that the normalization on the sister chromatid exchanges. The cell fusion experiments suggest that the normalization of the sister chromatid exchange frequencies in BS cells can be achieved by factors conserved in the cells of various mammalian species. These findings are compatible with the concept that BS is a recessive genetic mutation at regulatory levels of the DNA repair function.

Near-ultraviolet sensitivity of skin fibroblasts of patients with bloom's syndrome

Near-ultraviolet survival of colony forming ability was compared between fibroblast strains from normal individuals and seven strains from Bloom's Syndrome patients using monochromatic light at 313 nm. Six BS strains possessed abnormal survival properties. Two strains lacked the low dose shoulder, which is characteristic for normal fibroblasts, but their overall sensitivity was in the normal range. Four BS strains were hypersensitive and possessed biphasic survival curves with a sharp initial drop. They mimick in culture the characteristic solar sensitivity of BS patients.

Mitotic crossing-over and segregation in man.

Although clear genetic evidence of mitotic crossing-over is lacking in man, observations of mitotic chiasmata in normal cells (0.1–1 per 1000) and in Bloom's syndrome (BS) cells (5–150 per 1000) demonstrate its occurrence. That mitotic chiasmata are true exchanges is concluded from the occurrence of heteromorphic “bivalents” and the pattern of sister chromatid exchanges in mitotic “bivalents”. Several observations demonstrate that chiasmata are different in principle from chromatid translocations which simply happen to take place at homologous loci. For example, the ratio of adjacent exchanges to mitotic chiasmata is 1/20–1/60, whereas this ratio is approximately 1:1 for chromatid translocations. Furthermore, mitotic chiasmata make up a very high proportion of total quadriradials (QRs): 48% in normal untreated cells and 90% in BS cells. Close proximity of homologous chromosomes promotes mitotic crossing-over. Thus in normal diplochromosomes, the incidence is increased a hundred-fold as compared to diploid cells. However, closeness of homologues is not the only factor promoting crossing-over; the BS gene specifically promotes exchanges between homologous segments as shown by the roughly 15-fold increase of chiasmata in BS diplochromosomes as compared to normal diplochromosomes. Mitotic chiasmata are distributed extremely nonrandomly in different chromosomes and chromosome segments. The preferred sites are short Q-dark regions, 3p21, 6p21, 11q13, 12q13, 17q12, and 19p13 or q13 being veritable hot spots. Our preferred hypothesis is that the hot spots have higher gene densities than other regions. Consequently they are active and extended in interphase which would promote their pairing and chiasma formation. Segregation after mitotic corssing-over in satellite stalks can be demonstrated by means of distinct satellites. In a BS patient there were 31 different patterns for Q-bright satellites in 58 cells. Segregation after presumed crossing-over has also been seen in three dicentric chromosomes with one centromere inactivated. Recombination in satellite stalks in BS resulted in 12/58 cells homozygous for Q-bright satellites. In two of these cells, two chromosomes were homozygous for Q-bright satellites, and in one cell, three chromosomes were homozygous. This high degree of homozygosity which obviously applies to other chromosome regions too, may explain the high incidence of malignant disease in BS on the assumption that cancer is caused by recessive genes.

Diphtheria toxin resistance in human fibroblast cell strains from normal and cancer-prone individuals

Mutants resistant to diphtheria toxin (Dip r) have been selected from a variety of human fibroblast cell strains derived from both normal subjects and individuals with known genetic predisposition to cancer such as xeroderma pigmentosum, Fanconi anemia and Bloom's syndrome. Treatment with N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) led to a marked increase in the frequency of Dip r mutants in various cell strains. The increase in the frequency of Dip r mutants occurred in a linear dose-dependent manner in response to MNNG and ethyl methanesulfonate, in one of the cell strains examined. The rate of muation to diphtheria toxin as determined by fluctuation analysis was very similar in various cell strains (1–3 × 10 −7 mutations/cell/generation), except for the strain GM1492 (8.8 × 10 −7 mutations/cell/generation) which is derived from a Bloom syndrome patient.

Effects of X-irradiation in G1 and G2 on Bloom's syndrome and normal chromosomes

The X-ray sensitivity of chromosomes from a Bloom's syndrome patient and a normal control was compared in G1 and G2. There was no significant difference in the number of aberrations induced by irradiation in G1. An increased sensitivity of the BS chromosomes was found in G2. The frequency of mitotic chiasmata in the BS cells was not increased by the G2-irradiation, even though the frequency of chromatid translocations was much increased. This provides further evidence for the fundamental difference of these two phenomena. Evidence from the types of aberrations induced suggests that there is no appreciably increased frequency of pairing of homologous chromosomes in the BS cells over that in normal cells.