Relationship between group G chromosomes, especially chromosome 21, and the sensitivity of human cells to Coxsackie B viruses]

The R-method of differential chromosome staining by length was applied to comparative karyological studies on the culture of J 96 human cells susceptible to enteroviruses, and on the J 41 cell line derived from this culture and possessing high specific resistance to Coxsackie B viruses. Karyotype of the J 41 cell line was shown to be deprived of chromosome G21 (P less than 0.0001). The number of other chromosomes varied from cell to cell, but they are constantly present in the majority of cells of both the J 96 and J 41 cell lines. A conclusion is drawn that chromosome G21 incorporates gene(s) which controls the human cells susceptibility to Coxsackie B viruses.     

Clastogenic effects of bleomycin, cyclophosphamide, and ethyl methanesulfonate on resting and proliferating human B- and T-lymphocytes

The effects of bleomycin (BM), cyclophosphamide (CP), and ethyl methanesulfonate (EMS) on the frequencies of chromosomal aberrations were tested in mitogen-stimulated highly purified human B- and T-lymphocytes. In unstimulated G0/G1 B- and T-lymphocytes the clastogen induction of chromosome fragments was investigated in prematurely condensed chromosomes (PCC) induced by cell fusion with xenogenic mitotic cells. BM, CP (with metabolic activation), and EMS induced a significant increase in chromosome aberrations in proliferating human B- and T-lymphocytes. There were no significant differences in the BM-induced aberration rates between the cell populations. CP and EMS induced more aberrations in T- than in B-lymphocytes. In the PCC tests, BM-exposed G0/G1 lymphocytes showed dose-dependent high yields of chromosome fragments. No significant differences between B- and T-lymphocytes were observed. CP and EMS induced no clear increase in fragments in either cell population.     

Micronuclei frequency induced by bleomycin in human peripheral lymphocytes: correlating BLHX polymorphism with mutagen sensitivity

Mutagen sensitivity assay, by measuring chromosome damage induced by an in vitro treatment of peripheral lymphocytes with bleomycin, has been proposed as a biomarker for assessing cancer susceptibility. Recently, a single nucleotide polymorphism (SNP A1450G) of the gene for bleomycin hydrolase (BLHX), a specific neutral cysteine protease able to metabolise bleomycin, was proposed as a plausible candidate to variation in mutagen sensitivity. To shed more light on the effect of BLHX genotype on the expression of chromosome damage induced in vitro by bleomycin, we determined mutagen sensitivity for 45 non-smoker healthy volunteers. The level of bleomycin-induced chromosome damage was assessed as frequencies of micronuclei (MN) in cytokinesis-blocked lymphocytes. The subjects were genotyped for the BLHX gene, to determine the possible effect of this polymorphism on mutagen sensitivity. No difference in the spontaneous value of MN was detected between the homozygotes wild-type (A/A) and the carriers of variant alleles A/G heterozygotes or G/G homozygotes (MN/1000 binucleated (BN) cells: 6.69+/-2.53 and 6.37+/-4.87, respectively). A substantial effect of BLHX polymorphism in predetermining individual mutagen sensitivity status was observed: subjects with the BLHX A/A genotype displayed significantly lower mean levels of bleomycin-induced MN frequency than the carriers of A/G or G/G variant alleles combined (12.00+/-3.76 MN/1000 BN vs. 16.37+/-8.86 MN/1000 BN, respectively; P=0.029). The multiple regression analysis, including BLHX genotype and age, confirmed the significant effect of BLHX variant alleles (A/G, G/G) on the chromosome damage induced by bleomycin (P=0.01), whereas age correlated only with the spontaneous MN frequency.     

The response of normal and ataxia-telangiectasia cells to bleomycin: relationships between chromosome damage, cell cycle delay and cell killing

In agreement with our earlier observation (Scott and Zampetti-Bosseler, 1982) on X-irradiated normal and ataxia-telangiectasia (A-T) fibroblasts, we now report that after bleomycin or neocarzinostatin treatment also, A-T cells exhibit less G2 delay than normal cells. We confirm that A-T cells sustain more chromosome damage and lethality than normal cells after bleomycin. These observations support the hypothesis (Painter and Young, 1980) that A-T cells are defective in the recognition of certain lesions which normally lead to delays in progression through the cell cycle, during which they are repaired, and which, if unrepaired, lead to cell-lethal chromosome damage. However, we find that after bleomycin, as opposed to X-rays, the contribution of this type of lesion to cell death is minimal. The predominant lesions leading to cell death after bleomycin are not manifested at chromosome aberrations and do not lead to G2 delay or DNA-synthesis inhibition. A-T cells are defective in the recognition and/or repair of both types of lesion.     

Synergistic effect of CCNU and bleomycin on human lymphocytes exposed at late G1 and G2 states of the cell cycle.

The combined action of the antitumor antibiotic bleomycin and chloroethylnitrosourea (CCNU) was studied in human lymphocytes in vitro. All the experiments were carried out with 20 micrograms/ml bleomycin for a given treatment time. By adding 0.7 and 3.5 micrograms/ml CCNU at late G1-S phase we have demonstrated a considerable increase in both percent of aberrant cells and production of dicentrics and rings (5-fold, p less than 0.001). At late S-G2 the combined treatment led to a significant enhancement of breaks per cell (p less than 0.0001) and cells with more than 12 aberrations. A possible explanation could be the known repair-inhibitory potential of CCNU, but its pure clastogenic action still has to be considered. The results presented here point out the need for seeking chemotherapeutic regimens with reduced concentrations of the drugs in combination.     

Relationships between chromosome damage, cell cycle delay and cell killing induced by bleomycin or X-rays

The extent of mitotic delay and chromosome aberration induction by X-rays and bleomycin has been compared in normal human foetal fibroblasts at doses giving approximately equal levels of cell killing, assayed as colony-forming ability. Bleomycin induced much less G2 delay and chromosome damage than X-rays. We conclude that the major mechanism of cell killing by bleomycin does not involve chromosome damage but the cells pass through a number of division cycles before dying and a common DNA lesion is involved in G2 delay and chromosome damage.     

Influence of ataxia telangiectasia gene dosage on bleomycin-induced chromosome breakage and inhibition of replication in human lymphoblastoid cell lines

Long-term lymphoblastoid cell lines, obtained by E-B virus transformation of peripheral blood lymphocytes, retain many of the features of hypersensitivity to environmental agents found in primary cultures and fibroblast strains from patients with genetic diseases. Primary lymphocyte cultures from patients with ataxia telangiectasia, a cancer-prone genetic disease, have increased sensitivity to chromosomal damage induced by the radio-mimetic drug, bleomycin. In order to study the expression of ataxia telangiectasia gene dosage in lymphoblastoid cell lines, we examined chromosomal aberrations in lines containing two, one, or no alleles for ataxia telangiectasia. These were derived from ataxia telangiectasia homozygotes, from ataxia telangiectasia obligate heterozygotes, and from presumably normal donors, respectively. Chromosome preparations were made 46 h after a 2 h exposure to bleomycin and scored for chromosome breakage, for the relative rate of cell replication as assessed by sister chromatid differentiation patterns, and for the frequency of sister chromatid exchanges. Baseline frequencies of chromosome breakage and sister chromatid exchanges, and baseline rates of cell replication were similar in all nine lymphoblastoid cell lines. Following treatment with 25 or 250 mU/ml bleomycin, all the lymphoblastoid cell lines showed increased chromosome breakage and decreased cell replication. The lymphoblastoid cell lines from the ataxia telangiectasia homozygotes had significantly increased chromosome breakage and decreased rate of cell replication after either bleomycin dose in comparison with the normal or with the ataxia telangiectasia heterozygous lines. Sister chromatid exchange frequencies were not altered by bleomycin exposure.     

Effects of bleomycin on Down's syndrome lymphocytes in culture

Blood lymphocytes from 3 Down's syndrome (DS) and 3 age- and sex-matched normal probands were studied for the induction of chromosomal aberrations and sister-chromatid exchange (SCEs). Treatment with bleomycin (30 and 60 ng) at the initiation of culture showed a dose-dependent increase in the incidence of dicentric and ring chromosome aberrations. In contrast, the cells which were treated for the last 24 h in culture with bleomycin did not show an increase in chromosome-type aberrations. The proportion of metaphases in M1, M2, and M3 in cultures was not different between DS and normal cells. Sister-chromatid exchange frequency did not show significant changes between DS and normal individuals.     

Responses of Huntington's disease and ataxia telangiectasia lymphoblastoid cells to bleomycin

Ionizing radiation sensitive, mutant human lymphoblastoid cell lines derived from patients with Huntington's disease (HD), or ataxia telangiectasia (AT) both showed cross sensitivity to bleomycin, as assayed by reduced cell viability and increased frequency of chromosome aberrations compared to normal controls. In contrast to AT cells which failed to show inhibition of DNA synthesis after exposure to ionizing radiation, or bleomycin treatment, the sensitive cells from HD patients had depressed rates of DNA synthesis after damage with these agents, similar to that seen in normal cells. In terms of progression through the cell cycle bleomycin damaged AT cells moved from G1 into S and from S to G2 + M at almost the same rate as untreated cells. Bleomycin treated HD cells showed a large proportion of cells blocked in G1, cells were slowed down in S, the rate of entry to G2 + M was reduced and only 5% of cycling cells reached G2. Progress through the cell cycle in normal cells exposed to bleomycin showed a partial block in G1 and the rate of entry to G2 + M was reduced. These differences in response of normal, AT and HD cells to ionizing radiation and bleomycin treatment indicates that the defect underlying the sensitivity is different in HD cells from that in AT cells.     

FISH is more sensitive than Southern analysis at identifying increased levels of cyclin D1 gene amplified in breast cancer cell lines

Cyclin D1 is involved in regulating the transition of G1 to S-phase in the cell cycle through phosphorylation of the retinoblastoma susceptibility product (pRB). Amplification and overexpression of the cyclin D1 gene (CCND1) have been reported in human breast cancers and are suggested to play important roles in the pathogenesis of the disease process. Although cyclin D1 is potentially an important gene, relatively little is known about the distribution of its amplification in breast cancer cell lines. In this study, a cyclin D1 cosmid probe was isolated and used with fluorescence in situ hybridization (FISH) to identify the gene in chromosomal spreads of 12 breast cancer cell lines. Nine cell lines showed increased gene copy levels of cyclin D1, including Five cell lines had more than six copies of cyclin D1 on sister chromatids and four had more than four copies but less than six copies grouped at the chromosome 11 q13 band. Three cell lines had two “normal” chromosome 11 and one and two additional derivative chromosome 11’s with three and four 11q13 sites which lacked amplification of cyclin D1 on any of these sites. Using progesterone receptor (PR) gene as an internal control, a 2.0-fold or greater increase in cyclin D1 gene signals, was observed in five of the ten cell lines by Southern hybridization, the Amplification level of cyclin D1 varied from 2.3 to 19.6-fold. Three cell lines with low amplification of cyclin D1 showed overexpression of the gene by Northern analysis. Our experiments demonstrated that FISH was more sensitive than Southern blot at demonstrating low levels of gene amplification and, additionally, permitted assessment of the distribution of cyclin D1 gene among chromosomes.     

The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. I. Initial damage

We investigated the role of initial DNA and chromosome damage in determining the radiosensitivity difference between the variant murine leukemic lymphoblast cell lines L5178Y-S (sensitive) and L5178Y-R (resistant) and the difference in cell cycle-dependent variations in radiosensitivity of L5178Y-S cells. We measured initial DNA damage (by the neutral filter elution method) and chromosome damage (by the premature chromosome condensation method) and compared them with survival (measured by cloning) for both cell lines synchronized in G1 or G2 phase of the cell cycle (by centrifugal elutriation) and irradiated with low doses of X rays (up to 10 Gy). The initial yield of DNA and chromosome damage in G2 L5178Y-S cells was almost twice that in G1 L5178Y-S cells and G1 or G2 L5178Y-R cells. In all cases DNA damage expressed as relative elution corresponded with chromosome damage (breaks in G1 chromosomes, breaks and gaps in G2 chromosomes). Also we found that the initial DNA and chromosome damage did not determine cell age-dependent radiosensitivity variations in L5178Y-S cells, as there was less initial damage in the more sensitive G1 phase than in the G2 phase. L5178Y-R cells showed only small changes in survival or initial yield of DNA and chromosome damage throughout the cell cycle. Because survival and initial damage in sensitive and resistant cells irradiated in G2 phase correlated, the difference in radiosensitivity between L5178Y-S and L5178Y-R cells might be determined by initial damage in G2 phase only.     

Initial chromosome damage but not DNA damage is greater in ataxia telangiectasia cells.

Cells derived from individuals with ataxia telangiectasia (AT) exhibit increased sensitivity to ionizing radiation and certain drugs (e.g., bleomycin, neocarzinostatin, and etoposide) as evidenced by decreased survival and increased chromosome aberrations at mitosis when compared with normal cell lines. To understand better the basis of this sensitivity, three AT and two normal lymphoblastoid cell lines were fractionated into cell cycle phase-enriched populations by centrifugal elutriation and then examined for their survival and their relative initial levels of DNA damage (neutral DNA filter elution) and chromosome damage (premature chromosome condensation). AT cells exhibited decreased levels of survival in all phases of the cell cycle; however, AT cells in early G1 phase were especially sensitive compared with normal cells in G1 phase. While AT and normal cells exhibited similar levels of initial DNA double-strand breaks in exponential populations as well as throughout the cell cycle, AT cells showed nearly twofold higher initial levels of chromosome damage than normal control cells in G1 and G2 phase. These results suggest that there is a higher rate of conversion of DNA double-strand breaks into chromosome breaks in AT cells, perhaps due to a difference in chromatin organization or stability. Thus one determining component of cellular radiosensitivity might include chromatin structure.     

Premature chromosome condensation in humans associated with microcephaly and mental retardation: a novel autosomal recessive condition

We report a novel autosomal recessive disorder characterized by premature chromosome condensation in the early G2 phase. It was observed in two siblings, from consanguineous parents, affected with microcephaly, growth retardation, and severe mental retardation. Chromosome analysis showed a high frequency of prophase-like cells (>10%) in lymphocytes, fibroblasts, and lymphoblast cell lines with an otherwise normal karyotype. (3)H-thymidine-pulse labeling and autoradiography showed that, 2 h after the pulse, 28%-35% of the prophases were labeled, compared with 9%-11% in healthy control subjects, indicating that the phenomenon is due to premature chromosome condensation. Flow cytometry studies demonstrate that the entire cell cycle is not prolonged, compared with that in healthy control subjects, and compartment sizes did not differ from those in healthy control subjects. No increased reaction of the cells to X-irradiation or treatments with the clastogens bleomycin and mitomycin C was observed, in contrast to results in the cell-cycle mutants ataxia telangiectasia and Fanconi anemia. The rates of sister chromatid exchanges and the mitotic nondisjunction rates were inconspicuous. Premature entry of cells into mitosis suggests that a gene involved in cell-cycle regulation is mutated in these siblings.     

Vitamin C intake influences the bleomycin-induced chromosome damage assay: implications for detection of cancer susceptibility and chromosome breakage syndromes

Supplementation with 1 g of vitamin C (ascorbic acid) per day decreased the amount of chromosome damage induced in lymphocytes by an exposure to bleomycin during the last 5 h of cell culture. We did not see such changes in lymphocytes from control individuals samples at the same time but not taking vitamin C supplements. This bleomycin assay has been proposed as a test for cancer susceptibility. A similar assay for genetic instability may be useful in detecting heterozygotes for chromosome-breakage syndromes (for example, Fanconi anemia or ataxia telangiectasia). Even though our sample size is small and our results should be interpreted cautiously, statistically significant effects were found with vitamin C supplementation. It would, therefore, be prudent to consider dietary and perhaps other lifestyle factors when interpreting of results from this bleomycin assay and related assays for genetic instability.     

Mouse dendritic cells express the P2X7 purinergic receptor: characterization and possible participation in antigen presentation.

Immune cells express P2 purinoceptors of the P2Y and P2X subtypes. In the present work, we show that three dendritic cell (DC) lines, D2SC/1, CB1, and FSDC, representative of immature DCs, express the P2X7 (formerly P2Z) receptor, as judged from RT-PCR amplification, reactivity to a specific antiserum, and pharmacological and functional evidence. Receptor expression is higher in FSDC cells, a cell line that is functionally more mature than D2SC/1 and CB1. From the wild-type DC population, we selected cell clones lacking the P2X7R (P2X7less). We also used a P2XR blocker, oxidized ATP, to irreversibly inhibit the P2X7R. Ability of P2X7less FSDCs or of oxidized ATP-inhibited FSDCs to stimulate Ag-specific TH lymphocytes was severely decreased although Ag endocytosis was minimally affected. During coculture with TH lymphocytes, wild-type FSDC secreted large amounts of IL-1beta. Release of this cytokine was reduced in P2X7less DCs. These data show that DCs express the P2X7 purinoceptor and suggest a correlation between P2X7R expression and Ag-presenting activity.     

B cell lines as models for inherited phagocytic diseases: abnormal superoxide generation in chronic granulomatous disease and giant granules in Chediak-Higashi syndrome

Epstein Barr virus (EBV)-transformed B cell lines (BCL) were developed from peripheral blood lymphocytes of individuals with Chediak-Higashi syndrome (CHS) and chronic granulomatous disease (CGD), and were compared to EBV BCL from normals. The cells that were studied expressed both B cell surface antigens and EBV nuclear antigens. BCL from normals contained small cytoplasmic granules. When stimulated with phorbol myristate acetate (PMA), these B cells reduced nitroblue tetrazolium (NBT) and generated significant amounts of superoxide. However, two other phagocytic stimuli, A23187 and f-methionine-leucine-phenylalanine, failed to stimulate BCL oxygen metabolism. BCL from seven individuals with CGD failed to either reduce NBT or generate superoxide on PMA stimulation, although the lines were morphologically similar to BCL from normals. In contrast, CHS-BCL generated superoxide more rapidly than did normals, and contained the characteristic giant granules of CHS. Thus, continuous BCL evidencing the phenotypic abnormalities of inherited human phagocytic diseases can provide large numbers of cells for biochemical and genetic study.     

Telomerase RNA deficiency in peripheral blood mononuclear cells in X-linked dyskeratosis congenita

Compromised renewal and eventual failure of the hematopoietic system in dyskeratosis congenita (DC) have been proposed to arise from a deficiency in telomerase function. Previously, cultured cell lines from patients with X-linked DC were shown to accumulate less telomerase RNA than cell lines from unaffected family members. Here, we report that telomerase RNA deficiency is also present in the circulating lymphocytes of DC patients. We have compared the accumulation levels of telomerase RNA and a panel of other small RNAs in peripheral blood mononuclear cells from an X-linked DC patient and an unaffected maternal carrier and similarly analyzed cultured lymphoblasts from an X-linked DC patient and maternal carrier in a second family. The DC-patient lymphoid cells show a specific defect in telomerase RNA accumulation with or without cell culture. Our findings support the clinical significance of telomerase deficiency and encourage the use of telomerase activation as a disease therapy.     

Increased proliferation, lytic activity, and purity of human natural killer cells cocultured with mitogen-activated feeder cells.

The addition of mitogen-prestimulated periferal blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) cultures to enriched populations of natural killer (NK) cells obtained from PBL of normal donors in the presence of rIL-2 resulted in highly significant increases in proliferation, purity, and cytolytic activity of cultured NK cells. Two sources of enriched NK cell preparations were used: (i) Adherent-lymphokine activated killer (A-LAK) cells obtained by adherence to plastic during 24 hr activation with 10(3) Cetus U/ml rIL-2; and (ii) NK cells negatively selected from PBL by removal of high-affinity rosette-forming cells and CD3+ lymphocytes. Coculture of A-LAK cells for 14 days with autologous or allogeneic Con A-activated PBL (10(6) cells/ml) or selected EBV-transformed LCL (2 x 10(5) cells/ml) as feeder cells increased fold expansion by a mean +/- SEM of 629 fold +/- 275 (P less than 0.019) and 267 fold +/- 54 (P less than 0.0001), respectively, compared to 55 +/- 20 in A-LAK cultures without feeder cells. The addition of either activated PBL or EBV lines to A-LAK cultures also led to a significant increase in the percentage of NK cells (CD3- CD56+) (84 +/- 2.4 and 84 +/- 2.6%, respectively, P less than 0.0001 for both), compared to 53 +/- 7.2% in cultures without feeders. The presence of feeder cells in cultures of A-LAK cells also led to significantly higher anti-tumor cytolytic activity compared to control cultures, as measured against NK-sensitive (K562) and NK-resistant (Daudi) target cells. Mitogen-stimulated CD4+ PBL purified by positive selection on antibody-coated flasks were better feeders than CD8+ or unseparated PBL. In the presence of feeder cells, it was possible to generate up to 6 x 10(9) activated NK cells from 2 x 10(8) fresh PBL by Day 13 of culture. Enhanced NK cell proliferation in the presence of feeder cells was not attributable to a detectable soluble factor. The improved method for generating A-LAK or activated-NK cells should facilitate cellular adoptive immunotherapy by providing sufficient numbers of highly enriched CD3- CD56+ effector cells with high anti-tumor activity.     

Spontaneous and induced chromosomal instability in Werner syndrome

Summary In extension of a previous study, spontaneous and clastogen-induced chromosome damage was analyzed in cultures of peripheral blood lymphocytes from six further patients with Werner syndrome (WS) and six healthy controls. In addition, sister chromatid exchange (SCE) was estimated in four of these cases. Lymphocytes of patients with various other diseases were used for another series of control experiments. Diepoxybutane (DEB), 4-nitroquinoline-1-oxide (NQO), and bleomycin (BLM) were the standard clastogens throughout the study. While the spontaneous frequency of chromosomal breakage was significantly higher in lymphocytes from all the patients than in the control cells, the basis SCE rate was un-affected in WS cells. Sensitivity of WS cells to the chromosome-damaging action of BLM did not differ from that of control cells, and their sensitivity to DEB was slightly greater than that of control lymphocytes. However, NQO induced a more distinct increase of both break and interchange aberrations in the WS cells than in control cells or cells from patients with other diseases. This effect was not found for the SCE rate. Our data demonstrate the exceptional cytogenetic features of this syndrome: Although the spontaneous and the DEB- and NQO-induced chromosomal breakage rate would suggest that WS is like a classic chromosomal instability syndromes, the lack of sensitivity of WS cells to bleomycin and their stable SCE frequency compared with that of control cells clearly delimitate this syndrome from other entities.     

Ultraviolet mutagenesis in human lymphocytes: The effect of cellular transformation

We have assessed the role of cellular transformation in ultraviolet (uv)-induced mutagenic events in human cells. To maintain uniformity of genetic background and to eliminate the effect of DNA repair, primary nontransformed lymphocytes (T-cells) and Epstein-Barr virus-transformed lymphocytes (B-cells) from one patient (XP12Be) with the DNA repair-deficient disorder xeroderma pigmentosum (group A) were transfected with the mutagenesis shuttle vector pZ189. Parallel control experiments were performed with primary, nontransformed lymphocytes from a normal individual and with a repair-proficient Epstein-Barr virus-transformed lymphocyte line (KR6058). pZ189 was treated with uv and introduced into the four cell lines by electroporation. Plasmid survival and mutations inactivating the marker supF suppressor tRNA gene in the recovered pZ189 were scored by transforming an indicator strain of Escherichia coli. Plasmid survival was reduced and mutation frequency elevated equally with both XP-A cell lines compared to both normal cell lines. Base sequence analysis of more than 250 independent plasmids showed that while the G:C----A:T base substitution mutation was found in at least 60% of plasmids with single or tandem mutations with all four cell lines, the frequency with the transformed XP-A (93%) cells was significantly higher (P less than 0.01) than that with the nontransformed XP-A cells (77%). In addition, with the transformed XP-A cells, there were significantly fewer plasmids with transversions and with mutations at a transversion hotspot (base pair 134) than with plasmids recovered from nontransformed XP-A cells. Interleukin-2 and phytohemagglutinin (used to maintain growth of the nontransformed lymphocytes) treatment of transformed XP12Be cells did not change overall plasmid survival or mutation frequency, but increased the transversion frequency and induced a mutational hotspot (at base pair 159), while another mutational hotspot (at base pair 123) disappeared. Thus we have demonstrated that in repair-deficient human cells, cellular transformation, while not affecting overall postuv plasmid survival and mutation frequency, does increase the susceptibility to G:C----A:T transition mutations, a type of mutation associated with uv-induced neoplasia.