Louis-Bar syndrome: spontaneous and induced chromosomal aberrations in lymphocytes and micronuclei in lymphocytes,oral mucosa and hair root cells

Summary Louis-Bar (L-B) syndrome, also called ataxia-telangiectasia, is cytogenetically characterized by an increased frequency of spontaneous and induced chromosomal aberrations (CA) in cultured lymphocytes and skin fibroblasts. However, it is not yet clear whether the chromosomal instability is also present in uncultured cells. The spontaneous and bleomycin-induced CA in peripheral lymphocytes of 8 L-B patients were evaluated. The micronucleus test was also performed, for the first time in lymphocytes by the cytokinesis-block method, and in uncultured cells of the oral cavity and hair root. The spontaneous frequency of CA and micronuclei in lymphocytes was about 3 times higher in L-B patients than in controls, these two cytogenetic parameters being highly correlated. Moreover, the induction by bleomycin of CA was higher in patients than in controls. The micronuclei in buccal and hair root cells of patients were normal. It remains to be determined whether the different responses obtained with cultured and uncultured cells are the result of the different L-B gene expression of chromosomal instability or whether they arise because of a particular cell sensitivity to culture conditions. The spontaneous and induced CA in lymphocytes of heterozygotes cultured in the presence of L-B serum were studied to evaluate a possible increased sensitivity of heterozygotes to a possible diffusible clastogenic factor present in the plasma of L-B patients. We could not demonstrate the presence of any factor that enhances CA in normal subjects or in heterozygote carriers.     

G2 chromosomal radiosensitivity in Fanconi's anemia

Both the peripheral lymphocytes from 4 patients affected with the inherited disease Fanconi's anemia (FA), and tissue-culture fibroblasts from skin biopsies from 3 patients similarly affected were found to be about twice as sensitive to the induction of chromatid-type chromosomal aberrations by X-rays administratered in the G2 phase of the cell cycle as cells from normal controls. Using tritiated thymidine labelling of peripheral lymphocytes and of cultured fibroblasts, it was determined that 3 affected patients and 3 normal controls all had similar percent labeled mitoses (PLM) curves, so the increased induced aberration yields seen in the FA cells do not appear to be simply a consequences of a longer than normal G2 phase of the cell cycle.     

Normal red blood cells partially decrease diepoxybutane-induced chromosome breakage in cultured lymphocytes from Fanconi anaemia patients

Objectives: Fanconi anaemia (FA) is a cancer‐prone chromosome instability syndrome characterized by hypersensitivity to DNA cross‐linking agents, such as diepoxybutane (DEB). Previous studies have shown that normal red blood cells (RBC) can protect cultured lymphocytes against chromosomal breaks induced by DEB. The present study was designed to analyse influence of RBCs from normal individuals on frequency of DEB‐induced chromosome breaks in lymphocyte cultures from FA patients. Materials and methods: A comparative study was performed between DEB‐induced chromosome breaks in cultures of FA lymphocytes with either autologous or heterologous RBCs. A further comparative study was carried out between whole blood cultures from FA patients performed on two occasions, before and 1 week after transfusion of RBCs. Results: It was observed that normal RBCs compared to FA RBCs, partially reduced chromosome breaks in cultured FA lymphocytes. A significant reduction in DEB‐induced breaks was also observed in FA cultured lymphocytes obtained 1 week after transfusion of RBCs, in comparison to those observed in the same patients before RBC transfusion. Conclusions: This study shows that DEB‐induced chromosome instability in FA lymphocytes is partially reduced by normal RBCs. This effect may have some clinical relevance in vivo, whenever FA patients receive a RBC transfusion.     

Ca antagonist verapamil and tumor promoter 12-O-tetradecanoylphorbol-13- acetate (TPA) induce chromosomal aberrations in human lymphocytes

Lymphocytes from 8 healthy donors were cultured for 3 days in the presence of phytohemagglutinin. Addition of the Ca antagonist verapamil or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increased chromosomal aberrations in comparison with control cultures. The effects of TPA and verapamil were additive. Although the Ca ionophore A23187 had no effect per se, it did abolish the effect of verapamil. Five patients with supraventricular tachycardia were treated clinically with verapamil for 1 week. Their lymphocytes were cultured for 3 days in vitro in the presence of phytohemagglutinin. These lymphocytes showed higher chromosomal aberrations in comparison with lymphocytes isolated from the patients before treatment. The clinical significance is still unclear. We suggest that calcium ions may be necessary for the intactness of chromosomes of human lymphocytes and presumably of other cells.     

Chromosomes and Transformation of Lymphocytes in Lymphoproliferative Disorders

In chronic lymphocytic leukaemia the majority of circulating lymphocytes which responded to phytohaemagglutinin in vitro were found to have normal karyotypes. A minor population of cells in patients treated with chemotherapy had an increased number of chromosomal rearrangements as compared with cells from normal controls and untreated patients with chronic lymphocytic leukaemia. Probably bonemarrow and lymph-node cells also had a normal karyotype.In the other lymphoproliferative disorders the peripheral blood lymphocytes had either normal karyotypes or chromosomal abnormalities attributable to treatment, even in those cases where the tumour cells of involved lymph nodes were known to have abnormal karyotypes.It was possible that circulating tumour cells were present in one case.     

Bleomycin-induced chromosomal aberrations in Down's syndrome lymphocytes

Blood samples from 4 Down's syndrome (DS) patients with a 47,XY,21 + karyotype and from 4 normal male probands were cultured for 72 h in the presence of BrdU and lymphocytes analysed at their first mitosis for chromosomal aberrations. The frequencies of spontaneous aberrations and the proportions of cells in the first or later mitoses in culture were not different between the groups. Treatment with various doses of bleomycin in vitro resulted in similar delays in cell development for both DS and normal lymphocytes and dose-dependent increases in the incidence of chromosome-type aberrations. However, the induction of both dicentric aberrations and acentric fragments was significantly enhanced in DS cells relative to cells of normal karyotype.     

Molecular cytogenetic study of patients with Pallister-Killian syndrome

The Pallister-Killian syndrome (PKS) is characterized by tissue limited chromosomal mosaicism, i.e. the presence of a supernumerary metacentric chromosome [i(12p)] often confined to skin fibroblasts while the karyotype of cultured lymphocytes is normal. In the present study, chromosome painting by chromosomal in situ suppression (CISS) hybridization and interphase cytogenetic procedures employing biotinylated or digoxigenin labelled probes was carried out. These probes comprised a chromosome 12 specific library (LA 12NSO1) and chromosome 12 centromere specific -satellite (pSP12-1). They were used to analyse and quantify the presence of i(12p) in lymphocytes, granulocytes/monocytes, skin fibroblasts and buccal mucosal cells from five patients and one aborted fetus with PKS, and ten normal donors. CISS hybridization on mitotic skin fibroblasts reliably indicated the presence of i(12p) cells, even when metaphases of poor quality were included in the analysis. Two of the five patients showed i(12p) in a small proportion (0.5%) of the cultured lymphocytes too. The interphase cytogenetics procedure did not reveal the isochromosome in lymphocytes or granulocytes/monocytes in any of the patients. Two of the six patients had a twofold increase in the number of buccal mucosal cells with three hybridization signals over control values. However, for mucosal cells, methodological improvements are required. For cytogenetic diagnosis of PKS, cultured fibroblasts subjected to chromosome painting by CISS hybridization with a chromosome 12 specific library probe are recommended.     

Bimolane induces multiple types of chromosomal aberrations in human lymphocytes in vitro

Bimolane has been commonly used in China for the treatment of psoriasis and various types of cancer. Patients treated with bimolane have been reported to have an increased risk of developing therapy-related leukemias. Although bimolane has been identified as a human leukemia-inducing agent, little is known about its genotoxic effects, and a systematic study of the types of chromosomal alterations induced by this compound has not been performed. In this study, a combination of immunochemical, molecular and conventional cytogenetic techniques has been used to study the chromosomal alterations induced by bimolane in cultured human lymphocytes. Immunochemical staining with the CREST antibody indicated that bimolane induces micronuclei (MN) originating primarily from chromosome breakage. Interestingly fluorescence in situ hybridization (FISH) with differentially labeled chromosomes 1 and 9 centromeric probes indicated that bimolane also caused non-disjunction and polyploidy. Consistent with this, an expedited analysis of Giemsa-stained metaphase chromosomes in bimolane-treated lymphocytes revealed a high frequency of polyploidy/hyperdiploidy as well as dicentric chromosomes, and premature centromeric division (PCD). In addition, bimolane was also found to produce binucleated cells, possibly through an interference with normal functioning of intermediate filaments. As a follow-up to these studies, three different types of commercially available bimolane formulations obtained from different Chinese manufacturers were also evaluated. The effects seen with the formulated bimolane were similar to those seen with the synthesized compound. Our studies indicate that bimolane effectively induces a variety of cellular and chromosomal changes in cultured lymphocytes and that similar alterations occurring in bone marrow stem cells could contribute to the development of the secondary cancers seen in bimolane-treated patients.     

The role of foetal red blood cells in protecting cultured lymphocytes against diepoxybutane-induced chromosome breaks

Diepoxybutane (DEB) is an established mutagen that induces chromosome damage following in vitro treatment of peripheral blood lymphocytes. It is widely used to identify patients with Fanconi Anemia (FA), a clinical situation that is characterized, besides the hypersensitivity to DEB, by an elevated foetal haemoglobin (HbF) content in the peripheral blood. In a previous study, we showed that red blood cells (RBC) from normal individuals can protect cultured lymphocytes against chromosomal breaks induced by DEB and demonstrated the particular role of haemoglobin in the protective effect. In the present work, we studied the influence of RBC extracted from umbilical cord blood of neonates (F cells) on the frequency of DEB-induced chromosome breaks in lymphocyte cultures from normal individuals. Simultaneously, we determined individual GSTT1 and GSTM1 genotypes and the activity of Pi-class glutathione S-transferase (GSTP), catalase and superoxide dismutase (SOD) in adult and foetal RBC. Our results show that F cells, in comparison with adult RBC, elicit a better protection of cultured lymphocytes from normal individuals against chromosome breaks induced by DEB. Variability in the protective effect among RBC from different individuals was observed; we confirmed that the GSTT1 genotype modulates this inter-individual variability, but it is not sufficient to explain all of the protective effect of F cells. Our results suggest that the increased protective effect of F cells can be, at least in part, correlated with an increase in the activity of glutathione S-transferase, catalase and superoxide dismutase, in particular Cu/Zn SOD, in F cells compared with adult RBC.     

Hypersensitivity to ionizing radiation, in vitro, in a new chromosomal breakage disorder, the Nijmegen Breakage Syndrome

The Nijmegen Breakage Syndrome (NBS) is a new chromosomal instability disorder different from ataxia telangiectasia (AT) and other chromosome-breakage syndromes. Cells from an NBS patient appeared hypersensitive to X-irradiation. X-rays induced significantly more chromosomal damage in NBS lymphocytes and fibroblasts than in normal cells. The difference was most pronounced after irradiation in G2. Further, NBS fibroblasts were more readily killed by X-rays than normal fibroblasts. In addition, the DNA synthesis in NBS cells was more resistant to X-rays and bleomycin than that in normal cells. The reaction of NBS cells to X-rays and bleomycin was similar to that of cells from patients with ataxia telangiectasia. Our results indicate that NBS and AT, which also have similar chromosomal characteristics, must be closely related.     

The effect of caffeine posttreatment of X-ray-induced chromosomal aberrations in human blood lymphocytes in vitro

Summary The potentiating effect of caffeine on X-ray-induced chromosomal aberrations in human blood lymphocytes has been investigated, with special reference to cell cycle stages (G0 and G2). Both quantitative and qualitative differences in the yield of chromosomal aberrations were detected in caffeine-posttreated cells, depending on the cell stage irradiated. The studies on caffeine potentiating effects on X-irradiated G0 lymphocytes from normal adults, newborns, Down syndrome patients, and an ataxia telangiectasia patient pointed to interindividual variations in the response to caffeine potentiation among normal probands and a very profound effect in ataxia cells.     

Generation of specific helper cells and suppressor cells in vitro for the IgE and IgG antibody responses.

Normal splenic lymphocytes from BDF1 mice were cultured on ovalbumin (OA)-bearing syngeneic peritoneal adherent cells for 5 days and their subsequent helper function was tested by an adoptive transfer technique. Lymphocytes harvested from the culture were mixed with DNP-KLH-primed spleen cells and transferred into irradiated syngeneic mice followed by challenge with DNP-OA. The results showed that the cultured lymphocytes has helper function for both IgE and IgG anti-DNP antibody responses. Depletion of mast cells and T cells in the peritoneal adherent cell preparations did not affect the generation of helper cells in the culture. The helper function of the cultured lymphocytes was abolished by the treatment with anti-theta antiserum and complement and was specific for ovalbumin. The OA-specific helper T cells were generated in vitro by the culture of a T cell-rich fraction of normal spleen on T cell-depleted OA-bearing peritoneal cells. If the normal splenic lymphocytes or T cell-rich fraction were cultured with 10 mug/ml of OA in the absence of macrophages, cultured lymphocytes lacked helper function. The transfer of splenic lymphocytes or splenic T cells cultured with soluble OA to normal non-irradiated mice, however, suppressed both IgG and IgE antibody responses of the recipients to subsequent immunization with DNP-OA. The suppressor cells were sensitive to anti-theta antiserum and complement and their activity was specific for OA. The cultured cells transferred into normal mice did not suppress anti-hapten antibody response to DNP-KLH. Normal lymphocytes cultured on OA-bearing macrophages and had helper function in adoptive transfer experiments failed to suppress antibody response of non-irradiated recipients to DNP-OA. The results indicate that OA-bearing macrophages primed T cells and generated helper T cells, whereas the culture of normal lymphocytes with soluble OA in the absence of macrophages generated suppressor T cells.     

Cytogenetic studies using various clastogens in two patients with Werner syndrome and control individuals

Summary Chromosome studies were performed on peripheral lymphocytes from two patients with Werner syndrome and two healthy control individuals to detect spontaneous and/or mutagen-induced chromosomal instability of this disease. Diepoxybutane, isonicotinic acid hydrazide, 4-nitro-quinoline-1-oxide, and bleomycin were used as standard clastogens. While the spontaneous frequency of chromosomal breakage was much higher in lymphocytes from both patients than in the control cells, the basic rate of sister chromatid exchange (SCE) was found to be in the control range. The sensitivity to clastogens of the patients' cells, however, was not substantially increased as compared with the controls if the degree of multiplication of the spontaneous breakage rate or SCE frequency was taken as the basis for comparison. No indication of a greater inhibition of proliferation by the clastogens in the patients' cells than in normal cells was observed using BrdU-labelled lymphocytes. Thus, the lymphocytes from both patients of the present study lacked essential features of the classical chromosome instability syndromes.     

The role of suppressor cells in the pathogenesis of common variable hypogammaglobulinemia and the immunodeficiency associated with myeloma.

The role of suppressor cells in the pathogenesis of immunodeficiency was analyzed using a technique that permits study of the differentiation of B lymphocytes into immunoglobulin-synthesizing plasma cells. Lymphocytes from normals synthesized 4,910 ng of IgM, 1,270 ng of IgA, and 1,625 ng of IgG per 2 X 10(6) cells when cultured for 7 days in the presence of pokeweed mitogen. In contrast the lymphocytes from patients with common variable hypogammaglobulinemia did not synthesize significant quantities of immunoglobulin. When lymphocytes from 9 of 13 patients with common variable hypogammaglobulinemia studied were cocultured with normal lymphocytes, the synthesis of immunoglobulin by the normal lymphocytes was depressed by 75-100%. A comparable suppression of immunoglobulin synthesis by normal lymphocytes was observed when they were cocultured with T cells from hypogammaglobulinemic patients. These studies suggest that in some patients the disease common variable hypogammaglobulinemia may not be due to an intrinsic defect of B cells alone but may be cuased or perpetuated by an abnormality of regulatory T cells that act to suppress B-cell maturation and antibody production. Peripheral blood lymphocytes from myeloma patients also had a drastically reduced capacity to produce polyclonal immunoglobulins. Three of 6 myeloma patients tested had circulating mononuclear cells that suppressed immunoglobulin production by cocultured normal lymphocytes. Purified T cells from myeloma patients did not mediate this suppressor effect. These observations suggest that one mechanism for the humoral immune deficiency observed in myeloma patients is a block of polyclonal B-cell maturation by suppressor cells.     

Hepatitis B virus infection in alcoholics]

Chromosomal analysis has been carried out in 4 patients with the symptoms of hepatic coma. An analysis included lymphocytes cultured from peripheral blood. Chromosomal disorders have been assessed with two techniques: structural chromosomal aberrations test, and sister chromatid exchange (SCE) test. It has been shown that the extend of chromosomal damage in the form of the gaps, breaks, acentric chromosomes as well as the presence of ring and dicentric chromosomes, and micronuclear cells have been higher in the examined patients. Such changes may evidence DNA repair disorders, and the presence of micronuclear forms may seem an unfavourable prognosis.     

Chromosome-breaking agent of low molecular weight in human systemic Lypus Erythematosus

Summary The serum of patients with systemic lupus erymathosus contains a chromosome-breaking agent of low molecular weight, which produces chromosomal breakage and rearrangement in lymphocyte cultures of healthy donors. This breakage factor is probably produced by or released from the cells of patients, since lymphocyte extracts and cocultivation of patients' lymphocytes with lymphocytes from healthy subjects results in an increased frequency of chromosome aberrations in the normal cells. The aberration rate induced by this clastogenic agent is reduced to normal values if the free radical scavenging enzyme superoxide dismutase is added to the culture medium at a final concentration of 0.05 mg/ml.     

T-lymphocyte colony formation by lymphocytes from patients with aplastic anaemia

T-lymphocyte colonies were cultured using lymphocytes from patients with aplastic anaemia and normal donors to assess their respective proliferative activities. Colony numbers from aplastic patient's cells were lower than from normal donors', though this was not significant. When lymphocytes from patients were co-cultured with normal lymphocytes, inhibition of T-colony formation was observed in 8 out of 12 experiments. As the degree of inhibition was greater than if patient cells grew no colonies, then, clearly, normal T-colony formation was inhibited. This ability of patients' lymphocytes to suppress lymphopoiesis might account for the low levels of patient T-colony formation, as well as low in vivo numbers of lymphocytes found in patients with aplastic anaemia. The role of patients' lymphocytes in causing marrow aplasia was investigated. Although the incorporation of patients' lymphocytes in normal granulocyte-macrophage (GM) colony-forming systems inhibited colony growth, in only 1 out of 8 patients was this inhibition significantly greater than that caused by the addition of normal lymphocytes to GM colony systems. Therefore, lymphocytes may not be the primary cause of aplastic anaemia, except for a few rare cases.     

Isolation and subregional mapping of a human cDNA clone detecting a common RELP on chromosome 12

Summary Peripheral blood lymphocytes from three patients with Down syndrome (DS; trisomy 21; aged 5–6 years) and three age-matched control children were studied for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs).Cells in G₀ were exposed to bleomycin (20–100 g/ml) for 3 h, and then cultured in medium containing 5-bromodeoxyuridine and phytohemagglutinin for 66 h. By the sister chromatid differential staining method, chromosome analyses were performed on metaphase cells that had divided one, two, or three or more times after treatment. The results indicate that DS cells exposed to bleomycin are hypersensitive to the production of dicentric and ring chromosomes compared to normal cells. Bleomycin also led to a dose-related increase in the frequency of SCEs, but no difference was found between the SCE frequencies in DS or normal lymphocytes exposed to bleomycin.     

Immunofluorescent study of non-histone protein-DNA complexes in cultured cells and lymphocytes

Antibody against non-histone chromosomal protein-DNA complex of C-6 cells derived from human lymphoblastoid cells were prepared. By immunofluorescent studies using the antibody, nuclear fluorescence, which was speckled or clumped in appearance, was observed in cultured human lymphoblastoid cell lines, cultured human epithelial cell lines and human peripheral lymphocytes. In the human peripheral lymphocytes, there was a distinct variation in intensity of the nuclear fluorescence among the cell population. On the contrary, no nuclear fluorescence was observed in cultured animal cell lines.