Chromosome instability in T-cells cultured in the presence of pancuronium or fentanyl.

BACKGROUND: Genomic instability is recognized as a cause of cellular apoptosis and certain drugs that exhibit a proapoptotic effect are also able to induce chromosome damage. Since we found in recent experiments that drugs such as pancuronium and fentanyl exerted an apoptogenic effect on T cells, we studied the capacity of those agents to promote chromosome instability, i.e. chromosome aberrations (CA) and telomeric associations (tas) in peripheral blood lymphocytes. METHODS: Lymphocytes from healthy donors were cultured with pancuronium or fentanyl, using two different concentrations for each drug: 20 and 200 ng/ml for pancuronium and 10 and 30 ng/ml for fentanyl, respectively. Cells were exposed to each concentration of these drugs either for 24 or 48 h. The higher concentration chosen was the same at which we detected the proapoptotic effect in our previous works. Cytogenetic analysis was performed by means of a standard technique and chromosome aberrations or telomeric associations were blindly evaluated by two independent observers. RESULTS: The chromosome aberrations we observed in treated cells were not significantly different from control lymphocytes. However, an unusual rate of telomeric associations (P < 0.001) was detected in cells exposed to both pancuronium and fentanyl, at each concentration tested and at each exposure time of the study. CONCLUSIONS: Fentanyl and pancuronium do not have a direct clastogenic effect on T cultures, but at the same concentrations at which we demonstrated their apoptogenic power, these drugs are able to increase genomic instability through inducing an elevated rate of telomeric associations. Such a capacity could exploit in peripheral T cells the same mitochondrion-mediated signal pathway of apoptosis death.

Telomerase activity, apoptosis and cell cycle progression in ataxia telangiectasia lymphocytes expressing TCL1.

Individuals affected by ataxia telangiectasia (AT) have a marked susceptibility to cancer. Ataxia telangiectasia cells, in addition to defects in cell cycle checkpoints, show dysfunction of apoptosis and of telomeres, which are both thought to have a role in the progression of malignancy. In 1-5% of patients with AT, clonal expansion of T lymphocytes carrying t(14;14) chromosomal translocation, deregulating TCL1 gene(s), has been described. While it is known that these cells can progress with time to a frank leukaemia, the molecular pathway leading to tumorigenesis has not yet been fully investigated. In this study, we compared AT clonal cells, representing 88% of the entire T lymphocytes (AT94-1) and expressing TCL1 oncogene (ATM(-) TCL1(+)), cell cycle progression to T lymphocytes of AT patients without TCL1 expression (ATM(-) TCL1(-)) by analysing their spontaneous apoptosis rate, spontaneous telomerase activity and telomere instability. We show that in ATM(-) TCL1(+) lymphocytes, apoptosis rate and cell cycle progression are restored back to a rate comparable with that observed in normal lymphocytes while telomere dysfunction is maintained.

Telomeric associations and chromosome instability in ataxia telangiectasia T cells characterized by TCL1 expression.

T-cell tumors in ataxia telangiectasia (AT), such as T-PLL/T-CLL, are first preceded by the development of a large clone of T-lymphocytes, characterized by chromosomal rearrangements, which usually involve specific regions such as the 14q11 region. Malignancy develops years later, after additional chromosomal changes resulting from the genomic instability consequent to ATM disruption and to the activation of the TCL1 oncogene. Here we report the results of a cytogenetic follow-up of an AT patient (AT94-1), still without signs of hematological abnormalities, bearing a T-lymphocyte clone characterized by the t(14;14)(q11;q32) rearrangement and having TCL1 expression. We demonstrated that in clonal cells TCL1 expression correlates with increasing genomic instability and in time this mainly induces chromosomal rearrangements and telomeric associations (tas). Chromosome 21 is not randomly involved; in particular, an i(21q) indicates that it is a subclone prone to additional genetic changes and could represent an early chromosomal rearrangement involved in tumorigenesis. With regard to the increase in tas, we observed that: (i) it is inversely correlated with the proliferative ability of AT94-1 lymphocytes in PHA-stimulated short-term cultures (cell aging in vitro); (ii) this increase is not due to changes either in cell radiosensitivity (measured as bleomycin (BML)-sensitivity) or due to an illegitimate recombination (measured as adriamycin-sensitivity), which may not be sufficient for tumor development.

Chromosomal alterations and male infertility.

Reduced male fertility can be caused by genetic factors affecting gamete formation or function; in particular, chromosome abnormalities are a possible cause of male subfertility as shown by their higher frequency in infertile men than in the general male population. Meiotic studies in a number of these males have shown spermatogenesis breakdown, often related to alterations in the process of chromosome synapsis. Indeed, any condition that can interfere with X-Y bivalent formation and X-chromosome inactivation is critical to the meiotic process; furthermore, asynapsed regions may themselves represent a signal for the meiotic checkpoint that eliminates spermatocytes with synaptic errors. We performed cytogenetic, hormonal and seminal studies in 333 infertile patients selected because azoospermic, severely oligozoospermic or normozoospermic with failure to fertilize the partner's oocytes in an in vitro fertilization (IVF) program. Our findings: 1) confirm the high incidence of chromosomal anomalies among infertile males; 2) highlight the relevance in male infertility of quantitative/positional modifications of the constitutive heterochromatin; and 3) underline the relevance of cooperation between andrologists and cytogenetists prior to every kind of assisted reproduction, above all prior to intracytoplasmic sperm injection, in which selective hurdles eliminating abnormal germ cells are bypassed.

Effects of poly(ADP-ribose) polymerase inhibition on cell death and chromosome damage induced by VP16 and bleomycin.

Poly(ADP-ribose) polymerase (PARP) is a DNA-binding protein involved in cellular response to various genotoxic agents. To understand the role of PARP in the mechanisms which lead from specific DNA damage to cell death, we studied the effects of PARP inhibition in human lymphoblasts damaged with bleomycin (BLM) and VP16. These agents can induce DNA breakage but through different mechanisms, enabling the study of the different effects of PARP in inducing apoptosis in damaged cells. We demonstrate that in lymphoblasts VP16 treatment induces apoptosis to a greater extent than BLM treatment, and that PARP inhibition reduces VP16-induced apoptosis whereas it has no effect on BLM-induced apoptosis. After VP16 treatment with PARP inhibition, a reduction in the depletion of the proliferative compartment and a G2/M phase arrest are observed. Therefore, the increase in cell viability and the reduction in chromosome damage may both be the result of a prolonged DNA repair time. Hence, PARP appears to play a significant role in VP16-induced apoptosis and not in BLM-induced apoptosis. Since apoptosis is important in tumor treatment these findings might be useful when considering the combined employment of PARP inhibition with antineoplastic drugs.

Erythrocyte uroporphyrinogen decarboxylase activity: diagnostic value and relationship with clinical features in hereditary porphyria cutanea tarda.

A marked discrepancy between mild and late clinical features and a nearly complete absence of erythrocyte uroporphyrinogen decarboxylase activity (Ery-UROD activity) was observed in a case of inherited porphyria cutanea tarda. The entity and time of appearance of clinical features, the onset of clinical symptoms after exposure to contributing factors, the effectiveness of phlebotomies and heterozygosity of the mother alone for uroporphyrinogen decarboxylase (UROD) deficiency were typical for familial porphyria cutanea tarda (F-PCT), whereas the extremely low UROD activity was peculiar to hepatoerythropoietic porphyria (HEP). These observations indicate that: 1) Ery-UROD activity may not always be useful to discriminate between F-PCT and HEP; 2) Ery-UROD activity does not always correlate with clinical symptoms; 3) in inherited UROD deficiency, the genetic defect may be heterogeneous. Finally, the observed discrepancy may provide additional evidence for the existence of tissue-specific isozymes.

Effects of topoisomerase II inhibition in lymphoblasts from patients with progeroid and "chromosome instability" syndromes.

DNA topoisomerase II is involved in DNA topologic changes through the formation of a cleavable complex. This is stabilized by the antitumor drug VP16, which results in DNA breakage, aberrant recombination, and cell death. In this work, we compare the chromosomal damage induced by VP16 with that induced by bleomycin (BLM) in lymphoblasts from patients affected by the chromosome breakage syndromes ataxia telangiectasia (AT), xeroderma pigmentosum (XP), and Bloom syndrome (BS), and by the progeroid syndromes Werner (WS) and Cockayne (CS). Patients affected by AT, XP, BS, and WS have a greatly enhanced risk of developing cancer. The results show that AF and WS cells are hypersensitive to VP16, as revealed in the higher proportion of metaphases showing exchange figures and more than two breaks. All lines except AT and one CS line showed normal sensitivity to BLM. Our data on the sensitivity to VP16 of all these mutant cells underline the fact that VP16 damage is amplified only in cells that have abnormal illegitimate recombination (i.e., AT and WS).

VP16 hypersensitivity and increased faulty recombination in ataxia telangiectasia lymphocytes characterized by the tandem translocation t(14;14)(q11;q32).

Ataxia telangiectasia (AT) patients show variable degrees of immunodeficiency and a higher than normal predisposition to lymphoid malignancies. AT cells are characterized by spontaneous chromosome instability resulting in chromosome breakage and in non random chromosome rearrangements. Sequential cytogenetic studies on T-lymphocytes from an AT patient showed the progressive development of a clone bearing a tandem translocation t(14;14)(q11;q32). The abnormal clone had spontaneous chromosome rearrangements. Compared to non clonal cells, the abnormal clone displayed a higher frequency of spontaneous chromosome rearrangements. In only the clonal cells we observed two particular and predominant rearrangements: isodicentric chromosomes and telomeric associations which may derive from faulty recombination. Chromosome instability induced by the etoposide VP16, a DNA topoisomerase II inhibitor, was evaluated in terms of chromosome breakage and SCE frequency. T-lymphocytes from the AT patient showed hypersensitivity to VP16 significantly higher than normal T-lymphocytes. The chromosome instability induced by VP16 is significantly higher in clonal than in non clonal cells, whilst the chromosome instability induced by the radiomimetic drug bleomycin is not significantly different in the two AT lymphocyte subpopulations. The different spontaneous chromosome instability in clonal and non clonal cells together with their different behavior after treatment with only VP16, suggest that clonal cells bearing the tandem translocation could have increased faulty recombination. Given the presence of translocations t(14;14)(q11;q32) in T-prolymphocytic leukemias and T-cell tumors of non AT patients, our findings suggest that VP16 could be considered an antineoplastic treatment particularly indicated in these patients.

Expression of genes carried by pR plasmid in damaged E. coli and mouse cells.

In LTA mouse cells pR plasmid constitutively expresses itself resulting in protection against typical SOS inducers (UV, 4NQO) and in sensitization to different DNA-damaging agents (MNNG, cisDDP, BLM and geneticin (G418). The pR sensitizing effect is specific to mammalian cells, since the plasmid can only protect prokaryotic cells against the damaging agents tested. The pR protecting effect requires the expression of both the uvp1 and uvp2 (mucAB) regions in bacteria as well as in mouse cells. The coordinated function of these regions could result in protection against typical SOS inducers through an SOS/SOS-like pathway. The sensitization conferred by pR plasmid depends mostly on the expression of the mucAB genes, as shown by the survival of mouse cells transfected with different pR::Tn5 mutants. In particular, BLM and G418 survival data demonstrate that, inserted into the pR plasmid, the ble and neo genes of the Tn5 transposon express themselves. This was confirmed by the presence of Tn5 transcripts in untreated mouse cells. The comparison between the pR effects in bacterial and mouse cells shows that during evolution the repair pathways against UV damage are better conserved than those against other kinds of damage.

Neurological and cytogenetic study in early-onset ataxia-telangiectasia patients.

The clinical diagnosis of ataxia-telangiectasia (AT) is difficult before the age of 4 years. We report clinical and cytogenetic data on three early-onset, early-diagnosed AT patients at the age of 12, 18 and 22 months, respectively. Postural instability of the trunk, characterized by motor impersistence, was the earliest neurological sign detected as early as 1 year of life. Dystonic movements and postures of arms and trunk and a subtle disorder of eye movement (blinking before gaze changing, increased latency and dysmetry of saccades) were observed during the 2nd year of life. All patients exhibited an unusual temper tantrum. We also observed an increased bleomycin-induced chromosomal instability in patient's cells in the early stages of the disease before all the clinical hallmarks were apparent. Our data suggest that detection of clinical indications, leading to early laboratory confirmation of AT, can reduce the age at diagnosis.

Heterogeneity in ataxia-telangiectasia: classical phenotype associated with intermediate cellular radiosensitivity.

We identified a subgroup of ataxia-telangiectasia (AT) patients (2 sibs and 1 unrelated case) characterized by typical clinical manifestations of the disease and cellular radiosensitivity intermediate between classical AT and normal subjects. Our data and a literature review of the intermediate radiosensitivity AT cases show that radioresistant DNA synthesis, cellular radiosensitivity (measured in terms of survival and chromosome breakage), and the clinical hallmarks behave independently. This raises a number of interesting questions about the correlation between radiobiological and clinical features, and about the nature of the AT gene(s).

Human cells (normal and ataxia telangiectasia) transfected with pR plasmid are hypersensitive to DNA strand-breaking agents.

Ataxia telangiectasia (AT) cells are known to be hypersensitive to ionizing radiations and to drugs such as bleomycin and epipodophyllotoxin VP16, a topoisomerase II poison. Both of these produce DNA double-strand breaks even if through different mechanisms. In this work we analyzed the sensitivity to bleomycin and to epipodophyllotoxin of AT cells after transfection with pR plasmid. This plasmid, interacting with bacterial SOS repair pathways, expresses itself in mammalian cells conferring cell resistance to the SOS inducers UV and 4NQO and cell sensitivity to different drugs such as bleomycin. This effect is presumably due to the interaction of pR products with double-strand breaks. Our findings indicate that pR plasmid, in both AT lines tested (AT5BIVA fibroblasts and ATL6 lymphoblasts), expresses itself (increasing UV protection) and amplifies the already enhanced AT cell sensitivity to both bleomycin and VP16.

Establishment of a new Epstein-Barr virus-immortalized cell line from chronic lymphocytic leukemia with trisomy of chromosome 12 that produces monoclonal IgM against a sheep RBC antigen.

Leukemia cells from a patient with chronic lymphocytic leukemia (CLL) were found to bind sheep RBC (SRBC) through their monoclonal surface IgM. A lymphoblastoid cell line was obtained by immortalization of leukemic cells with Epstein-Barr virus (EBV). Cultured leukemic cells were found to have a supernumerary chromosome 12, an abnormality typical of CLL of the B cell type. To our knowledge, this is the first EBV-immortalized cell line from B-CLL cells of known SRBC specificity and the third reported CLL cell line carrying trisomy of chromosome 12.

The pR plasmid: a tool for discriminating between DNA lesions induced by different types of cytotoxic agents in cultured mammalian cells.

The LA-D cells, obtained by cotransformation of LTA mouse cells (tk- aprt-) with pR plasmid and with tk gene as selective marker, are significantly more resistant to UV light and 4-nitroquinoline-N-1-oxide than LTA control cells. In this work, we report that the LA-D cells exhibit different degrees of response to various DNA-damaging agents: wild-type survival to mitomycin, increased sensitivity to bleomycin, cis-diamminedichloroplatinum and N-methyl-N'-nitro-N-nitrosoguanidine. The pR plasmid could, therefore, play an important role in the DNA-repair mechanisms that modulate the cytotoxic effect of the DNA-inhibitory agents. The possible interactions between pR plasmid products and the different repair enzymes involved are discussed.

Variant of ataxia-telangiectasia with low-level radiosensitivity.

In the present study we examined cells from several patients clinically diagnosed as having ataxia-telangiectasia (AT), for the capacity of their cells to inhibit DNA synthesis following exposure to gamma irradiation, and for the rate of spontaneous or bleomycin-induced chromosomal aberrations. Cells from two patients showed normal inhibition of DNA synthesis and levels of induced chromosomal aberrations intermediate between normal and AT cells. These two patients had only minimal immunologic impairment. These findings appear to define one distinct subset of AT.

Premature centromere splitting in a presumptive mild form of Roberts syndrome.

Cytogenetic investigations performed on lymphocytes from a 29-year-old woman with no severe anomalies, allowed us to recognize a mild form of Roberts syndrome. The proposita's metaphases showed a consistent centromere splitting, especially affecting chromosomes 16, 19, 21, and 22. This centromere separation sequence seems to be unique to Roberts syndrome cells. The experiments also showed that no diffusible factor, involved in the mechanism of sister chromatid pairing-disjunction, exists.

Cytogenetic investigations on Werner's syndrome, Acrogeria and Keratosis Palmo-Plantaris.

The frequencies of sister chromatid exchanges and of aspecific chromosome aberrations have been investigated in the lymphocytes from a Werner patient, from an Acrogeria patient and from three members of a family with Keratosis Palmo-Plantaris. These investigations point out that: 1) the SCE frequency is significatively enhanced in Werner as in KPP lymphocytes, 2) the frequency of aspecific chromosome aberrations is increased only in Werner lymphocytes, without evidence of variegated translocation mosaicism. The findings confirm that SCE and chromosome aberrations do not necessarily result from the same genetic damage and that SCE may represent the cytological evidence of unexcised non fatal DNA lesions, which occasionally may be responsible for carcinogenesis.