Raffaella Elli

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).

Relationship between recovery from sublethal damage by dose fractionation and the restoration of viability after delayed plating in diploid Saccharomyces cerevisiae.

Recovery from radiation damage has been observed in several organisms subjected to several different types of irradiation. However, there are two methodologically distinct phenomena subsumed under the general heading of recovery: the first, recovery from sublethal damage, measured by a modification of cellular response during an incubation period between two dose fractions (1-8); and the second, recovery from lethal damage,4 measured in yeasts and bacteria, by an increase in colony-forming ability on delayed plating after a single dose of irradiation (9-24). These two phenomena are obviously operationally distinct because sublethally damaged cells are able to form colonies if plated immediately after the irradiation, whereas lethally damaged cells are not.

The hemoglobin of amphibia. X. Sedimentation behaviour of frog, triton and axolotl hemoglobins.

Abstract 1. 1. Axolotl mexicanum hemoglobin: the oxygenated derivative remains as a tetramer as a function of pH, while part of the deoxygenated derivative undergoes a concentration-dependent polymerization to octomers below pH 6. 2. 2. Rana esculenta hemoglobin: the oxygenated derivative does not polymerize as a function of pH. The deoxygenated derivative partly polymerizes to octomers over the pH range 5–8. 3. 3. The polymerization of the deoxygenated Rana esculenta and Axolotl hemoglobin is reversible on reoxygenation of the solutions. 4. 4. Triturus cristatus hemoglobin: both the oxygenated and deoxygenated derivatives remain as tetrames over the pH range 5–8.

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. Environ. Mol. Mutagen. 32:56–63, 1998.

Recovery from X-ray-induced Sub-lethal Damage in Saccharomyces Cerevisiae Cells of Different Ploidy

SummaryExperiments of dose fractionation have been performed on a polyploid series (from 2n to 4n) of yeast cells, in order to check for the recovery from sub-lethal damage and to investigate whether there is a correlation between kinetics of recovery and ploidy. Cells in a resting state or in log phase of growth were used.The data obtained show that recovery takes place in all the tested strains and display a similar type of kinetics, independently from the degree of ploidy. In the case of resting cells we get a curve describing only the recovery process, while, when growing cells are used, this curve is modified by the concomitant progression of the cells toward division. In this latter case, the value of complete recovery is attained by the three strains in different time intervals, probably related to the specific rates of growth.

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 patients 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.

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.

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.

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.

The rep region of pR plasmid regulates the expression of SOS system.

SummaryBy using an artificial hybrid between phage λ and the pR plasmid, we have shown that the rep region of the pR plasmid encodes a function which regulates the expression of the muc genes (plasmid genes that are under the negative control of lexA and responsible for an increased rate of spontaneous mutagenesis and resistance to UV and chemicals). Expression of the muc genes was monitored by a fusion between the muc promoter and the lacZ structural gene. When E. coli cells containing such a fusion are infected by the hybrid λpR phasmid, β-galactosidase activity is enhanced, indicating that pR encodes an antagonist of lexA. By deletion mapping we have located the gene encoding the antagonist of lexA (bat) in the rep region of the plasmid. The bat gene product can also antagonize the λcI repressor as shown by the observation that λpR phasmids are virulent on a homoimmune lysogen. We have exploited this latter property to carry out genetic and functional analysis of the bat region. This region is organized as a classical operon where the expression of the bat structural gene is negatively regulated by a repressor gene that encodes a proteic product.