Chromosomal aberrations in Wilms' tumour

In 26 consecutive patients operated for Wilms' tumour samples from the tumour were genetically analyzed. Clonal acquired chromosome aberrations were found in 13 patients and a constitutional trisomy 18 as the sole change in 1. The chromosome number was altered in 13 patients. Numerical changes occurred in 16 patients and breakpoint of chromosome 1 in 6 patients. There was no structural alteration of chromosome 11. The observed cytogenetic heterogeneity illustrates the complexity of genetic changes involved in the genesis and progression of Wilms' tumour. To further elucidate the phenotypic impact of chromosomal aberrations the correlation to histology and the clinical course will be important.     

An analysis of the cellular distribution of chromosome aberrations induced in human lymphocytes after a single irradiation and under conditions of preliminary adaptive exposure

Irradiation by an adaptive dose 0.05 Gy at the G0 stage decreased the number of chromosome aberrations induced in lymphocytes by a challenge dose 0.5 Gy at the G2 stage. Adaptive response was not observed at the G1 stage, when the cells were exposed to adaptive dose 0.05 Gy and challenge dose 1.0 Gy respectively after 24 h and 29 h incubation with PHA. In lymphocytes exposed to 1.0 Gy at the G1 stage, cellular distribution of chromosomal aberrations followed the Poisson distribution, while in lymphocytes exposed to 0.5 Gy at the G2 stage, the distribution of aberrations differed from the Poisson distribution and was nearer to the degenerated Poisson distribution. The adaptive dose 0.05 Gy did not alter the distribution of chromosome aberrations induced by the challenge dose at the G1 or the G2 stages. The role of independent and whole-cellular repair in the formation of chromosome and chromatid aberration is discussed.     

Chromosomal aberrations in lymphocytes predict human cancer: a report from the European Study Group on Cytogenetic Biomarkers and Health (ESCH)

Chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), and micronuclei (MN) in peripheral blood lymphocytes have for decades been used as cytogenetic biomarkers to survey genotoxic risks in the work environment. The conceptual basis for this application has been the idea that increased cytogenetic damage reflects an enhanced cancer risk. Nordic and Italian cohorts have been established to evaluate this hypothesis, and analyses presented previously have shown a positive trend between CA frequency and increased cancer risk. We now report on a pooled analysis of updated data for 3541 subjects examined for CAs, 2703 for SCEs, and 1496 for MN. To standardize for interlaboratory variation, the results for the various cytogenetic end points were trichotomized on the basis of the absolute value distribution within each laboratory as "low" (1-33 percentile), "medium" (34-66 percentile), or "high" (67-100 percentile). In the Nordic cohort, there was an elevated standardized incidence ratio (SMR) for all cancer among subjects with high CA frequency [1.53; 95% confidence interval (CI), 1.13-2.05] but not for those with medium or low CA frequency. In the Italian cohort, a SMR in cancer of 2.01 (95% CI, 1.35-2.89) was obtained for those with a high CA frequency level, whereas the SMRs for those with medium or low did not noticeably differ from unity. Cox's proportional hazards models gave no evidence that the effect of CAs on total cancer incidence/mortality was modified by gender, age at test, or time since test. No association was seen between the SCEs or the MN frequencies and subsequent cancer incidence/mortality. The present study further supports our previous observation on the cancer predictivity of the CA biomarker, which seems to be independent of age at test, gender, and time since test. The risk patterns were similar within each national cohort. This result suggests that the frequency of CAs in peripheral blood lymphocytes is a relevant biomarker for cancer risk in humans, reflecting either early biological effects of genotoxic carcinogens or individual cancer susceptibility.     

Spontaneous chromosome aberrations in the lymphocytes of human peripheral blood]

The authors carried out cytogenetic studies on lymphocytes from human peripheral blood of 105 persons--49 women and 56 men in order to examine spontaneous frequency of the structural chromosomal abberations in the somatic cells. There were 191 cells with abberations (1,17%), out of 16,267 analyzed metaphasic plates. They found 0,39% of chromatid fragments, 0,71% of chromosomal fragments, 0,06% dicentricets, 0,01% of symetric and asymetric chromatid exchanges and 0,006% of rings. There were no statistically significant differences in the discovery of the spontaneous abberations in the examined persons at various age and sex.     

Dose dependent of sister chromatid exchanges in humans lymphocytes induced by in vitro alpha-particle irradiation

AIM: To study the effect of (-)-stepholidine (SPD) on serum prolactin (PRL) level and elucidate its pharmacological action on dopamine D2 receptors. METHOD: After i.p. administration of dopamine receptor agonist, antagonist, or SPD, the serum PRL levels were determined by radioimmunoassay. RESULTS: SPD (24 mg.kg-1, i.p.) caused a rapid rise in serum PRL level, lasting more than 1 h. SPD 0.2-40 mg.kg-1 raised serum PRL level in a dose-dependent manner with ED50 of 3.7 mg.kg-1 (95% confidence limits, 2.6-4.3 mg.kg-1) and PRL maximal level of 448 +/- 64 micrograms.L-1. Pergolide 2 mg.kg-1 i.p. caused a decrease (P < 0.01 vs saline) of PRL level, which was partially attenuated by SPD of 5 mg.kg-1 and completely abolished by 10 mg.kg-1. CONCLUSION: SPD is a dopamine D2 receptor antagonist.     

Chromosome aberrations in human fibroblasts induced by monoenergetic neutrons. I. Relative biological effectiveness

The relative biological effectiveness (RBE) of neutrons for many biological end points varies with neutron energy. To test the hypothesis that the RBE of neutrons varies with respect to their energy for chromosome aberrations in a cell system that does not face interphase death, we studied the yield of chromosome aberrations induced by monoenergetic neutrons in normal human fibroblasts at the first mitosis postirradiation. Monoenergetic neutrons at 0.22, 0.34, 0.43, 1, 5.9 and 13.6 MeV were generated at the Accelerator Facility of the Center for Radiological Research, Columbia University, and were used to irradiate plateau-phase fibroblasts at low absorbed doses from 0.3 to 1.2 Gy at a low dose rate. The reference low-LET, low-dose-rate radiation was 137Cs-gamma rays (0.66 MeV). A linear dose response (Y = alphaD) for chromosome aberrations was obtained for all monoenergetic neutrons and for the gamma rays. The yield of chromosome aberrations per unit dose was high at low neutron energies (0.22, 0.34 and 0.43 MeV) with a gradual decline with the increase in neutron energy. Maximum RBE (RBEm) values varied for the different types of chromosome aberrations. The highest RBE (24.3) for 0.22 and 0.43 MeV neutrons was observed for intrachromosomal deletions, a category of chromosomal change common in solid tumors. Even for the 13.6 MeV neutrons the RBEm (11.1) exceeded 10. These results show that the RBE of neutrons varies with neutron energy and that RBEs are dissimilar between different types of asymmetric chromosome aberrations and suggest that the radiation weighting factors applicable to low-energy neutrons need firmer delineation. This latter may best be attained with neutrons of well-defined energies. This would enable integrations of appropriate quality factors with measured radiation fields, such as those in high-altitude Earth atmosphere. The introduction of commercial flights at high altitude could result in many more individuals being exposed to neutrons than occurs in terrestrial workers, emphasizing the necessity for better-defined estimates of risk.     

Chromosomal mutations induced by triplex-forming oligonucleotides in mammalian cells

Specific recognition of a region of duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Based on this, we have investigated the ability of the triplex-directed approach to induce mutations at a chromosomal locus in living cells. A mouse fibroblast cell line was constructed containing multiple chromosomal copies of the lambdasupFG1 vector carrying the supFG1 mutation-reporter gene. Cells were treated with specific (psoAG30) or control (psoSCR30) psoralen-conjugated TFOs in the presence and absence of UVA irradiation. The results demonstrated a 6- to 10-fold induction of supFG1 mutations in the psoAG30-treated cells as compared with psoSCR30-treated or untreated control cells. Interestingly, UVA irradiation had no effect onthe mutation frequencies induced by the psoralen-conjugated TFOs, suggesting a triplex-mediated but photoproduct-independent process of mutagenesis. Sequencing data were consistent with this finding since the expected T.A-->A.T transversions at the predicted psoralen crosslinking site were not detected. However, insertions and deletions were detected within the triplex binding site, indicating a TFO-specific induction of mutagenesis. This result demonstrates the ability of triplex-forming oligonucleotides to influence mutation frequencies at a specific site in a mammalian chromosome.     

Relative involvement of chromosome #21 in radiation induced exchange aberrations in lymphocytes of Down syndrome patients

The reaction of chemical carcinogens with DNA appears to be one of the earliest events in the initiation phase of cancer. These DNA reactions can be base- and position-specific, are affected by sequence context, and are repaired at different rates depending on whether or not they are on the transcribed or nontranscribed strand of DNA and which nucleotide sequence is modified. Thus, measurement of total genomic DNA reaction of carcinogens is only a crude first step in dissecting out which are the critical lesions for cancer initiation. On the other hand, we know that DNA adducts, which have been primarily characterised in experimental studies, appear to have similar structures in human DNA arising from occupational or environmental exposures. A number of different methods have been developed to detect and measure DNA adducts in man. These include physico-chemical methods such as mass spectrometry, 32P-postlabelling, fluorescence and accelerator mass spectrometry (AMS) and biological methods such as immunoassay. All these methods have their strengths and weaknesses. Human studies, using 32P-postlabelling, demonstrate that this method can be used to examine the effect of potential chemoprotective agents on DNA adduct level. AMS has been used to measure DNA adducts in human tissue after patients have ingested trace quantities of the food mutagens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, a heterocyclic amine formed during the cooking of meat and the naturally occurring mycotoxin, aflatoxin B1. These studies can assist in assessing the risks associated with low-level exposure to food genotoxins.     

Apoptosis induced by fast neutrons versus 60Co gamma-rays in human peripheral blood lymphocytes

Gunshot and air weapon wounds in children have become a significant source of morbidity and mortality in our community in the last four years. Ninety five children, 15 years of age and younger, were admitted to the Pediatric Surgical Service for gunshot and air mechanism wounds during this period. Review of the circumstances of injury revealed that more than 50% were accidental 71% were male patients, and 65% were caused by gunshots and outside of the home. Thoracic region was affected in 42%; abdomen, 20%; skull, face and neck, 13.4%; lower extremities, 13.4%; upper extremities 9%; genitalia, 2.0%; and perineal region, 0.8%. Rapid resuscitation and triage of major injury allowed us a survival of 97%. Social service intervention and educational task can offer significant benefit to these children but, ultimately gun control laws with strict enforcement are needed to stop this type of violence toward children.     

Kinetics of the formation of chromosome aberrations in X-irradiated human lymphocytes, using PCC and FISH

In order to study the initial frequencies and define kinetics of the formation of chromosomal exchanges in X-irradiated human lymphocytes, the premature chromosome condensation (PCC) technique was employed in combination with fluorescence in situ hybridization (FISH) with a composite probe for human chromosome 8 and a pan-centromeric probe for the whole genome. Human lymphocytes were X-irradiated (0.5, 1, 2, 3, 4 and 6 Gy), fused with mitotic Chinese hamster ovary (CHO) cells immediately or 1, 3, 6, 12 and 18 h after irradiation. Immediately after irradiation chromosomal breaks, dicentrics and translocations showed a linear dose-response. Unrejoined chromosome breaks were the most frequent types of aberrations (about 85%) observed. About 15% of total aberrations were chromosome exchanges of 65% of these were translocations and 35% were dicentrics. The chromosomal exchanges initially observed were mostly incomplete, with no complex exchanges at doses of 1 and 2 Gy, at higher doses (3-6 Gy) complex exchanges were observed and their frequencies increased with increasing post incubation time. Following different recovery times, repair kinetics of breaks for different doses of irradiation was studied. The shapes of the curves obtained for breaks as well as chromosome exchanges were linear-quadratic. The linear yield component, alpha, is formed entirely in the fast process that can be manifested in the early plateau, while component beta developed slowly in the subsequent hours. The kinetics of breaks rejoining was exponential, almost 50% of breaks rejoined after 1 h and at 18 h about 20% of breaks remained. At low doses of 1 and 2 Gy most of the exchanges were formed immediately and at higher doses, the frequency of exchanges increased with kinetics similar to that observed for the rejoining of breaks. However, the kinetics was different for different doses of irradiation. The frequency of dicentrics increased at doses above 2 Gy following 3 h recovery time, but for the translocations effect was pronounced even at 1 h recovery time. The frequency of incomplete exchanges (i.e., terminal translocations) decreased with post irradiation time and at 18 h was 30-40% less than the frequency obtained immediately after irradiation. The increase in the total translocations as a function of time between irradiation and fusion was due to a rapid increase in complete exchanges (i.e., reciprocal translocations). The frequency of ring chromosomes immediately after irradiation, also increased linearly, however, it was 3-5 times lower than dicentrics and remained almost constant in number for different doses and at different post-irradiation times.     

Anti-CD38 prevents the development of the adaptive response induced by X-rays in human lymphocytes

Irradiation of human lymphocytes (1 cGy X-rays, 37 degrees C) evoked an approximately 30% decrease in the frequency of micronuclei upon subsequent X-irradiation (1.5 Gy). The response was reflected in a lower micronucleus frequency but not in the DNA repair rate measured by the comet assay directly after the challenge dose. Treatment of lymphocytes with anti-CD38 antibody 1 h before irradiation with the adaptive dose prevented the development of the adaptive response measured as micronuclei frequency, but adaptation was not reflected in a lower rate of DNA repair, measured by the alkaline version of the 'comet' assay. In lymphocytes that were anti-CD38-treated and irradiated and or irradiated with the adaptive dose the rate of DNA repair was not changed. However, the mean DNA damage level in adapted anti-CD38-treated lymphocytes was significantly lower than that in the control lymphocytes at all time points. We conclude that ligation of CD38 by antibody initiates signalling that prevents the development of the adaptive response induced by X-rays. Lower chromosome damage revealed by the cytokinesis block-micronucleus test in the adapted lymphocytes is unrelated to DNA repair rate.     

Caspase-independent cell death induced by anti-CD2 or staurosporine in activated human peripheral T lymphocytes

We examined the effects of the cell-permeable, broad spectrum peptide caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD.fmk), and BOC-Asp(OMe)-fluoromethyl ketone (BOC-D.fmk), on apoptosis induced by anti-CD2, anti-Fas, and the protein kinase inhibitor staurosporine in activated human peripheral T lymphocytes. We monitored ultrastructural, flow cytometric, and biochemical apoptotic changes, including externalization of phosphatidylserine, cleavage of poly(ADP-ribose) polymerase (PARP) and lamins, activation of caspase-3 and caspase-7, decrease in mitochondrial membrane potential, and DNA fragmentation. Z-VAD.fmk and BOC-D.fmk completely inhibited all the biochemical and ultrastructural changes of apoptosis in anti-Fas-treated cells. In marked contrast, neither Z-VAD.fmk nor BOC-D.fmk inhibited CD2- or staurosporine-mediated cell shrinkage, dilatation of the endoplasmic reticulum (seen in anti-CD2-treated cells), externalization of phosphatidylserine, and loss of mitochondrial membrane potential that accompanied cell death. However, these inhibitors did inhibit the cleavage of PARP and lamins and the formation of hypodiploid cells, and partially inhibited chromatin condensation. These results demonstrate that in activated T cells, anti-CD2 and staurosporine induce a caspase-independent cell death pathway that exhibits prominent cytoplasmic features of apoptosis. However, caspase activation is required for the proteolytic degradation of nuclear substrates such as PARP and lamins together with the DNA fragmentation and extreme chromatin condensation that occur in apoptotic cells.     

Telomerase activity is induced in human peripheral B lymphocytes by the stimulation to antigen receptor

To understand the molecular events for the proliferation of B cells, we studied the induction of telomerase activity in vitro after stimulation to B-cell antigen receptor (BCR) on human peripheral B cells. Although unstimulated purified B cells of tonsils and peripheral blood from healthy volunteers do not express detectable telomerase activity, anti-IgM beads induce telomerase activity in these B cells. Soluble anti-IgM antibody (Ab) alone does not induce telomerase activity, but the second signal, given by either one of the cytokines of interleukin-2 (IL-2), IL-4, and IL-13 or by anti-CD40 monoclonal Ab (MoAb), is effective as the costimulation for the induction of the activity. Stimulation with anti-IgM Ab and anti-CD40 MoAb induces telomerase activity in most mature B cells of the tonsils and peripheral blood. The stimuli to both IgM and IgD receptors similarly induce the activity. Induction of telomerase activity is accompanied with the proliferation of B cells, but is not absolutely correlated with the extent of B-cell growth. Phorbol dibutylate (PDB) plus calcium (Ca) ionophore (PDB/Ca), which replace the activation through BCR and the costimulatory molecules, also induce telomerase activity. Moreover, it is suggested that phosphoinositide (PI) 3-kinase plays a role for the induction of telomerase activity in B cells stimulated with anti-IgM Ab and anti-CD40 MoAb. These results suggest that telomerase activity is induced in the B-cell activation of the antigen specific immune response.     

Alterations induced by Trypanosoma cruzi in activated mouse lymphocytes

Although a number of immunological anomalies have been shown to occur during the acute period of Trypanosoma cruzi infection, the contribution of the parasite has not been clarified. In this work, we co-cultured activated splenic mononuclear cells (SMC) from normal outbred (CD1) or inbred (CBA/J) mice with purified T. cruzi trypomastigotes and studied ensuing T- and B-lymphocyte alterations. In the presence of parasites, phytohaemagglutinin-stimulated SMC from either mouse background manifested a marked reduction in both lymphoproliferative capacity (i.e., 3H-thymidine incorporation) and cell membrane level of interleukin-2 receptors (IL-2R; determined by flow cytometry) relative to SMC from parasite-free cultures. Thus, substantial proportions of activated SMC either became unable to express detectable levels of IL-2R or expressed this receptor in significantly lower numbers than control SMC. Supernatants from T. cruzi suspensions reproduced these suppressive effects on phytohaemagglutinin-stimulated SMC from normal or chronically infected CD1 or CBA/J mice. Similar results were obtained with SMC activated with a bacterial lipopolysaccharide. Since IL-2R expression is required for activated lymphocytes to progress through the cell cycle and multiply to mount effective immune responses, impaired IL-2R expression by T. cruzi provides a plausible hypothesis for the wide-ranged immunosuppression that occurs in the infected host.     

R.b.e. for d(42MeV)-Be neutrons based on chromosome-type aberrations induced in human lymphocytes and scored in cells at first division

The irradiation of human lymphocytes with five doses of 250 kV X-rays and five doses of d(42MeV)-Be neutrons was performed in order to obtain dose-response curves for both radiation qualities. By using the FPC technique, aberration scoring was confined to first division cells only and dose-response curves were obtained at three sampling times. Sampling time independence was observed for chromosome-type aberrations and common curves could be fitted, confirming homogeneity of the initial lymphocyte population. R.b.e. values between 1x1 and 8x8 were obtained for dicentric yields between 0x01 and 2x0 per cell. Some variabilities were encountered for other aberration types. Mitotic delay showed an r.b.e. of 1x75. There was no evidence for any induction of SCE by either X-rays or neutrons, up to the highest doses used.     

Sister chromatid exchanges and cell division delays induced by diethylstilbestrol, estradiol, and estriol in human lymphocytes

It had been found previously that exposure of human lymphocytes in vitro to diethylstilbestrol (DES), a synthetic estrogen and known human carcinogen, led to the induction of sister chromatid exchanges. More sister chromatid exchanges were induced in cells from pregnant women than from men. To see if the effects of DES could be induced by other estrogens, lymphocytes from a man and a pregnant woman were treated in vitro with the natural estrogens estradiol and estriol. These did not induce sister chromatid exchanges. To see if the presence of exogenous female hormones might be responsible for the increase in DES-induced sister chromatid exchanges seen in cells from pregnant women, lymphocytes from a man and a pregnant woman were also treated in vitro simultaneously with DES, estradiol, estriol, and progesterone. Treatments with these exogenous hormones did not alter the number of DES-induced sister chromatid exchanges. Our previous studies showed that DES also inhibits in vitro proliferation of lymphocytes. The results reported here show that estradiol also strongly inhibits this proliferation but that estriol is only a weak inhibitor. The cause of delayed cell proliferation induced by DES and estradiol was 2-fold: some of the cells were delayed in phytohemagglutinin-mediated blast cell transformation but, additionally, most cells had a prolonged cell cycle because of an extended G2 phase. These studies also showed that DES, but not estradiol or estriol, induced a low level of polyploidy in human lymphocytes.