Topoisomerase IIα levels and G2 radiosensitivity in T-lymphocytes of women presenting with breast cancer.

Previous studies from our laboratory have identified a link between intracellular topoisomerase IIα (topo IIα) levels and chromosomal radiosensitivity, as measured by the frequencies of chromatid breaks in the so-called G2-assay. Lower topo IIα levels were associated with reduced chromosomal radiosensitivity in cultured human cells. These findings supported a model, in which it is proposed that such chromatid breaks are the result of radiation-induced errors made by topoisomerase IIα during decatenation of chromatids. Studies from our and other laboratories, using the G2-assay, have shown that phytohaemagglutinin (PHA)-stimulated peripheral blood T-lymphocytes from 40% of female breast cancer cases show elevated chromatid break frequencies when exposed to a small standard dose of ionizing radiation, i.e. elevated above the 90th percentile of a group of female control samples. In the present study we have used a modified G2-assay to test whether elevated frequency of chromatid breaks in breast cancer cases is linked with elevated intracellular topo IIα level in PHA-stimulated T-lymphocytes, and also whether there is a general correlation between chromosomal radiosensitivity and topo IIα level. Our results confirm previous studies that 40% of breast cancer cases show elevated radiosensitivity as compared with controls. Also, the mean chromatid break frequency in breast cancer cases was significantly higher than in controls (P = 0.0001). We found that the mean topo IIα level in the cohort of breast cancer cases studied was significantly raised, as compared with controls (P = 0.0016), which could indicate a genetic propensity towards a raised intracellular production of topo IIα in these individuals. There was no direct correlation between chromosomal radiosensitivity and topo IIα level for individual samples either in the breast cancer cohort or in controls. However, a comparison between control and breast cancer samples shows a higher mean topo IIα level in breast cancer samples that correlates with the elevated mean chromatid break frequency seen in these patient samples. We found no meaningful correlations between either chromatid break frequency or topo IIα level and either tumour grade or hormone status. We conclude that elevated intracellular topo IIα level is likely to be a significant factor in determining the chromosomal response of stimulated T-lymphocytes from certain breast cancer cases.

Mechanisms of the formation of radiation-induced chromosomal aberrations.

Although much is now known about the mechanisms of radiation-induction of DNA double-strand breaks (DSB), there is less known about the conversion of DSB into chromosomal aberrations. In particular the induction and 'rejoining' of chromatid breaks has been a controversial topic for many years. However, its importance becomes clear in the light of the wide variation in the chromatid break response of human peripheral blood lymphocytes from different individuals when exposed to ionizing radiation, and the elevation of the frequency of radiation-induced chromatid breaks in stimulated peripheral blood lymphocytes of around 40% of breast cancer cases. A common assumption has been that chromatid breaks are merely expansions of initiating DSB, although the classic 'breakage-first' hypothesis (Sax, Ref. 44) was already challenged in the 50's by Revell [30] who maintained that chromatid breaks were formed as a result of an incomplete exchange process initiated by two interacting lesions of an unspecified nature. Here we argue that both these models of chromatid break formation are flawed and we suggest an alternative hypothesis, namely that a radiation-induced DSB initiates an indirect mechanism leading to a chromatid break. This mechanism we suggest involves the nuclear enzyme topoisomerase IIalpha and we present evidence from topoisomerase IIalpha expression variant human cell lines and from siRNA treatment of human cells that supports this hypothesis.

Suppression of topoisomerase IIalpha expression and function in human cells decreases chromosomal radiosensitivity.

The mechanism behind chromatid break formation is as yet unclear, although it is known that DNA double-strand breaks (DSBs) are the initiating lesions. Chromatid breaks formed in cells in the G2-phase of the cell-cycle disappear ('rejoin') as a function of time between radiation exposure and cell fixation. However, the kinetics of disappearance of chromatid breaks does not correspond to those of DSB rejoining, leading us to seek alternative models. We have proposed that chromatid breaks could be formed indirectly from DSB and that the mechanism involves topoisomerase IIalpha. In support of this hypothesis we have recently shown that frequencies of radiation-induced chromatid breaks are lower in two variant human promyelocytic leukaemic cell lines with reduced topoisomerase IIalpha expression. Here we report that suppression of topoisomerase IIalpha in human hTERT-RPE1 cells, either by its abrogation using specific siRNA or by inhibition of its catalytic activity with the inhibitor ICRF-193, causes a reduction in frequency of chromatid breaks in radiation-exposed cells. The findings support our hypothesis for the involvement of topoisomerase IIalpha in the formation of radiation-induced chromatid breaks, and could help explain inter-individual variation in human chromosomal radiosensitivity; elevation of which has been linked with cancer susceptibility.

An improved assay for radiation-induced chromatid breaks using a colcemid block and calyculin-induced PCC combination.

We report on a new method for the study of radiation-induced chromatid breaks in stimulated human peripheral blood T lymphocytes, involving a combination of a 1-h colcemid block and a short (15 min) calyculin A treatment. We find that this procedure eliminates the problem of centromere splitting when calyculin A is used alone for a longer period and produces metaphase spreads with superior quality. By this procedure, the chromosomes and the chromatid breaks are expanded and thereby make for improved break scoring. In a comparison of the new technique with the conventional colcemid block method, we show a close proportionality between the frequencies of chromatid breaks scored with the two methods. The frequency of chromatid breaks with the new method was found to be significantly higher than that with colcemid alone, adding a higher sensitivity to the assay as an additional advantage.

Adaptive response: some underlying mechanisms and open questions: [review]

Organisms are affected by different DNA damaging agents naturally present in the environment or released as a result of human activity. Many defense mechanisms have evolved in organisms to minimize genotoxic damage. One of them is induced radioresistance or adaptive response. The adaptive response could be considered as a nonspecific phenomenon in which exposure to minimal stress could result in increased resistance to higher levels of the same or to other types of stress some hours later. A better understanding of the molecular mechanism underlying the adaptive response may lead to an improvement of cancer treatment, risk assessment and risk management strategies, radiation protection, e.g. of astronauts during long-term space flights. In this mini-review we discuss some open questions and the probable underlying mechanisms involved in adaptive response: the transcription of many genes and the activation of numerous signaling pathways that trigger cell defenses - DNA repair systems, induction of proteins synthesis, enhanced detoxification of free radicals and antioxidant production.

Radiation sensitivity of leukocytes from healthy individuals and breast cancer patients as measured by the alkaline and neutral comet assay.

Initial radiation-induced DNA damage, dose-response curves and kinetics of DNA repair in leukocytes from healthy volunteers and breast cancer patients, was assessed using alkaline and neutral comet assay after exposure to (60)Co gamma rays. Both versions of comet assay showed higher levels of baseline DNA damage in leukocytes of breast cancer cases than in controls. Gamma ray induced initial DNA damage in leukocytes of cancer cases was not significantly different from that of healthy donors. A similar dose-response was obtained with both versions of comets for two groups. After a repair time of 24h, following irradiation, samples from the healthy individuals showed no residual DNA damage in their leukocytes, whereas breast cancer patients revealed more than 20%. Although similar initial radiosensitivity was observed for both groups but the repair kinetics of radiation-induced DNA damage of leukocytes from breast cancer cases and healthy subjects was statistically different.

A comparison of G2 phase radiation-induced chromatid break kinetics using calyculin-PCC with those obtained using colcemid block.

To study the possible influence of cell-cycle delay on cells reaching mitosis during conventional radiation-induced chromatid break experiments using colcemid as a blocking agent, we have compared the chromatid break kinetics following a single dose of gamma rays (0.75 Gy) in metaphase CHO cells using calyculin-induced premature chromosome condensation (PCC), with those using colcemid block. Calyculin-induced PCC causes very rapid condensation of G2 cell chromosomes without the need for a cell to progress to mitosis, hence eliminating any effect of cell-cycle checkpoint on chromatid break frequency. We found that the kinetics of the exponential first-order decrease in chromatid breaks with time after irradiation was similar (not significantly different) between the two methods of chromosome condensation. However, use of the calyculin-PCC technique resulted in a slightly increased rate of disappearance of chromatid breaks and thus higher frequencies of breaks at 1.5 and 2.5 h following irradiation. We also report on the effect of the nucleoside analogue ara A on chromatid break kinetics using the two chromosome condensation techniques. Ara A treatment of cells abrogated the decrease in chromatid breaks with time, both using the calyculin-PCC and colcemid methods. We conclude that cell-cycle delay may be a factor determining the absolute frequency of chromatid breaks at various times following irradiation of cells in G2 phase but that the first-order disappearance of chromatid breaks with time and its abrogation by ara A are not significantly influenced by the G2 checkpoint.

Repair and chromosomal damage.

Chromosomal aberrations in somatic cells link DNA damage with radiation-induced cell killing and individual susceptibility to oncogenesis, and are also potential markers of cancer susceptibility. While there is general acceptance that the DNA double-strand break (DSB) is the principal initiating lesion the complexity of the relationship between the induced frequency and the rates of repair and misjoining of DSB, and the production of chromosome and chromatid aberrations has led to much controversy. The principal models of chromosome aberrations are: the classical 'breakage-and-reunion' or 'breakage-first' model of Sax [Genetics 25 (1940) 41-68], the 'mis-recombination' model of Chadwick and Leenhouts [Mutat Res 404 (1998) 113-117] and the 'transcription-based' model of Radford [Int J Radiat Biol 78 (2002) 1081-1093]. Chromatid aberrations have also been variously interpreted on the 'breakage-first model', Revell's 'exchange' model [Proc R Soc B 150 (1959) 563-589] and the 'signal' model [Int J Radiat Biol 73 (1998) 243-251]. Recent evidence argues strongly for different mechanisms for chromosome (formed in G1 or Go) and chromatid (formed in G2) aberrations, i.e. there is little or no correspondence in the relative frequencies between chromosome and chromatid aberrations. The balance of evidence indicates that chromosome aberrations may be formed by a breakage-first type mechanism. Elevated frequencies of chromosomal aberrations occur to various extents in cell lines mutated in genes involved in both non-homologous DSB end-joining and homologous recombinational rejoining of DSB. Chromatid breaks, seem to be formed by a more complex mechanism since there is a lack of correspondence between the rates of DSB rejoining and chromatid break 'disappearance' (assumed by some to represent DSB repair). Thus, a model based on the dissociation of DSB rejoining from chromatid break rejoining is required to explain these data. A substantial proportion (approximately 20%) of both spontaneous and induced chromatid breaks visibly involve inter-chromatid rearrangements (determined using harlequin staining of chromatids). It is postulated that the remaining proportion may also involve rearrangements, but within a single chromatid (i.e. intra-chromatid rearrangements). Disappearance of chromatid breaks with time is postulated to result from the completion of rearrangements, i.e. rather than simply from repair of DSB.

Interaçäo social e inferência lógica / Social interaction and logical inferences

Em um estudo de intervençäo, investiga o efeito da interaçäo social no nível de desempenho de crianças, em uma tarefa lógico-inferencial envolvendo deduçäo sobre números. Os Ss säo 72 crianças (M=36; F=36) que freqüentavam o curso de alfabetizaçäo em escola particular, com idades variando entre 4 anos e 1 mês e 5 anos e 5 meses. Subdivide-os em 3 grupos (um de controle e dois experimentais) cada um composto por 12 crianças. Submete-os ao experimento teste da balança em 4 fases, cuja tarefa consiste em julgar o n§ de balas dentro de uma caixa opaca (caixa alvo A) comparando o seu peso com o de outras caixas (caixas comparativas 1 [Ca1] e 2 [Ca2] com a ajuda de uma balança. Verifica se esta simples tarefa inferencial melhorava em funçäo da interaçäo social. Os resultados indicam que crianças que passaram por uma experiência de cooperaçäo com crianças mais habilidosas nesta tarefa melhoravam mais do que outras que näo passaram por este tipo de experiência. Conclui que a interaçäo social pode melhorar a habilidade das crianças em operar inferências lógicas

Correspondência: um esquema quantitativo básico / Correspondence: a basic quantitative strategy

Os problemas de comparaçäo em aritmética säo difíceis para crianças de 5 a 7 anos. Uma estratégia eficaz usada por algumas crianças nessa idade consiste em aplicar a correspondência termo-a-termo aos conjuntos comparados. Analisamos a eficácia do treinamento no uso da correspondência espacial ou temporal sobre a resoluçäo de problemas comparativos. 180 crianças em dois níveis de instruçäo (pré-escolar e primeira série) e de idade (5/6 e 6/7 anos) de três escolas particulares de Recife foram randomicamente distribuidas entre um grupo de controle e dois experimentais, os quais recebiam instruçäo no uso da correspondência espacial ou temporal, respectivamente. Oobservaram-se ganhos significativos apenas no grupo instruído em correspondência espacial, näo havendo interaçäo entre idade e treinamento. As crianças desse grupo mostraram desempenho superior áquele tipicamente observado nessa faixa etária, o que indica a possibilidade de instruçäo em resoluçäo de problemas desde o pré-escolar