Assessment of BCL2/J(H) translocation in healthy individuals exposed to low-level radiation of 137CsCl in Goiânia, Goiás, Brazil.

Healthy radio-exposed individuals who received low levels of Cesium-137 radiation during the accident that occurred in Goiânia in 1987, their families and controls were tested for the detection of t(14;18)-rearranged B cells in peripheral blood by using a highly sensitive, real-time quantitative PCR method. The chromosomal translocation t(14;18)(q32;q21) is characteristic of follicular lymphoma and is a frequent abnormality observed in other types of non-Hodgkin's lymphoma. This translocation leads to constitutive activation of the BCL2 oncogene by the enhancers of the immunoglobulin heavy-chain locus. In healthy individuals, the same translocation may also be found in a small fraction of peripheral blood lymphocytes, and positive cells might serve as an indicator for environmental exposure to carcinogens and possibly correlate with the cumulative risk of developing t(14;18)- positive non-Hodgkin's lymphoma. Twenty healthy radio-exposed individuals, 10 relatives and 10 non-exposed healthy individuals were tested for the detection of this translocation. Only 1 non-exposed individual was positive for the chromosomal translocation, and healthy radio-exposed individuals presented lower levels of cells bearing the BCL2/J(H) rearrangement when compared to the levels of the patients with follicular lymphoma before treatment. However, evaluation of more cells would be required to confirm the total absence of circulating cells bearing BCL2/J(H) rearrangement.

Interphase chromosome positioning affects the spectrum of radiation-induced chromosomal aberrations.

In interphase, chromosomes occupy defined nuclear volumes known as chromosome territories. To probe the biological consequences of the described nonrandom spatial positioning of chromosome territories in human lymphocytes, we performed an extensive FISH-based analysis of ionizing radiation-induced interchanges involving chromosomes 1, 4, 18 and 19. Since the probability of exchange formation depends strongly on the spatial distance between the damage sites in the genome, a preferential formation of exchanges between proximally positioned chromosomes is expected. Here we show that the spectrum of interchanges deviates significantly from one expected based on random chromosome positioning. Moreover, the observed exchange interactions between specific chromosome pairs as well as the interactions between homologous chromosomes are consistent with the proposed gene density-related radial distribution of chromosome territories. The differences between expected and observed exchange frequencies are more pronounced after exposure to densely ionizing neutrons than after exposure to sparsely ionizing X rays. These experiments demonstrate that the spatial positioning of interphase chromosomes affects the spectrum of chromosome rearrangements.

Gadolinium enhances the sensitivity of SW-1573 cells for thermal neutron irradiation.

Gadolinium neutron capture therapy (Gd-NCT) is an experimental cancer treatment based on the physical principal that neutron capture by gadolinium-157 ensures the release of focal high-dose radiation, such as gamma-rays and electrons. Survival and induction of chromosomal aberrations of human SW-1573 cells was studied after thermal neutron irradiation without and with gadolinium. After neutron irradiation with Gd-DTPA, an alpha-enhancement factor of 2.3 was obtained compared to thermal neutron irradiation alone. Gd-DTPA could not radioenhance the cells for gamma-ray irradiation. Induction of colour junctions and chromosome fragments by thermal neutron irradiation and Gd-NCT were studied using PCC-FISH. Correlations (r2-value) between survival and chromosome aberrations ranged from 0.81 to 0.94 for colour junctions and from 0.78 to 0.98 for chromosome fragments of chromosomes 18 and 2 respectively. Thermal neutron irradiation with or without gadolinium induced more chromosome aberrations than gamma-ray irradiation. After correction for chromosome length it appeared that both chromosomes were equally sensitive to radiation. It is concluded that Gd-NCT at a non-toxic concentration of gadolinium is effective in inducing cell death and chromosome aberrations in in vitro cell cultures.

Importance of TP53 and RB in the repair of potentially lethal damage and induction of color junctions after exposure to ionizing radiation.

Repair of potentially lethal damage (PLD) was investigated in cells with functional G1-phase arrest with wild-type TP53 and wild-type RB and in cells in which G1-phase arrest was abrogated by inactivation of TP53 or RB. Confluent cultures of cells were plated for clonogenic survival assay either immediately or 24 h after irradiation. Induction of color junctions, an exchange between a painted and unpainted chromosome, was studied in chromosomes 18 and 19 after irradiation with 4 Gy gamma rays. Significant repair of PLD was found in cells carrying both wild-type TP53 and wild-type RB. In cells in which TP53 or RB was inactivated, the survival curves from immediately plated and delayed-plated cells were not significantly different. The numbers of radiation-induced color junctions in chromosomes 18 and 19 were similar in all cell lines. From this study we conclude that a functional G1-phase arrest is important for repair of PLD and that TP53 and RB do not affect the frequencies of induction of color junctions in chromosome 18 or 19.

Significant large-scale chromosome territory movement occurs as a result of mitosis, but not during interphase.

PURPOSE: To investigate large-scale relative movement (position change) of interphase chromosome territories (ICT), as indicated by the separation of chromosome derivatives following exposure to ionizing radiation. MATERIALS AND METHODS: A simple experiment was conducted to investigate large-scale movement of ICT, using whole chromosome 18 painting to measure the frequency of fluorescent ICT in irradiated lymphocytes, cultured over 9 days (seven cell cycles). After chromosome 18 painting, derivative chromosome territory separation was indicated by the observation of three fluorescent ICT in aberrant cells compared with the presence of two fluorescent ICT in normal cells. The frequencies of interphase nuclei containing three fluorescent chromosome territories for unirradiated resting lymphocytes and for lymphocytes acutely irradiated with 2.25 or 5.5 Gy 137Cs gamma-rays were measured for each culture time point of 0, 1, 2, 3, 4, 7 and 9 days. RESULTS: No significant difference was observed in the frequency of three ICT between the average of the controls and irradiated cells cultured for 0, 1 and 2 days. However, irradiated cells cultured for >or=3 days showed a significant increase in the frequency of three fluorescent ICT over those cultured for

Gene amplification and microsatellite instability induced in tumorigenic human bronchial epithelial cells by alpha particles and heavy ions.

Gene amplification and microsatellite alteration are useful markers of genomic instability in tumor and transformed cell lines. It has been suggested that genomic instability contributes to the progression of tumorigenesis by accumulating genetic changes. In this study, amplification of the carbamyl-P-synthetase, aspartate transcarbamylase, dihydro-orotase (CAD) gene in transformed and tumorigenic human bronchial epithelial (BEP2D) cells induced by either alpha particles or (56)Fe ions was assessed by measuring resistance to N-(phosphonacetyl)-l-aspartate (PALA). In addition, alterations of microsatellite loci located on chromosomes 3p and 18q were analyzed in a series of primary and secondary tumor cell lines generated in nude mice. The frequency of PALA-resistant colonies was 1-3 x 10(-3) in tumor cell lines, 5-8 x 10(-5) in transformed cells prior to inoculation into nude mice, and less than 10(-7) in control BEP2D cells. Microsatellite alterations were detected in all 11 tumor cell lines examined at the following loci: D18S34, D18S363, D18S877, D3S1038 and D3S1607. No significant difference in either PALA resistance or microsatellite instability was found in tumor cell lines that were induced by alpha particles compared to those induced by (56)Fe ions.

Chromosomal breakpoint positions suggest a direct role for radiation in inducing illegitimate recombination between the ELE1 and RET genes in radiation-induced thyroid carcinomas.

The RET/PTC3 rearrangement is formed by fusion of the ELE1 and RET genes, and is highly prevalent in radiation-induced post-Chernobyl papillary thyroid carcinomas. We characterized the breakpoints in the ELE1 and RET genes in 12 post-Chernobyl pediatric papillary carcinomas with known RET/PTC3 rearrangement. We found that the breakpoints within each intron were distributed in a relatively random fashion, except for clustering in the Alu regions of ELE1. None of the breakpoints occurred at the same base or within a similar sequence. There was also no evidence of preferential cleavage in AT-rich regions or other target DNA sites implicated in illegitimate recombination in mammalian cells. Modification of sequences at the cleavage sites was minimal, typically involving a 1-3 nucleotide deletion and/or duplication. Surprisingly, the alignment of ELE1 and RET introns in opposite orientation revealed that in each tumor the position of the break in one gene corresponded to the position of the break in the other gene. This tendency suggests that the two genes may lie next to each other but point in opposite directions in the nucleus. Such a structure would facilitate formation of RET/PTC3 rearrangements because a single radiation track could produce concerted breaks in both genes, leading to inversion due to reciprocal exchange via end-joining.

In vitro radiation-induced neoplastic progression of low-grade uroepithelial tumors.

Recent interest has focused on the identification of molecular genetic mechanisms in multistep neoplastic transformation. In vitro exposure of simian virus 40 (SV40)-immortalized human uroepithelial cells (SV-HUC) that are environmentally relevant to bladder carcinogens has been shown to produce tumorigenic transformation, as assessed by the ability of cells exposed to a carcinogen to form xenograph tumors with heterogeneous cancer phenotypes ranging from very aggressive, invasive high-grade carcinomas to superficial low-grade indolent tumors. In addition, exposure of a low-grade indolent tumor generated in the SV-HUC system, MC-T11, to the same carcinogens results in neoplastic progression as assessed by the production of high-grade aggressive cancers. In the present study, we show neoplastic progression of MC-T11 after in vitro exposure to a single dose of 6 Gy X rays. In addition, we show that the chromosome deletions, including losses of 4q, 11p, 13q and 18, observed in these radiation-induced tumors are similar to those observed in carcinogen-induced tumors, thus supporting the hypothesis that the experimental cell system, not the transforming agent, dictates the genetic losses required for tumorigenic transformation and progression.