J. N. Lucas

Rapid Translocation Frequency Analysis in Humans Decades after Exposure to Ionizing Radiation

This paper presents an analysis of the utility of fluorescence in situ hybridization (FISH) with whole-chromosome probes for measurement of the genomic frequency of translocations found in the peripheral blood of individuals exposed to ionizing radiation. First, we derive the equation: Fp = 2.05fp(1-fp)FG, relating the translocation frequency, Fp, measured using FISH to the genomic translocation frequency, FG, where fp is the fraction of the genome covered by the composite probe. We demonstrate the validity of this equation by showing that: (a) translocation detection efficiency predicted by the equation is consistent with experimental data as fp is changed; (b) translocation frequency dose-response curves measured in vitro using FISH agree well with dicentric frequency dose-response curves measured in vitro using conventional cytogenetic procedures; and (c) the genomic translocation frequencies estimated from FISH measurements for 20 Hiroshima A-bomb survivors and four workers exposed to ionizing radiation during the Y-12 criticality accident are approximately the same as the translocation frequencies measured using G-banding. We also show that translocation frequency dose response curves estimated using FISH are similar for Hiroshima A-bomb survivors and for first division lymphocytes irradiated in vitro. We conclude with a discussion of the potential utility of translocation frequency analysis for assessment of the level of acute radiation exposure independent of the time between analysis and exposure.

Rapid human chromosome aberration analysis using fluorescence in situ hybridization

We have used in situ hybridization of repeat-sequence DNA probes, specific to the paracentromric locus 1q12 and the telomeric locus 1p36, to fluorescently stain regions that flank human chromosome 1p. This procedure was used for fast detection of structural aberrations involving human chromosome 1p in two separate experiments. In one, human lymphocytes were irradiated with 0, 0.8, 1.6, 2.4 and 3.2 Gy of 137Cs gamma-rays. In the other, human lymphocytes were irradiated with 0, 0.09, 0.18, 2.0, 3.1 and 4.1 Gy of 60Co gamma-rays. The frequencies (per cell) of translocations and dicentrics with one breakpoint in 1p and one elsewhere in the genome were determined for cells irradiated at each dose point. These frequencies both increased with dose, D, in a linear-quadratic manner. The delta, alpha, and beta coefficients resulting from a fit of the equation f(D)=delta + alphaD + betaD2 to the translocation frequency dose-response data were 0.0025, 0.0027 and 0.0037 for 137Cs gamma-rays, and 0.0010, 0.0041, and 0.0057 for 60Co gamma-rays. The delta, alpha, and beta coefficients resulting from a fit to the dicentric frequency dose-response data were 0.0005, 0.0010 and 0.0028 for 137Cs gamma-rays and 0.0001, 0.0002 and 0.0035, for 60Co gamma-rays. Approximately 32,000 metaphase spreads were scored in this study. The average analysis rate was over two metaphase spreads per minute. However, an experienced analyst was able to find and score one metaphase spread every 10s. The importance of this new cytogenetic analysis technique for biological dosimetry and in vivo risk assessment is discussed.

Two-color hybridization with high complexity chromosome-specific probes and a degenerate alpha satellite probe DNA allows unambiguous discrimination between symmetrical and asymmetrical translocations

This report describes a fluorescence in situ hybridization approach to chromosome staining that facilitates detection of structural aberrations and allows discrimination between dicentric chromosomes and symmetrically translocated chromosomes. In this approach, selected whole chromosomes are stained in one color by hybridization with composite probes whose elements have DNA sequence homology along the length of the target chromosomes. In addition, all chromosomes are counterstained with a DNA specific dye so that structural aberrations between target and non-target chromosomes are clearly visible. Discrimination between dicentric chromosomes and symmetrical translocations is accomplished by hybridization with a second probe that is homologous to DNA sequences found in the centromeric region of all chromosomes. The centromeric marker is visualized in a different color, so that the number of centromeres per aberrant chromosome can be rapidly determined in the microscope by changing excitation and fluorescence filters.

A comparison of the yields of translocations and dicentrics measured using fluorescence in situ hybridization.

Chromosome aberration analysis using fluorescence in situ hybridization (FISH) methods without centromere-specific markers results in the misscoring of a substantial fraction of the dicentrics as translocations; that is, too many translocations and too few dicentrics are scored. Such misscoring has led to considerable confusion in the rapidly emerging literature on FISH-based cytogenetics. Here, we demonstrate that the problem is fully resolved when centromeric probes are used.

The persistence of chromosome translocations in a radiation worker accidentally exposed to tritium

The chromosome translocation frequency in lymphocytes of an individual accidentally exposed to tritium six years previously was measured using chromosome painting. Comparisons with results from cytogenetic studies shortly after the accident indicate that the translocation frequency has remained unaltered in this individual for six years.

A New Method for Improving Metaphase Chromosome Spreading

The success of complex molecular cytogenetic studies depends on having properly spread chromosomes. However, inconsistency of optimum chromosome spreading remains a major problem in cytogenetic studies.

Distinct profiles of critically short telomeres are a key determinant of different chromosome aberrations in immortalized human cells: whole-genome evidence from multiple cell lines.

Chromosomal aberrations are common in cancers. However, the search for chromosomal aberrations leading to development of specific solid tumors has been severely hindered because the majority of solid tumors have complex chromosomal aberrations that differ within the same tumor types. A similar phenomenon exists in immortalized cell lines. The underlying mechanisms driving these diverse aberrations are largely unknown. Telomeres play crucial roles in protecting the integrity of eucaryotic chromosomes and maintaining genomic stability of human cells. Telomere lengths on individual chromosomes in normal human somatic cells are heterogeneous and undergo progressive shortening with aging process. In this study, for the first time, a molecular cytogenetic method using sequential telomere quantitative fluorescence in situ hybridization and spectral karyotyping on the same human metaphases was applied successfully to examine the dynamic profiles of individual telomere shortening and their relationship to chromosome aberrations in multiple human cell lines undergoing immortalization. Human ovarian surface epithelial cells and esophageal epithelial cells were immortalized by the expression of HPV16 E6 and E7, which drive cells to proliferate by inactivating p53 and Rb genes. In these cell lines, we consistently detected large-scale differences in telomere signal intensities not only among nonhomologous chromosome arms but also between some homologous chromosome arms. The cell lines derived from different donors had different profiles of critically short telomeres (lacking telomere signals). Strikingly, the different profiles of chromosomal structural aberrations in multiple immortalized cell lines were highly significantly associated with the distinct distributions of critically short telomeres in whole-genome. Since cellular immortalization is one of the hallmarks of cancer, our findings suggest that distinct profiles of critically short telomeres in different human individuals might play an essential role in determining the complex and individual-specific chromosomal structural aberrations in human solid tumors.

Translocations between two specific human chromosomes detected by three-color “chromosome painting”

Translocations between two specific chromosomes are important markers for many human malignancies. Previously, the detection of translocations involving random breakpoints between two specific chromosomes could only be accomplished by banding techniques, which are severely labor intensive and require highly trained technicians. The three-color chromosome painting approach described in this paper was developed in our laboratory to detect translocations between two specific human chromosomes rapidly and accurately, while simultaneously revealing the nonhybridized chromosomes. Because this method efficiently detects translocations involving breakpoints anywhere on the targeted chromosomes, it is ideal as a screening tool for chromosome-specific translocations.

Ratios of radiation-produced chromosome aberrations as indicators of large-scale DNA geometry during interphase

Chromosome aberrations produced by ionizing radiation are assumed to develop from DNA double-strand breaks (DSBs) which interact pairwise. Stable chromosome aberrations that exemplify inter- and intra-chromosomal exchanges are, respectively, translocations and pericentric inversions. By comparing the number of these for each chromosome one can infer results on the randomness of DSB induction or exchange formation and on large-scale chromosome geometry. We analyze frequencies of translocations and pericentric inversions in lymphocytes from 38 A-bomb survivors, using G-banding. A total of 636 translocations and 102 pericentric inversions were found. The 636/102 ratio of translocations to pericentric inversions is approximately 14 times smaller than predicted by a random model, in general agreement with earlier results and results on the ratio of dicentrics to centric rings for in vitro irradiation. Presumably the excess of intra-chromosomal exchanges is due to a spatial proximity effect, implying a localization of chromosomes within the cell nucleus during and shortly after irradiation. The distribution of the pericentric inversions among different chromosomes indicates this proximity effect is roughly the same for all chromosomes, regardless of DNA content; i.e., the ratio of pericentric inversions for two different chromosomes approximately equals the ratio given by a model which takes into account chromosome lengths and centromere locations but otherwise assumes randomness. Possible exceptions are chromosomes 7 and 12, which show some excess of pericentric inversions. The percentage of translocations involving each chromosome corresponds roughly to the percentage expected assuming randomness, except that for chromosome 1 there is a significant excess.

Dose reconstruction for individuals exposed to ionizing radiation using chromosome painting

To be most useful, a biomarker for dose reconstruction should employ an end point that is highly quantitative, stable with time and easily measured. Reciprocal translocations have been shown to be a promising biomarker that is linked to both prior exposure and risk, and they can be measured easily and quantitatively using fluorescence in situ hybridization. In contrast to other biomarkers that are available, the frequency of reciprocal translocations in individuals exposed to whole-body radiation is stable with time after exposure, has rather small interindividual variability and can be measured accurately at low levels of exposure. Results from recent studies demonstrate that measurements of reciprocal translocation frequencies, facilitated by chromosome painting, can be used to reconstruct radiation dose for individuals exposed in the distant past.