Pairing of heterochromatin in response to cellular stress.

We previously reported that exposure of human cells to DNA-damaging agents (X-rays and mitomycin C (MMC)) induces pairing of the homologous paracentromeric heterochromatin of chromosome 9 (9q12-13). Here, we show that UV irradiation and also heat shock treatment of human cells lead to similar effects. Since the various agents induce very different types and frequencies of damage to cellular constituents, the data suggest a general stress response as the underlying mechanism. Moreover, local UV irradiation experiments revealed that pairing of heterochromatin is an event that can be triggered without induction of DNA damage in the heterochromatic sequences. The repair deficient xeroderma pigmentosum cells (group F) previously shown to fail pairing after MMC displayed elevated pairing after heat shock treatment but not after UV exposure. Taken together, the present results indicate that pairing of heterochromatin following exposure to DNA-damaging agents is initiated by a general stress response and that the sensing of stress or the maintenance of the paired status of the heterochromatin might be dependent on DNA repair.

Mitomycin C-induced pairing of heterochromatin reflects initiation of DNA repair and chromatid exchange formation.

Chromatid interchanges induced by the DNA cross-linking agent mitomycin C (MMC) are over-represented in human chromosomes containing large heterochromatic regions. We found that nearly all exchange breakpoints of chromosome 9 are located within the paracentromeric heterochromatin and over 70% of exchanges involving chromosome 9 are between its homologues. We provide evidence that the required pairing of chromosome 9 heterochromatic regions occurs in G(0)/G(1) and S-phase cells as a result of an active cellular process initiated upon MMC treatment. By contrast, no pairing was observed for a euchromatic paracentromeric region of the equal-sized chromosome 8. The MMC-induced pairing of chromosome 9 heterochromatin is observed in a subset of cells; its percentage closely mimics the frequency of homologous interchanges found at metaphase. Moreover, the absence of pairing in cells derived from XPF patients correlates with an altered spectrum of MMC-induced exchanges. Together, the data suggest that the heterochromatin-specific pairing following MMC treatment reflects the initiation of DNA cross-link repair and the formation of exchanges.

Human-hamster hybrid cells used as models to investigate species-specific factors modulating the efficiency of repair of UV-induced DNA damage.

The human-Chinese hamster hybrid cell line XR-C1#8, containing human chromosome 8, was used as a model system to investigate the relative importance of cellular enzymatic environment and chromosomal structure for modulating the efficiency of repair of UV-induced DNA damage. The hybrid cells were irradiated with UVC light and the extent of cytogenetic damage, detected as frequencies of sister chromatid exchanges (SCEs), was compared between the human and the hamster chromosomes. The combination of immunofluorescent staining for SCEs and chromosome painting with fluorescence in situ hybridization allowed the simultaneous analysis of SCEs in the human and hamster chromosomes. The aim of the present study was to determine if the differences in biological response to comparable UV treatments observed between human and hamster cells were maintained in the hybrid cells in which human and hamster chromosomes are exposed in the same cellular environment. The analysis of replication time of human chromosome 8 indicated the active status of this chromosome in XR-C1#8 hybrid cells. The frequencies of SCEs for human chromosome 8 and a hamster chromosome of comparable size were 0.35 +/- 0.52, 0.80 +/- 0.73, 1.24 +/- 2.24 and 0.36 +/- 0.12, 0.71 +/- 0.2, 0.97 +/- 0.27, respectively, after irradiation with 0, 5, and 10 J/m2. The persistence of UV-induced SCEs after three cell cycles was also analyzed, both for the human and hamster chromosomes. The observed frequencies of SCEs were 0.40 +/- 0.57, 0.62 +/- 1.05, 0.58 +/- 0.83 and 0.26 +/- 0.08, 0.67 +/- 0.18, 0.69 +/- 0.24, in human and hamster chromosomes respectively, after treatment with 0, 10, and 20 J/m2 of UVC light. No significant differences could be observed between the human and hamster chromosomes. These results suggest that the enzymatic environment of human and hamster cells has the main role, in comparison to the structural organization of human and hamster chromosomes, for determining the different level of repair of UV-induced DNA damage observed in these two species.

Ionizing radiation-induced instant pairing of heterochromatin of homologous chromosomes in human cells.

Using fluorescence in situ hybridization with human band-specific DNA probes we examined the effect of ionizing radiation on the intra-nuclear localization of the heterochromatic region 9q12-->q13 and the euchromatic region 8p11.2 of similar sized chromosomes 9 and 8 respectively in confluent (G1) primary human fibroblasts. Microscopic analysis of the interphase nuclei revealed colocalization of the homologous heterochromatic regions from chromosome 9 in a proportion of cells directly after exposure to 4 Gy X-rays. The percentage of cells with paired chromosomes 9 gradually decreased to control levels during a period of one hour. No significant changes in localization were observed for chromosome 8. Using 2-D image analysis, radial and inter-homologue distances were measured for both chromosome bands. In unexposed cells, a random distribution of the chromosomes over the interphase nucleus was found. Directly after irradiation, the average inter-homologue distance decreased for chromosome 9 without alterations in radial distribution. The percentage of cells with inter-homologue distance <3 micro m increased from 11% in control cells to 25% in irradiated cells. In contrast, irradiation did not result in significant changes in the inter-homologue distance for chromosome 8. Colocalization of the heterochromatic regions of homologous chromosomes 9 was not observed in cells irradiated on ice. This observation, together with the time dependency of the colocalization, suggests an underlying active cellular process. The biological relevance of the observed homologous pairing remains unclear. It might be related to a homology dependent repair process of ionizing radiation induced DNA damage that is specific for heterochromatin. However, also other more general cellular responses to radiation-induced stress or change in chromatin organization might be responsible for the observed pairing of heterochromatic regions.

Formation of chromosome aberrations: insights from FISH.

Most of the mutagenic and carcinogenic agents induce chromosome aberrations in vivo and in vitro. Conventional solid staining (such as Giemsa) has been employed to evaluate the frequencies and types of spontaneous and induced chromosomal aberrations. Recently, molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH) using chromosome specific or chromosome region-specific DNA libraries have become available, which have increased the resolution of the detection of aberrations. This has lead to a better understanding on the mechanisms of formation of chromosome aberrations, especially following treatment with ionizing radiation. The present paper reviews briefly the results obtained using FISH technique both from basic and applied studies.

Intrachanges as part of complex chromosome-type exchange aberrations.

The chromosome-type exchange aberrations induced by ionizing radiation during the G(0)/G(1) phase of the cell cycle are believed to be the result of illegitimate rejoining of chromosome breaks. From numerous studies using chromosome painting, it has emerged that even after a moderate dose of radiation, a substantial fraction of these exchanges is complex. Most of them are derived from the free interaction between the ends of three or more breaks. Other studies have demonstrated that chromosomes occupy distinct territories in the interphase nucleus. Since breaks that are in close proximity have an enhanced interaction probability, it seems likely that after ionizing radiation many of the interacting breaks will be present within one chromosome or chromosome arm. Unfortunately, the majority of these intrachanges remain undetected, even when sophisticated molecular cytogenetic detection methods (i.e. mFISH) are applied to paint all chromosome pairs in distinct colors. In the present paper, we evaluate the limitations of full-color painting for the detection of complex exchanges and the correct interpretations of break interactions.