The use of fluorescence in situ hybridisation combined with premature chromosome condensation for the identification of chromosome damage

Jw Evans,D Pinkel,Jm Brown,Ja Chang,Aj Giaccia

doi:10.1038/bjc.1991.123

Abstract

The technique of fusing mitotic cells to interphase cells, thereby producing condensation of the chromosomes of the interphase cell (so-called 'premature chromosome condensation' or PCC), has allowed detection of the initial number of chromosome breaks and their repair following ionising radiation. However, the difficulty and tedium of scoring all the chromosome fragments, as well as the inability to readily detect exchange aberrations, has limited the use of PCC. We describe here the use of the recently developed technique of fluorescence in situ hybridisation with whole chromosome libraries to stain individual human chromosomes (also called 'chromosome painting') with the PCC's and show that this overcomes most of the limitations with the analysis of PCC's. First, by focusing on a single chromosome, scoring of breaks in the target chromosome is easy and rapid and greatly expands the radiation dose range over which the PCC technique can be used. Second, it allows the easy recognition of exchange type aberrations. A number of new applications of this technology, such as predicting the radiosensitivity of human tumours in situ, are feasible.

Highlights

In this paper we describe the combination of the premature chromosome condensation (PCC)
In the present paper we demonstrate the use of chromosome painting with specific full length human chromosome probes to PCC's derived from diploid human cells
Since CHO cells do not cross hybridise to human probes, we performed the initial experiments with CHO mitotic cells in order that only human PCC's would be visualised

Summary

Methods

The normal human fibroblast cell line AG1522 was obtained from Dr Michael Cornforth (Los Alamos National Laboratory, Los Alamos, NM 87545). HeLa and HT1080, were obtained from Dr Joel Bedford (Colorado State University, CO 80523) and the American Culture Collection, respectively. AG1 522 cells were maintained at low passage in a-MEM (GIBCO, Grand Island, NY), supplemented with 15% foetal bovine serum (FBS). HeLa and HT1080 cells were grown in the same medium supplemented with 10% FBS. Mitotic HeLa and HT1080 cells were partially synchronised with 2 mM hydroxyurea (HU, Sigma, St. Louis, MO) for 12 h (Cornforth & Bedford, 1983a). HU was washed from the medium, and cells were allowed to progress through the cell cycle for 7 h.

Results

Discussion

Conclusion