PCC + CHROMOSOME PAINTING IN RADIATION AND DRUG STUDIES

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The overall goal of this project is to use the technique of fluorescence in situ hybridization (FISH, or "chromosome painting") combined either with premature chromosome condensation (PCC) or with metaphase spreads as a means of predicting the response of solid tumors to radiation. No method of predicting the radiation sensitivity of individual human tumors is presently available, and recently published data show that other factors, in addition to the intrinsic radiosensitivity of the tumor cells, play a major role in the in vivo response of human tumors. Since these factors likely involve the tumor milieu (e.g., cell-cell contact and tumor hypoxia), an in situ assay of radiosensitivity is required. This should be made following the first one of few doses in the radiotherapy regimen. Although an analysis based on chromosome damage is the only suitable damage is impractical. By allowing the visualization of chromosomes in interphase cells, the PCC technique overcomes the need to culture the tumor cells in vitro, but the technical problem remains of counting a small excess number of breaks over the often large pretreatment chromosome number. However, the combination of FISH with PCC enormously simplifies the problem by focusing the analysis on a single chromosome. It also allows exchange aberrations to be scored easily. The purpose of this project is to develop the PCC + FISH technique to the point at which it could be used clinically as a predictive assay. Initially, the simplest system will be used, that of G1 diploid human AG1522 fibroblasts, whose survival and PCC induction rate and repair following irradiation are known. Next, aneuploid human tumor cells (squamous carcinoma and melanoma), covering a wide range of radiation sensitivities, will be used, and finally the in vivo response of these tumor lines will be determined. In all cases, the hypothesis will be tested that chromosome damage after repair (breaks and exchanges) scored by PCC + FISH closely predict the radiation sensitivity as determined by clonogenic assay. The FISH technology will also be used to estimate radiation sensitivity from stable reciprocal translocations in metaphase in the same cell lines as used with PCC + FISH above. The ability to combine whole chromosome painting of 1-4 chromosomes with a second color hybridization to the repeat sequences common to the centromeres allows the unambiguous discrimination between symmetrical and asymmetrical translocations. Since the frequency of stable translocations should correlate with initial chromosome damage and since these translocations are not preferentially lost from the irradiated tumor cell population by cell death an estimate of tumor cell killing following 1-5 dose fractions should be possible. Each of these two methods has its advantages, and a careful study of the two should establish which is superior for routine use to determine tumor radiosensitivity in situ.