CELLULAR HETEROGENEITY IN DNA-DAMAGE AND GROWTH-INHIBITION INDUCED BYICI D1694, THYMIDYLATE SYNTHASE INHIBITOR, USING SINGLE-CELL ASSAYS

Heterogeneity in the response of the HCT-8 (human ileocecal adenocarci noma) tumor cell line to a new thymidylate synthase inhibitor, ICI D16 94, was investigated in terms of induction of DNA single-strand breaks and cytotoxicity, applying the single cell alkaline gel (SCG) electro phoresis assay and the individual colony formation assay (iCFA), respe ctively. ICI D1694 induced maximal total DNA single-strand breaks 24 h r after a 2-hr drug exposure with incomplete repair by 72 hr. The leve l of DNA damage was concentration dependent and paralleled cellular gr owth inhibition in vitro. The proportion of cells with DNA damage and the extent of DNA single strand breaks increased with drug concentrati on. At 1 mu M ICI D1694 (IC95), a significant level of DNA damage was detected in 58% of the cells; however, 25% of the cells had little or no damage. Using the iCFA system, it was observed that with 1 mu M ICI D1694, only 2.6% of the seeded cells maintained a colony growth rate similar to that of the control colonies, and 22% of the cells were gro wing significantly more slowly. In conclusion, the SCG assay and the i CFA identified subpopulations of cells that were unaffected by ICI D16 94. Although these cells represented only a small proportion of the to tal cell population, this phenomenon of heterogeneity in response to I CI D1694 might limit its therapeutic efficacy.