Z Gastroenterol 2010; 48 - P167
DOI: 10.1055/s-0030-1263611

MMR status determines short-term apoptosis while growth speed determines necrosis and long-term survival of colon carcinoma cells after 5-FU treatment

B Choudhary 1, ML Hanski 1, M Zeitz 1, C Hanski 1
  • 1Charité-Campus Benjamin Franklin, Department of Gastroenterology, Berlin, Germany

Introduction: Whether mutations in the hMLH1 gene, which is a component of the mismatch repair (MMR) system, increase or decrease the sensitivity of colon carcinomas to 5-fluorouracil (5-FU) is a matter of controversy. We investigated separately the effects of growth speed and of MMR status on apoptosis, necrosis and long-term survival of colon carcinoma cells after 5-FU treatment.

Materials and methods: We used HCT116 cell line which was either stably transfected with hMLH1 plasmid (MMR+) or with empty vector (MMR-) or the semiisogenic cell pair HCT116Chr3 (MMR+) and HCT116Chr2 (MMR-). Doubling time of MMR- and MMR+ cells was determined and cells were treated with 30µM 5-FU for 2 days. Long-term cell survival was determined 5–14 days after treatment by counting live cells. The colony forming ability of treated cells was measured in clonogenic assay. PARP cleavage (apoptosis) and histone H2AX phosphorylation (DNA double strand breaks) were determined in western blot. For detection of necrosis, LDH release was measured.

Results: MMR- or MMR+ cells growing at the same speed showed similar necrosis and long-term survival after treatment. However, MMR+ cells showed more DNA double strand breaks, more short-term apoptosis and less clonogenic survival afte 5-FU treatment than MMR- cells. The investigation of the effect of growth speed on cell survival after treatment showed that fast-growing cells were associated with high long-term necrosis and less long-term survival than slow-growing cells with the same MMR status. Thus, growth speed does not affect short-term apoptosis but it determines long-term necrosis which is associated with less long-term cell survival.

Conclusions: The result indicates that the MMR status determines short-term apoptosis and clonogenic survival whereas growth speed determines necrosis and long-term survival of colon carcinoma cells after 5-FU treatment. This suggests that the growth speed of a tumor would be a better predictor of the therapeutic effect of 5-FU in colon cancer patients than the MMR status and explains the contradictory data on the effect of the MMR status on the response to 5-FU.