Growth speed plays a greater role than MMR status in determining the long-term survival of colon carcinoma cells treated with 5-FU

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

Materials and methods: We used the HCT116 cell line which was either transfected with hMLH1 plasmid (MMR⁺) or with empty vector (MMR^-) or the semiisogenic cell pair HCT116Chr3 (MMR⁺) and HCT116Chr2 (MMR^-). MMR^- and MMR⁺ cells were treated with 30µM 5-FU for 48h and clonogenic survival, long term growth, DNA double strand breaks (determined by H2AX histone phosphorylation), apoptosis (determined by PARP cleavage or degradation), necrosis (determined by LDH release) and the activation of the signalling pathways were followed by Western blots. Growth of MMR⁺ cells was slowed down by reducing the FCS concentration to 4% FCS in the medium.

Results: The clonogenic survival, which reflects the survival of the single cell after 2 days of treatment, was higher in the MMR^- than in the MMR⁺ cells and was independent of growth speed. The lower clonogenic survival of MMR⁺ cells was associated with more DNA double strand breaks and more apoptosis. The long-term total cell survival determined by cell count 5–10 days after treatment was lesser in fast-growing MMR⁺ cells than in slow-growing MMR⁺ cells. It was similar in MMR⁺ and MMR^- cells growing at the same speed. The lesser long-term cell survival of fast-growing cells was associated with a higher necrosis in these cells after 5-FU treatment.

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