gms | German Medical Science

Introduction: The cellular mismatch repair (MMR) system plays an important role in surveillance and repair of damaged DNA. The protein hMLH1, a crucial component of the MMR system is mutated in approximately 50% of MMR-defective tumours. Our previous work showed that the hMLH1 status influences the extent of CPT-11-induced tetraploid cell cycle arrest in colon carcinoma cells (Magrini et al., Int. J. Cancer. 2002). Here, we generated an isogenic system of HCT116 cells differing only in hMLH1 status. Our aim was to investigate the effect of hMLH1 on the response of colon carcinoma cells to the CPT-11 and its metabolite SN-38.

Materials and methods: Stable mock- and hMLH1-transfectants were generated by introducing the hMLH1 cDNA into MMR-deficient HCT116 cells. HCT116 and HCT116+chr3 (MMR-proficient) cell lines were also used as a reference. Sensitivity to the MMR-activating compound methyl nitrosourea, (MNU) or to SN-38 was determined by clonogenic assay. The cell cycle distribution was analysed by FACS. To determine the response in vivo, tumour xenografts were generated from HCT116 and HCT116+chr3 cells in nude mice.

Results: The HCT116+chr3 cells were more sensitive to MNU in clonogenic assay confirming that the MMR proficiency increases sensitivity to MNU. The hMLH1-expressing clones were also more sensitive to MNU than the hMLH1-deficient ones thus showing that the MMR system in the clones was functional. Treatment with MNU led to a G2/M arrest only in the hMLH1-expressing cells. In response to SN-38, the hMLH1-transfectants underwent a long-term tetraploid cell cycle arrest and showed a slower rate of cell proliferation as compared to the mock-transfectants. Treatment of tumour xenografts with CPT-11 led to a longer delay in the exponential growth phase of the hMLH1-proficient tumours as compared to the hMLH1-deficient ones.

Conclusions: 1. The MMR system is functional in the HCT116 transfectants which we have generated. 2. The presence of hMLH1 sensitizes cells to SN-38. 3. hMLH1 lowers the rate of cell proliferation in response to SN-38 treatment. 4. The experiments with tumour xenografts confirm the in vitro data and show that hMLH1- tumours are more resistant to CPT-11 than hMLH1+ tumours. This isogenic system is suitable for the investigation of the role of hMLH1 in response to other chemotherapeutic agents.

*This work has been supported by the Ernst von Leyden Stipendium from the Berliner Krebsgesellschaft to Mandar Bhonde.