Fluorescence intensity was quantified (counts per pixel) as a function of the incubation time using IPLab Spectrum software (Scanalytics, Fairfax, VA, USA). Experiments were repeated five times. Statistical analysis Mean��s.d. values were calculated for all data sets. The two-sided paired t-test was used to compare the effect of loss of MLH1 or MSH2 selleck inhibitor on PDT sensitivity. IC50-values were calculated by log-linear interpolation. P<0.05 was considered to be a statistically significant difference. RESULTS Clonogenic cell survival after PDT A point investigated was whether the sensitivity of tumour cells to PDT is affected by the MMR status. 6-Thioguanine, to which repair-deficient cells were, as expected, 4.2- (IC50; HCT116+ch2) and 5.6-fold (IC50; HEC59) more resistant than the repair-proficient sublines, was included as a control.

Figure 1 shows the survival curves for the MLH1-deficient HCT116+ch2 and the MSH2-deficient HEC59 cells as well as the respective repair-proficient HCT116+ch3 and HEC59+ch2 cell lines after PDT as a function of the optical dose (J cm?2). The optical dose required to induce cell death in 50% of all cells (IC50) was 0.32��0.03Jcm?2 for the HCT116+ch2 and 0.39��0.20Jcm?2 for the HCT116+ch3 cells (P=0.57). Likewise, MSH2-deficient tumour cells showed no altered sensitivity to PDT. The corresponding IC50-values were 0.54��0.06Jcm?2 for the MMR-deficient HEC59 and 0.46��0.17J cm?2 for the repair-proficient HEC59+ch2 cells (P=0.24). Thus, loss of MMR does not result in resistance to PDT.

Figure 1 Clonogenic survival curves for PDT for the MLH1-deficient and -proficient colon carcinoma cell lines and the MSH2-deficient and -proficient endometrial carcinoma cell lines. Each point represents the mean��s.d. of at least three experiments performed … MMR protein expression in MCF-7 cells after repetitive PDT exposure The question was addressed as to whether repetitive treatments with PDT result in de novo loss of expression of MMR proteins in the parental human breast cancer MCF-7 cells. These cells express MLH1, PMS2, MSH2, and MSH6 in amounts that are readily detectable by immunoblot, and they have previously been reported to be sensitive to PDT (Hornung et al, 1998). Expression of MMR proteins in MCF-7 cells after five subsequent exposures to PDT and in untreated MCF-7 cells was determined by immunoblot analyses.

Figure 2 shows that MCF-7 cells repeatedly treated with PDT express parental levels of MLH1, MSH2, MSH6 and PMS2. The sensitivity of the MCF-7 cells after five PDT treatments was similar to that of MCF-7 cells after a single exposure. Therefore, repeated exposure of tumour cells to PDT does not result in loss of MMR proteins. Anacetrapib Figure 2 Immunoblot of untreated MCF-7 cells (ut) and MCF-7 cells after five subsequent PDT exposures (PDT). MCF-7 cells repeatedly treated with PDT express parental levels of MLH1, MSH2, MSH6 and PMS2. ��-tubulin was used as a loading control.