These proteins were often not detectable without PHA stimulation. (B) Dose response of fresh lymphocytes to PHA. Lymphocytes were stimulated with the indicated concentrations of PHA for 48 hrs. The expression of MLH1 and MSH2
proteins in fresh blood lymphocytes increased in a dose-dependent manner. (C) Dose response of immortalized lymphocytes to PHA. There was no effect of PHA on immortalized lymphocytes. MLH1 and MSH2 proteins were detectable even without PHA stimulation. Analysis of fresh lymphocytes (PHA treated) from a cohort of patients (N > 50 subjects) at high risk for LS, showed a bimodal distribution of MMR ratios (see histogram in Figure 3). The ratios ranged from 0.3 to 1.0 and peaks (mean ± SDE) were at 0.97 ± 0.02 and 0.81 ± 0.08. Stratification GS-9973 solubility dmso of results (shown as a scatter plot in Figure 3) shows that the MLH1 protein level is substantially reduced (“”plus”" symbols) in some fresh lymphocyte samples and MSH2 is reduced (“”diamond”" symbols) in other samples. In contrast, analysis of PHA stimulated fresh lymphocytes from normal controls revealed an MMR ratio close to 1.0 (Table 2). Analysis of normal controls and SW480 cells shows that the assay is highly reproducible (overall mean ± SDE = 0.96 ± 0.03). A selleck chemicals bimodal distribution was not seen for normal healthy control subjects. Figure 3 DNA mismatch repair protein
ratios for fresh lymphocyte samples from a population of individuals that were at high risk for having a germline MMR mutation. The left panel shows a scatter plot of MMR ratios. The “”+”" signs represent ratios where MLH1 was less than MLH2. The diamonds represent ratios many where MSH2 was less than MLH1. Because these plots were largely superimposable, we pooled them to establish the histogram shown in the right panel. The histogram shows that there is a bimodal distribution of MMR ratios. Moreover, the proportion of cases in the smaller mode (left most curve in right panel) is ~28%, which is very close to the proportion of patients (25%) at our recruitment site that have historically proved
to have a germline MMR mutation. Table 2 Reproducibility of the Western Blotting Assay* Cells Mean ± SDE SW480 0.989 ± 0.006 WBC Control 1 0.980 ± 0.018 WBC Control 2 0.967 ± 0.031 WBC Control 3 0.954 ± 0.059 WBC Control 4 0.921 ± 0.074 * Mean and standard deviation from MMR protein ratios determined from three different experiments on fresh WBCs from 4 control cases as well as SW480 colon cancer cells used as an internal control. Discussion A main finding of this study is that levels of MMR proteins can readily be measured in lymphocytes from fresh blood samples if the lymphocytes are first stimulated to proliferate by PHA. This supports our idea that a practical immunoassay for MMR proteins can be developed and used to screen for patients affected with the LS trait before they develop cancer.