In vitro expression levels of cell-cycle checkpoint proteins are associated with cellular DNA repair capacity in peripheral blood lymphocytes: a multivariate analysis

Abstract

DNA repair should occur after cells sense DNA damage signals and undergo cell-cycle arrest to provide sufficient time for DNA repair, and suboptimal DNA repair capacity (DRC) in peripheral lymphocytes has been suggested as a cancer susceptibility marker. Numerous studies showed a functional link between DNA damage sensing, cell-cycle checkpoint, and DNA repair. We hypothesized that in vitro cell-cycle checkpoint-related protein expression levels in stimulated lymphocytes predict DRC levels. To test this hypothesis, we performed the host-cell reactivation assay for DRC by transfecting stimulated peripheral blood lymphocytes from 120 normal donors with transient expression plasmids damaged by benzo[a]pyrene diol epoxide (BPDE). The same cells were assessed for protein expression induction of eight cell-cycle checkpoint-related genes using the reverse-phase protein lysate microarray assay. In multivariate linear regression analysis adjusting for age, sex, blastogenic rate, and sample storage duration, the association between DRC and expression levels of cell-cycle checkpoint-related proteins induced by BPDE-adducts was statistically significant for p27, CCND1, ATM, and MDM2 (P = 0.00, 0.03, 0.03, and 0.03, respectively), borderline for p73 and p21 (P = 0.07 and 0.09, respectively), but not for p53 and p16 (P = 0.13 and 0.18, respectively). Because the relative expression levels of all these eight proteins were highly correlated, we further performed the principal component analysis and identified ATM as the most important predictor of DRC, followed by MDM2 and p27. Our results provide population-based in vitro evidence demonstrating that cell-cycle checkpoint-related proteins play essential roles in regulating DNA repair, at least in unaffected human peripheral blood lymphocytes. Further studies are warranted to investigate the role of interindividual variation in the expression levels of these proteins in cancer susceptibility.

Figure 1

Quality evaluation by Western blotting of the antibody used in the reverse-protein microarray assay.

Figure 2

Protein microarrays fabricated by using proteins extracted from activated peripheral T lymphocytes. Three dilutions were used for each sample in duplicate. Specific antibodies were used to detect levels of the corresponding proteins. The names of the proteins were shown on top of each microarray.

Figure 3

Spearman correlations between the readings of the three dilutions (use MDM2 as an example): r = 0.94, P = 0.00 between dilution 1 and 2; r = 0.85, P = 0.00 between dilution 1 and 3; and r = 0.92, P = 0.00 between dilution 2 and 3. Each point represents a mean of duplicate experiments of MDM2 protein expression levels in three dilutions.

Figure 4

Correlation between p27 and ATM protein expression levels and DRC level in primary lymphoblasts from healthy subjects. Each point represents a mean of duplicate experiments of p27 or ATM protein expression levels and DRC measurements. (r = 0.19 and P = 0.05 for p27 vs. DRC and r = 0.20 and P = 0.03 for ATM vs. DRC)

Figure 5

Principal component analysis. A, first component; B, second component; C, third component. Each principal component is a weighted sum of the concentrations of the eight proteins. The weights (shown on y-axes) in each principal component form a unit-length vector, which means the sum of the squares of the weights is equal to one. These three principal components form three vectors that are perpendicular to each other in the eight-dimensional space, i.e., these vectors are linearly independent of each other.