Gene copy number loss and MSH2 protein expression in primary human leukemia cells. MSH2 protein (a) and mRNA (b) expression measured in primary acute lymphoblastic leukemia (ALL) cells isolated from bone marrow aspirates of 90 newly diagnosed patients with ALL constituting the discovery cohort. MSH2 protein levels (a) were determined by Western blot analysis and normalized to the GAPDH signal. MSH2-L was defined as cells with low MSH2 protein signals on a western blot of 1 million ALL cells (<10% RU). MSH2 mRNA expression (b) was determined by Affymetrix gene expression array. Differences in the MSH2 mRNA or protein levels were assessed using the exact Wilcoxon-Mann-Whitney test. The closed circles indicate values in individual cases. (c) Pathway of genes upstream of PKCζ and MSH2 degradation. Depicted in color are four genes that were deleted significantly more frequently in MSH2-L leukemias. (d) SNP array data were analyzed using dChip, revealing over-representation of mono-allelic copy number loss (yellow) in MSH2-L leukemia cells. (e) MSH2 protein levels in leukemia cells from the validation cohort (i.e., all cases with deletions of one or more of these genes with sufficient cells for western analysis, and a 2:1 matched cohort of ALL cases without these deletions). Quantification of the MSH2 protein levels normalized to the GAPDH signal in the validation cohort documents a significant difference between cases with deletions compared to those without deletions of the four genes in their ALL cells (P = 0.00026). Horizontal lines in panels a, b and e depict median values for each cohort.

Protein expression in primary leukemia cells with hemizygous deletions, treatment outcome and drug sensitivity according to leukemia cell MSH2 phenotype. (a) Western blot analysis of two cases with hemizygous deletions of FRAP1 and two cases with hemizygous deletions of PRKCZ show ~30–60% lower amounts of the corresponding protein compared to the matched cases that did not have these deletions. Kaplan-Meier analysis of overall survival (b) and cumulative incidence of hematological relapse (c) in children whose ALL cells had the MSH2-L phenotype compared to those with the MSH2-H phenotype. The difference in overall 10 year survival is statistically significant (P = 0.009, Log-rank Test), whereas the difference in cumulative incidence of a hematological relapse approached the conventional level of statistical significance (P = 0.06, Gray test). (d) CCRF-CEM cells in which PIK3C2B, HERC1, FRAP1 or PRKCZ were knocked-down exhibit significantly greater resistance to the thiopurines antileukemic agents 6-thioguanine (6-TG) and 6-mercaptopurine (6-MP). In contrast these cells exhibited increased sensitivity to the alkylating agent melphalan. Cells were treated for 72 hours in the presence of increasing concentrations of 6-TG, 6-MP or melphalan. Cell viability was determined by the MTT assay.

PRKCZ, PIK3C2B, HERC1, FRAP1 inhibition and MSH2 stability. Human leukemia cells (CCRF-CEM) were transduced with shRNA against PRKCZ, PIK3C2B, HERC1, FRAP1 or with the non-target control. Western blot analysis was used to determine the effect of these knockdowns on each protein knocked down and on MSH2 protein levels after HERC1 (a) PRKCZ (b) PIK3C2B (c) and FRAP1 (d) stable knockdown. (c) Phosphorylation of AKT (Ser 473) and P70S6 kinase (Thr389) were measured by phospho-specific antibodies in panels a, c, d, e. (e) Depicts the decrease in MSH2 protein levels following treatment with increasing concentrations of rapamycin to inhibit FRAP1. (f) MSH2 protein levels after PRKCZ, PIK3C2B, HERC1, or FRAP1 inhibition. MSH2 protein levels were quantified by densitometry, normalized to GAPDH signal and expressed as a percent of the control. Values are means ± SD of three independent experiments. MSH2 protein levels were significantly lower (P < 0.007) after each knockdown or after treatment with rapamycin (300 nM), when compared to controls. (g) To determine the half-life of MSH2 protein, CCRF-CEM cells were treated with 5μg ml⁻¹ cycloheximide to inhibit protein synthesis. Leukemia cells were harvested at indicated time points in SDS cell lysis buffer before being fractionated in 4–12% Nupage gels and immunoblotted with MSH2 and GAPDH antibodies. (h) The level of MSH2 protein on Western blots was quantified by densitometry and normalized to the GAPDH protein signal. The results expressed as a percent of the time zero value show a shorter half-life of MSH2 after PRKCZ, PIK3C2B, HERC1, FRAP1 inhibition. (i) Inhibition of FRAP1 by rapamycin (300 nM) resulted in accumulation of ubiquitinated MSH2 protein in CEM cells after 48 hours and (j) the lower MSH2 protein levels were reversed by the proteasome inhibitor MG132 (10 nM).

Increase in PP2A activity via inhibition or knockdown of FRAP1, HERC1 or PIK3C2B with rescue by okadaic acid (OA) and effects on MMR activity. (a) Phosphatase activity was assayed colorimetrically using threonine phosphopeptide, K-R-pT-I-R-R, as a substrate. Error bars represent standard deviations of three replicate experiments. PP2A activity was significantly higher than controls after each knockdown or rapamycin treatment (P < 0.01). (b) Increase of PP2A activity by FRAP1, HERC1 or PIK3C2B knockdown decreases PKCzeta activation (phosphorylation) and MSH2 protein levels, both of which are rescued by okadaic acid. OA+ indicates that cells were incubated 24h with 10 nM of okadaic acid. Whole cell extracts were resolved by SDS-PAGE and immunoblotted for MSH2, GAPDH, PKCζ and the active form of PKCζ (phosphorylation of threonine 410). (c) Phospho PKCζ signal was quantified by densitometry. Values are means ± SD of three independent experiments. (d) Using a M13mp2 DNA substrate containing a two base loop, the repair efficiency is expressed as a percentage: 100 × (ratio of mixed colonies in the control group – the ratio of mixed colonies in repaired group)/(ratio of mixed colonies in the control group). Each experiment was performed in triplicate, and data represent the mean ± S.D. of three separate determinations. DNA repair efficiency was significantly lower than controls after each knockdown (P < 0.02) and this effect was rescued by the addition of purified hMutSα. (e) Cytosolic extracts were depleted of MSH2 with antibody or were mock-depleted with mouse IgG. The data are averages from three (mock-depleted, MSH2-depleted) experiments. Error bars represent the mean ± S. D. of three independent experiments (f) MSH2 immuno-depletion decreases MMR activity. Mock-depleted and MSH2-depleted cytosolic extracts were used to determine MMR activity as described above. DNA repair efficiency was significantly lower after MSH2 depletion (P = 0.004).