Cross-talk between Chk1 and Chk2 in double-mutant thymocytes

Abstract

Chk1 is a checkpoint kinase and an important regulator of mammalian cell division. Because null mutation of Chk1 in mice is embryonic lethal, we used the Cre-loxP system and the Lck promoter to generate conditional mutant mice in which Chk1 was deleted only in the T lineage. In the absence of Chk1, the transition of CD4(-)CD8(-) double-negative (DN) thymocytes to CD4(+)CD8(+) double-positive (DP) cells was blocked due to an increase in apoptosis at the DN2 and DN3 stages. Strikingly, loss of Chk1 activated the checkpoint kinase Chk2 as well as the tumor suppressor p53 in these thymocytes. However, the developmental defects caused by Chk1 deletion were not rescued by p53 inactivation. Significantly, even though Chk1 deletion is highly lethal in proliferating tissues, we succeeded in using in vivo methods to generate Chk1/Chk2 double-knockout T cells. Analysis of these T cells revealed an interesting interaction between Chk1 and Chk2 functions that partially rescued the apoptosis of the double-mutant cells. Thus, Chk1 is both critical for the survival of proliferating cells and engages in cross-talk with the Chk2 checkpoint kinase pathway. These factors have implications for the targeting of Chk1 as an anticancer therapy.

Fig. 1.

Chk1 protein expression in various subsets of WT T cells and tissue-specific disruption of the Chk1 locus. (A) Chk1 protein expression in various T lineage cell populations. Surface staining of WT thymocytes with either anti-CD4 or anti-CD8 was followed by intracellular staining with anti-Chk1 Ab (green) or isotype control (purple) in samples of the thymus, spleen, or lymph node. To determine the Chk1 protein level in DN cells, thymocytes were stained with the anti-CD25, anti-CD44, and anti-Lin Abs. Lin⁺ cells were then gated out. (B) Southern blot analysis of DN thymocytes of Lck-Chk1^fl/fl and WT mice revealed proper integration of the floxed Chk1 allele and its complete excision after Cre expression. Thymocytes were depleted of CD4⁺ and CD8⁺ cells, and the Southern blot was performed as described previously (13).

Fig. 2.

Flow cytometric and histological analyses of the effect of Chk1 depletion on murine thymus. (A) Defective thymocyte production in Chk1 heterozygotes. Total thymocytes from Lck-Chk1^+/+, Lck-Chk1^fl/+, and Lck-Chk1^fl/fl mice (eight per group) were counted with a flow cytometer. Results shown are the mean number ± SD. P = 0.04 and P < 0.001, respectively. (B) Impaired development of Lck-Chk1^fl/fl thymocytes. Thymocytes from Lck-Chk1^+/+ and Lck-Chk1^fl/fl mice were stained with anti-CD4 and anti-CD8 Abs (Left) or with anti-Lin, anti-CD25, and anti-CD44 Abs (Right). The Lin⁻ population (only) was then examined by flow cytometry. (C) Reduced size and cellularity of Lck-Chk1^fl/fl thymus. Hematoxylin/eosin and anti-CD3 staining of transverse sections of the thymus from a Lck-Chk1^+/+ (Left) and a Lck-Chk1^fl/fl (Right) mouse are shown. Data are representative of three thymi examined per group.

Fig. 3.

T cells lacking Chk1 show increased cell death. (A) TUNEL staining. An increased number of TUNEL⁺ cells is present in the thymus of a Lck-Chk1^fl/fl mouse compared with a Lck-Chk1^+/+ mouse. Data are representative of three thymi examined per group. (B) DN thymocytes were prepared ex vivo; depleted of CD4⁺, CD8⁺, and B220⁺ cells with Dynabeads; and stained with anti-CD25, anti-CD44, and anti-Lin Ab followed by Annexin V. The number of Annexin V⁺ Lin⁻ cells was assessed by flow cytometry. Results shown are the mean ± SD of eight mice per group and are representative of at least three experiments. P < 0.01 for DN2, DN3E, and DN3L; P > 0.5 for DN4.

Fig. 4.

Decreased survival of T cell-specific Chk1-deficient mice. Kaplan–Meier plots of survival of Lck-Chk1^+/+ and Lck-Chk1^fl/fl mice in a WT or bcl2-tg background are shown. Lck-Chk1^+/+ (WT) mice (61), Lck-Chk1^fl/fl mice (40), bcl2-tg mice (48), and Lck-Chk1^fl/fl;bcl2-tg mice (28) were left untreated and monitored for survival. Survival is plotted against animal age in days.

Fig. 5.

Effect of different genetic backgrounds on the Chk1 T cell phenotype. (A) Partial rescue of T cell numbers in Lck-Chk1^fl/fl mice induced by loss of Chk2 or expression of bcl2. Lck-Chk1^fl/fl mice were backcrossed to mice of different genetic backgrounds, including Chk2-, p53-, Brca1-, p21-null, and bcl2-tg mice. The total number of T cells in the thymi of the indicated double-mutant offspring and controls was counted by flow cytometry. ∗, P < 0.05. (B) Flow cytometric analysis of DN thymocytes of different genetic backgrounds. After depletion of CD4⁺, CD8⁺, and B220⁺ cells with Dynabeads, staining was performed with anti-CD25, anti-CD44, and anti-Lin Abs. Lin⁻ cells of the indicated double-mutant mice and controls were analyzed for CD25 and CD44 expression. (C) Apoptosis in DN thymocyte subpopulations in Lck-Chk1^fl/fl;Brca^fl/fl double-mutant mice. Thymocytes from the indicated mutant mice and controls (at least three per group) were analyzed for DN stage and stained with Annexin V. Results shown are flow cytometric determinations of the percentages of Annexin V⁺ cells in the DN2, DN3E, and DN3L subpopulations.

Fig. 6.

Phospho-specific flow cytometric analysis of phosphoprotein expression in Chk1-deficient T cells. (A) Specificity of the anti-p-p53 Ab. Thymocytes from WT or p53-null mice that had been treated with 5 Gy or sham irradiation were stained with anti-CD25, anti-CD44, anti-Lin, and anti-p-p53 (Ser-15) Ab. Levels of phosphorylated p53 were determined by phospho-specific flow cytometry. (B) Phospho-specific flow cytometric analysis of DN2 and DN3 subpopulations in Lck-Chk1^fl/fl mice. Intracellular staining for p-p53 (Ser-9 and Ser-15), p-Chk1 (Ser-317), p-Chk2 (Thr-387), and p-Brca1 (Ser-1423) was performed for mice of the indicated genetic backgrounds. (C) Phospho-specific flow analysis of DN2 and DN3 thymocytes from Lck-Chk1^fl/fl mice in a Chk2-null background. Intracellular staining for p-Chk1 (Ser-317) and p-p53 (Ser-9 and 15) was performed for mice of the indicated genetic backgrounds.