Hematopoiesis and thymic apoptosis are not affected by the loss of Cdk2

Abstract

Cell cycle regulation is essential for proper homeostasis of hematopoietic cells. Cdk2 is a major regulator of S phase entry, is activated by mitogenic cytokines, and has been suggested to be involved in antigen-induced apoptosis of T lymphocytes. The role of Cdk2 in hematopoietic cells and apoptosis in vivo has not yet been addressed. To determine whether Cdk2 plays a role in these cells, we performed multiple analyses of bone marrow cells, thymocytes, and splenocytes from Cdk2 knockout mice. We found that Cdk2 is not required in vivo to induce apoptosis in lymphocytes, a result that differs from previous pharmacological in vitro studies. Furthermore, thymocyte maturation was not affected by the lack of Cdk2. We then analyzed the hematopoietic stem cell compartment and found similar proportions of stem cells and progenitors in Cdk2(-)(/)(-) and wild-type animals. Knockouts of Cdk2 inhibitors (p21, p27) affect stem cell renewal, but a competitive graft experiment indicated that renewal and multilineage differentiation are normal in the absence of Cdk2. Finally, we stimulated T lymphocytes or macrophages to induce proliferation and observed normal reactivation of Cdk2(-)(/)(-) quiescent cells. Our results indicate that Cdk2 is not required for proliferation and differentiation of hematopoietic cells in vivo, although in vitro analyses consider Cdk2 to be a major player in proliferation and apoptosis in these cells and a potential target for therapy.

FIG. 1.

Apoptosis of Cdk2^−/− thymocytes. (A) Apoptosis of thymocytes was analyzed by flow cytometry. Cells were stained with propidium iodide (blue), and caspase 3 activity (green and blue) was detected. Viable cells (red) are double negative. Cdk2^−/− and Cdk2^+/+ thymocytes were harvested and treated for 24 h with 1 μM dexamethasone (Dex) to induce apoptosis and/or 50 μM roscovitine, which inhibits Cdk2. Percentages of each population are indicated inside each gate. (B) A similar assay was reproduced in five independent experiments (n ≥ 6). Cdk4-null thymocytes were included (n = 2). Roscovitine (Rs) was added at 50 or 12.5 μM. Cdk2 inhibitor (inh) III was added at 20 μM, and Cdk1 inhibitor III was used at 7 μM. Percentages of viable nonapoptotic cells were plotted.

FIG. 2.

Molecular analysis of apoptosis in thymocytes. (A) Viability of thymocytes over a 24-h time period was determined. Wild-type untreated thymocytes (Cdk2^+/+) (contr), untreated Cdk2^−/− thymocytes, Cdk2^+/+ thymocytes treated with dexamethasone (Dex), Cdk2^−/− thymocytes treated with dexamethasone, Cdk2^+/+ thymocytes treated with dexamethasone and roscovitine (Rosc), and Cdk2^−/− thymocytes treated with dexamethasone and roscovitine were plotted. (B) Western blots of Cdk2^+/+ and Cdk2^−/− thymocyte extracts probed with antibodies against Cdk1, Cdk2, Cdk4, and p27. Molecular size markers are indicated on the left, in kilodaltons. (C) Kinase activity was determined by immunoprecipitating thymocyte extracts with the antibodies indicated on the right, followed by an in vitro histone H1 assay using radiolabeled ATP.

FIG. 3.

Alternative stimuli leading to apoptosis. (A) Caspase 3 activity in splenocytes was measured at day 0 and after 3 days of in vitro stimulation with anti-CD3 and anti-CD28. Percentages of apoptotic cells were plotted. Error bars represent an average of three independent experiments (n = 8). (B) Apoptosis in Cdk2^+/+ and Cdk2^−/− thymocytes was induced by treatment with etoposide (Etop) (2.5 or 10 μg/ml) or PMA (10 ng/ml) in the presence or absence of 50 μM roscovitine (Rs). Cell death was determined by a FACS-based caspase 3 assay (see Materials and Methods).

FIG. 4.

Analysis of thymocyte subpopulations in Cdk2^−/− mice. (A) Analysis of CD4 and CD8 receptor expression by flow cytometry in thymocytes to discriminate subpopulations in Cdk2^−/− mice (n = 9) compared to their wild-type littermates (n = 11). Dot plots of representative data are shown, and average percentages of each subpopulation acquired in three independent experiments are plotted. DN, double negative; DP, double positive. (B) Expression of specific markers (HSA, CD5, CD69, H57) in the indicated subpopulations. DN, double negative; DP, double positive.

FIG. 5.

Analysis of bone marrow cells in Cdk2^−/− mice. (A) Analysis of bone marrow subpopulations by flow cytometry in Cdk2^−/− mice (n = 5) compared to their wild type-littermates (n = 4). Dot plots of representative data are shown, and average percentages of each subpopulation acquired in three independent experiments are plotted. (B) CFU-c assays performed with Cdk2^−/− bone marrow cells (n = 5) and those from their wild-type littermates (n = 4) using the indicated cytokines. Each assay was performed in triplicate. Averages of colony numbers obtained from one experiment are plotted. Two other independent experiments yielded comparable results. (C) Competitive grafts in five recipients with Cdk2^−/− (n = 2) or wild-type (n = 2) bone marrow cells were performed, and cells were analyzed after 46 weeks. Repopulation of the indicated compartments is expressed by the percentage of the Ly5.1 subpopulation. Myeloid cells and B cells correspond to the Mac-1⁺/Ly5.1⁺ and B220⁺/Ly5.1⁺ subcompartments, respectively.

FIG. 6.

Analysis of splenocytes in Cdk2^−/− mice. (A) Analysis of splenocyte subpopulations by flow cytometry in Cdk2^−/− mice (n = 4) compared to their wild-type littermates (n = 3). Dot plots and subpopulation percentages of representative data are shown. (B) Analysis of IL-2R (CD25) expression in CD4- or CD8-positive cells by flow cytometry. Lymphocytes harvested from Cdk2^−/− (n = 12) or wild-type (n = 10) spleens were stimulated with anti-CD3 and anti-CD28 for 3 days. Histograms at day 0 and day 3 of a representative experiment are shown. (C) Expression of CD4 and CD8 receptors on unstimulated (day 0) or stimulated (day 3 after anti-CD3 and anti-CD28 treatment) lymphocytes was analyzed by flow cytometry. Total cell numbers of CD4⁺ or CD8⁺ lymphocytes were determined (percentage of population times spleen cellularity), and average values from three independent experiments are plotted. CD8/CD4 ratios are indicated on the graph. (D) CBA was performed with cell culture supernatants from LPS-stimulated macrophages to measure cytokine gene expression. Average values of cytokine expression in Cdk2^−/− (n = 2) or Cdk2^+/+ (n = 2) cells are plotted.