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.2002 Nov;22(22):7831-41.
doi: 10.1128/MCB.22.22.7831-7841.2002.

Activation of Akt/protein kinase B overcomes a G(2)/m cell cycle checkpoint induced by DNA damage

Affiliations

Activation of Akt/protein kinase B overcomes a G(2)/m cell cycle checkpoint induced by DNA damage

Eugene S Kandel et al. Mol Cell Biol.2002 Nov.

Abstract

Activation of Akt, or protein kinase B, is frequently observed in human cancers. Here we report that Akt activation via overexpression of a constitutively active form or via the loss of PTEN can overcome a G(2)/M cell cycle checkpoint that is induced by DNA damage. Activated Akt also alleviates the reduction in CDC2 activity and mitotic index upon exposure to DNA damage. In addition, we found that PTEN null embryonic stem (ES) cells transit faster from the G(2)/M to the G(1) phase of the cell cycle when compared to wild-type ES cells and that inhibition of phosphoinositol-3-kinase (PI3K) in HEK293 cells elicits G(2) arrest that is alleviated by activated Akt. Furthermore, the transition from the G(2)/M to the G(1) phase of the cell cycle in Akt1 null mouse embryo fibroblasts (MEFs) is attenuated when compared to that of wild-type MEFs. These results indicate that the PI3K/PTEN/Akt pathway plays a role in the regulation of G(2)/M transition. Thus, cells expressing activated Akt continue to divide, without being eliminated by apoptosis, in the presence of continuous exposure to mutagen and accumulate mutations, as measured by inactivation of an exogenously expressed herpes simplex virus thymidine kinase (HSV-tk) gene. This phenotype is independent of p53 status and cannot be reproduced by overexpression of Bcl-2 or Myc and Bcl-2 but seems to counteract a cell cycle checkpoint mediated by DNA mismatch repair (MMR). Accordingly, restoration of the G(2)/M cell cycle checkpoint and apoptosis in MMR-deficient cells, through reintroduction of the missing component of MMR, is alleviated by activated Akt. We suggest that this new activity of Akt in conjunction with its antiapoptotic activity may contribute to genetic instability and could explain its frequent activation in human cancers.

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Figures

FIG. 1.
FIG. 1.
Activated Akt attenuates G2 arrest in Rat1a cells following DNA damage. (A to D) Cell cycle evaluation of Akt effects. Cell cycle profiles of untreated Rat1a cells transduced with activated Akt (MyrAkt) or an empty control vector (A) and Rat1a cells treated for 4 days with 2 μM 6-TG and transduced with activated Akt (MyrAkt) or Myc plus Bcl-2 (Myc/Bcl-2) or an empty control vector (B). (C) Summary of three independent experiments performed as described for panel B. The percentages of cells in G1 and G2 are shown. (D) Twelve hours after 7-Gy gamma irradiation. Cells were PI stained and analyzed by flow cytometry. (E) The effect of activated Akt is not reestablished by the presence of dominant-negative p53. Rat1a cells transduced with an empty vector (upper panels) or activated Akt (middle panels) or dominant-negative p53 fragment (GSE56) (bottom panels) were either left untreated or treated with 2 μM 6-TG and analyzed by flow cytometry upon PI staining. (F) Activated Akt attenuates accumulation of 10.1 3T3 cells in the G2 phase of the cell cycle. 10.1 3T3 cells and 10.1 3T3 cells expressing activated Akt were exposed to 2 μM 6-TG as described for panels A to E and analyzed by flow cytometry upon PI staining.
FIG. 2.
FIG. 2.
Activated Akt maintains higher mitotic index and CDC2 activity upon exposure to DNA damage. (A) Cells with activated Akt maintain a higher mitotic index following gamma irradiation. Vector (pBP)- and MyrAkt (pBPMyrAkt)-transduced Rat1a cells were treated with the indicated doses of gamma irradiation and analyzed 4 and 6 h posttreatment. The mitotic indices were determined as described in Materials and Methods and are normalized to that of an untreated control for each cell line. The relative mitotic index of vector-transduced cells is shown as 100%. The difference between vector and MyrAkt cell lines is significant, withP = 0.05 (two-tailedt test). (B) Cells with activated Akt maintain a higher mitotic index following 6-TG treatment. The mitotic indices of vector (pBP)- and MyrAkt (pBPMyrAkt)-transduced Rat1a cells were measured following 3 days of 6-TG treatment as described in Materials and Methods. (C) Activated Akt reduces the decline in CDC2 activity following 6-TG treatment. CDC2 was immunoprecipitated from treated (MyrAkt) and untreated (control) cells as indicated, and its activity was measured and normalized for the total amount of this protein as described in Materials and Methods. Normalized activity is shown as a percentage of that in an untreated vector-transduced control.
FIG. 3.
FIG. 3.
(A) Akt enhances long-term survival upon 6-TG treatment. Rat1a cells transduced with MyrAkt or an empty control vector were treated with 2 μM 6-TG, as described in Materials and Methods, followed by methylene blue staining. (B) Expression of Bcl-XL does not reestablish the long-term effect of Akt. Rat1a cells transduced with MyrAkt or Bcl-XL or an empty vector were treated and visualized as described for panel A. The experiment shown is representative of eight independent experiments. (C) 6-TG resistance is coregulated with Akt expression. Rat1a pBPSTR-1 MyrAkt cells were treated as described for panel A in the presence (Akt OFF) or absence (Akt ON) of tetracycline. (D) Continuous Akt function is required for long-term 6-TG resistance. Cells were passed through 6-TG treatment in the Akt ON state as described for panel C and were retested for 6-TG resistance in the presence or absence of tetracycline. The Tet-inducible MyrAkt cell line has been described previously (16).
FIG. 4.
FIG. 4.
PTEN deficiency affects exit from G2 phase. (A and B) PTEN-deficient ES cells recover faster from gamma irradiation-induced G2 arrest. Wild-type and PTEN−/− ES cells were pulse labeled with BrdU and irradiated as described in Materials and Methods. Cell cycle distribution of BrdU-positive cells was monitored by flow cytometry at the indicated times posttreatment. Representative histogram plots (A) and changes over time in the G2-to-G1 ratio (B) are shown. (C) PTEN deficiency facilitates G2/M-to-G1 transition in untreated ES cells. Wild-type (WT) and PTEN−/− ES cells were labeled with BrdU, and the fraction of BrdU-positive cells in G1 was measured by flow cytometry at the indicated times. The results are representative of two independent experiments.
FIG. 5.
FIG. 5.
(A) Activation of Akt overcomes G2 cell cycle arrest induced by LY. Asynchronously growing HEK293 cells infected with pBabePuro (HEK293pBP) or pBabePuroMyrAkt (HEK293pBPmAkt) were either left untreated or treated with LY (20 μm) for 12 h and then subjected to flow cytometry analysis. (B) Akt accelerates the transition from G2/M to G1. HEK293pBP and HEK293mAkt cells were subjected to aphidicoline-mediated S-phase block and were released from the block as described in Materials and Methods. Samples were taken for flow cytometry analysis at the indicated time points. The profiles of asynchronously growing cells (control) are also shown. (C) Akt1−/− MEFs are attenuated in the transition from G2/M to G1. Asynchronously growing SV40-immortalized wild-type (left panels) and Akt1−/− (right panels) MEFs were pulse labeled with BrdU for 45 min and subjected to flow cytometry analysis at the indicated time points as described in Materials and Methods.x axis, DNA content;y axis, number of events.
FIG. 6.
FIG. 6.
Activated Akt attenuates G2 arrest following gamma irradiation in HCT116-ch3 cells and mimics MMR deficiency in HCT116 cells. (A) Immunoblotting analysis of expression of Akt in HCT116 and HCT116-ch3 transduced with empty vector (lanes 1 and 3) or activated Akt (lanes 2 and 4). β-Actin is shown as a loading control. (B) The isogenic cell lines HCT116 and HCT116-ch3, after being transduced with activated Akt or an empty control vector, were gamma irradiated (7 Gy), and their cell cycle profiles were determined by PI staining and flow cytometry. Ratios of G2-to-G1 fractions are shown. Cumulative data from two independent experiments are presented. (C) Akt activation increases long-term survival upon 6-TG treatment. A total of 1.5 × 106 HCT116 and HCT116-ch3 cells transduced with activated Akt or an empty control vector were continuously treated with 2 μM 6-TG. Colonies were visualized by crystal violet staining. Results of two independent experiments are shown. (D) Quantification of the results shown in panel B. Colony counts (presented as averages with standard deviations) are plotted on a logarithmic scale.
FIG. 6.
FIG. 6.
Activated Akt attenuates G2 arrest following gamma irradiation in HCT116-ch3 cells and mimics MMR deficiency in HCT116 cells. (A) Immunoblotting analysis of expression of Akt in HCT116 and HCT116-ch3 transduced with empty vector (lanes 1 and 3) or activated Akt (lanes 2 and 4). β-Actin is shown as a loading control. (B) The isogenic cell lines HCT116 and HCT116-ch3, after being transduced with activated Akt or an empty control vector, were gamma irradiated (7 Gy), and their cell cycle profiles were determined by PI staining and flow cytometry. Ratios of G2-to-G1 fractions are shown. Cumulative data from two independent experiments are presented. (C) Akt activation increases long-term survival upon 6-TG treatment. A total of 1.5 × 106 HCT116 and HCT116-ch3 cells transduced with activated Akt or an empty control vector were continuously treated with 2 μM 6-TG. Colonies were visualized by crystal violet staining. Results of two independent experiments are shown. (D) Quantification of the results shown in panel B. Colony counts (presented as averages with standard deviations) are plotted on a logarithmic scale.
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