The Akt and mitogen-activated protein kinase (MAPK) pathways have been implicated in tumor cell survival and contribute to radiation resistance. However, the molecular basis for link between MAPK and Akt in cell survival response to radiation is unclear. Here, we show that c-Src-Rac1-p38 MAPK pathway signals Akt activation and cell survival in response to radiation. Ionizing radiation triggered Thr308 and Ser473 phosphorylation of Akt. Exposure of cells to radiation also induced p38 MAPK and c-Jun NH2-terminal kinase activations. Inhibition of c-Jun NH2-terminal kinase suppressed radiation-induced cell death, whereas inhibition of p38 MAPK effectively increased sensitivity to radiation. Interestingly, inhibition of p38 MAPK completely attenuated radiation-induced Ser473 phosphorylation of Akt but did not affect Thr308 phosphorylation. Conversely, overexpression of p38 MAPK enhanced Ser473 phosphorylation of Akt in response to radiation. In addition, inhibition of p38 MAPK failed to alter phosphoinositide 3-kinase and phosphoinositide-dependent protein kinase activities. Ectopic expression of RacN17, dominant-negative form of Rac1, inhibited p38 MAPK activation and Ser473 phosphorylation of Akt. Following exposure to radiation, c-Src was selectively activated among Src family tyrosine kinases. Inhibition of c-Src attenuated Rac1 and p38 MAPK activations and Ser473 phosphorylation of Akt. Our results support the notion that the c-Src-Rac1-p38 MAPK pathway is required for activation of Akt in response to radiation and plays a cytoprotective role against radiation in human cancer cells. (Mol Cancer Res 2008;6(12):1872–80)

Sensitivity of tumor cells to radiation is a critical determinant of the probability of local control and ultimately of cure of cancers by radiation therapy. It has been shown that many factors affect susceptibility of tumor cells to ionizing radiation. Among them, intracellular signaling molecules seem to play an important role in determining the intrinsic radiosensitivity of tumor cells. Indeed, exposure of cells to ionizing radiation results in the simultaneous activation or down-regulation of multiple signaling pathways, which play critical roles in cell type-specific control of survival or death in response to ionizing radiation (1-4).

The serine-threonine kinase Akt plays a pivotal role in fundamental cellular functions, such as proliferation, migration, and survival, by phosphorylating various substrates, including Bad, IKK, Raf, mammalian target of rapamycin, and caspase-9 (5, 6). Moreover, Akt not only is cell survival kinase but also plays a central role in promoting tumorigenesis (7, 8). Overexpression of Akt is reported in several human cancers, including breast, colon, ovarian, prostate, and pancreas (9). Full activation of Akt requires multiple steps mediated by phosphoinositide 3-kinase (PI3K) and 3-phosphoinositide-dependent protein kinase (PDK) 1. Activation of the lipid kinase, PI3K, leads to generation of the second messenger, phosphatidylinositol-3,4,5-triphosphate, and subsequent recruitment of Akt to the plasma membrane. Membrane-bound PDK1 phosphorylates Thr308 in the pleckstrin homology domain of Akt, resulting in activation. Next, Ser473 is phosphorylated at the COOH-terminal domain for full activation of Akt by either autophosphorylation or an uncharacterized kinase, PDK2 (10, 11).

p38 mitogen-activated protein kinase (MAPK) responds strongly to a variety of stress signals including tumor necrosis factor, ionizing and UV irradiation, interleukin-1, chemotherapeutic drugs, and hyperosmotic stress (12-19). Indeed, activation of p38 MAPK has been shown to correlate well with the apoptotic cell death induced by these stress stimuli. In contrast, recent observations showed that p38 MAPK is a critical regulator for the cell survival and proliferation in response to cisplatin, doxorubicin, camptothecin, UV irradiation, and repetitive low-grade oxidative stress (20-22). Furthermore, other reports showed that p38 MAPK signaling is essentially required for the maintenance of a transformed cell phenotype in human malignancy (23, 24). These results are consistent with the fact that p38 MAPK pathway is involved in Akt activation as a survival response in human neutrophils (25) and that Akt phosphorylation elicited by angiotensin II is mediated by p38 MAPK signaling pathway (26).

Many reports showed that radiation-mediated activation of Akt signaling is an important mechanism in resistance of tumor cells to irradiation. Moreover, other reports also showed that MAPK pathway plays a role in cell survival against radiation-induced cell death. In the present study, we investigated the molecular basis for link between MAPK and Akt in cell survival in response to radiation. We show that the c-Src-Rac1-p38 MAPK pathway is required for the Akt activation in response to radiation and that the pathway plays a cytoprotective role against radiation-induced cell death. Improved understanding of the mechanisms involved in survival in response to radiation may ultimately afford novel strategies of intervention in specific pathways to favorably alter the therapeutic efficacy of human malignancy treatments.

Ionizing Radiation Triggers Phosphorylation of Akt as a Cell Survival Response to Radiation

Considerable evidence shows that the PI3K-Akt pathway enhances cell survival in response to various cellular stress conditions. We investigated whether the PI3K-Akt pathway is activated and involved in cervical cancer cell survival in response to radiation. Because full activation of Akt requires phosphorylation at two sites, Thr308 and Ser473, immunoblot analysis was conducted with antibodies specific for p-Thr308-Akt and p-Ser473-Akt in irradiated HeLa cells. Exposure of cells to radiation induced PI3K activation and subsequently increased kinase activity of Akt (Fig. 1A). Radiation also induced significant phosphorylation of Akt at Thr308 and Ser473 (Fig. 1A; Supplementary Fig. S1). Moreover, overexpression of dominant-negative form of Akt led to an increase in cell death in response to radiation (Fig. 1B). In addition, pretreatment with LY294002, an inhibitor of PI3K, attenuated radiation-induced increase in the kinase activity and Thr308 and Ser473 phosphorylation of Akt (Fig. 1C). These results indicate that PI3K-dependent Akt activation is involved in survival signaling of cervical cancer cells in response to radiation.

FIGURE 1.

Ionizing radiation induces phosphorylation of Akt as a cell survival response. A. Activation of PI3K and Akt in response to radiation. HeLa cells were exposed to 10 Gy γ-radiation. After 24, 48, and 72 h, cell lysates were immunoprecipitated with anti-PI3K (anti-p85) or Akt antibody. PI3K assay and Akt kinase assay were done on the immune complexes. A representative autoradiogram of the PI3K assay is shown, and the position of phosphatidylinositol phosphate is indicated. Akt kinase activity was determined by using GST-GSK3-β fusion protein as a substrate. Cell lysates were also subjected to Western blot analysis with anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-β-actin antibodies. β-Actin was used as a loading control. B. Effect of Akt inhibition on radiation-induced cell death. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of dominant-negative Akt (c-Myc tagged). After 24, 48, and 72 h, cell death was determined by flow cytometric analysis as described in Materials and Methods. Mean ± SE of three independent experiments. *, P < 0.05. After 48 h, cell lysates were also subjected to Western blot analysis with anti-c-Myc, anti-cleaved caspase-3, anti-poly(ADP-ribose) polymerase, and anti-β-actin antibodies. β-Actin was used as a loading control. C. Effect of the PI3K inhibition on radiation-induced Akt phosphorylation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of 10 μmol/L LY294002. After 48 h, cell lysates were immunoprecipitated with anti-Akt antibody, and Akt kinase assay was done on immune complexes. Akt kinase activity was determined by using GST-GSK3-β fusion protein as a substrate. Cell lysates were also subjected to Western blot analysis with anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-β-actin antibodies. β-Actin was used as a loading control.

FIGURE 1.

Ionizing radiation induces phosphorylation of Akt as a cell survival response. A. Activation of PI3K and Akt in response to radiation. HeLa cells were exposed to 10 Gy γ-radiation. After 24, 48, and 72 h, cell lysates were immunoprecipitated with anti-PI3K (anti-p85) or Akt antibody. PI3K assay and Akt kinase assay were done on the immune complexes. A representative autoradiogram of the PI3K assay is shown, and the position of phosphatidylinositol phosphate is indicated. Akt kinase activity was determined by using GST-GSK3-β fusion protein as a substrate. Cell lysates were also subjected to Western blot analysis with anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-β-actin antibodies. β-Actin was used as a loading control. B. Effect of Akt inhibition on radiation-induced cell death. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of dominant-negative Akt (c-Myc tagged). After 24, 48, and 72 h, cell death was determined by flow cytometric analysis as described in Materials and Methods. Mean ± SE of three independent experiments. *, P < 0.05. After 48 h, cell lysates were also subjected to Western blot analysis with anti-c-Myc, anti-cleaved caspase-3, anti-poly(ADP-ribose) polymerase, and anti-β-actin antibodies. β-Actin was used as a loading control. C. Effect of the PI3K inhibition on radiation-induced Akt phosphorylation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of 10 μmol/L LY294002. After 48 h, cell lysates were immunoprecipitated with anti-Akt antibody, and Akt kinase assay was done on immune complexes. Akt kinase activity was determined by using GST-GSK3-β fusion protein as a substrate. Cell lysates were also subjected to Western blot analysis with anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-β-actin antibodies. β-Actin was used as a loading control.

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p38 MAPK Is Involved in Akt Activation and Cell Survival in Response to Ionizing Radiation

MAPKs are implicated in the regulation of apoptotic cell death in response to various stimuli. To confirm the potential involvement of MAPK in ionizing radiation-induced cell death, we initially measured changes in MAPK activity after radiation treatment. As shown in Fig. 2A and Supplementary Fig. S1, irradiation of cells led to a dramatic increase in the phosphorylation of p38 MAPK and c-Jun NH2-terminal kinase (JNK), but gradual down-regulation of phosphorylated extracellular signal-regulated kinase (ERK). JNK and p38 MAPK phosphorylations were apparent at 24 h and peaked at 48 h after irradiation. The total MAPK cellular levels remained constant. Next, we investigated whether the changes in activities of MAPK are associated with radiation response in various human cervical cancer cells. To determine whether p38 MAPK and/or JNK are involved in radiation response, we employed specific inhibitors of these proteins in experiments with three human cervical cancer cell lines (HeLa, CaSki, and SiHa) and analyzed their effects on cell death in response to radiation. Treatment of SP600125 markedly inhibited radiation-induced cell death, whereas SB203580 effectively enhanced cell death in human cervical cancer cells (Fig. 2B; Supplementary Fig. S2A). Our findings indicate that JNK is involved in radiation-induced cell death, whereas p38 MAPK is implicated in cell survival in response to ionizing radiation.

FIGURE 2.

p38 MAPK is involved in Akt activation and cell survival in response to ionizing radiation. A. Regulation of MAPK activity after radiation treatment. HeLa cells were exposed to 10 Gy γ-radiation. After 24, 48, and 72 h, Western blot analysis with anti-p-ERK, anti-ERK, anti-p-p38 MAPK, anti-p38 MAPK, anti-p-JNK, and anti-JNK antibodies were done. B. Effects of MAPK inhibition on radiation-induced cell death. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of the 25 μmol/L PD98059, 30 μmol/L SB203580, or 5 μmol/L SP600125. Cell death was determined by flow cytometric analysis as described in Materials and Methods. Mean ± SE of three independent experiments. *, P < 0.05. C. Effects of MAPK inhibition on radiation-induced Akt phosphorylation. HeLa cells were exposed to 10 Gy γ-radiation in the presence of the 25 μmol/L PD98059, 30 μmol/L SB203580, or 5 μmol/L SP600125. After 48 h, cell lysates were subjected to Western blot analysis with anti-p-ERK, anti-p-JNK, anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-β-actin antibodies. β-Actin was used as a loading control. Cell lysates were also immunoprecipitated with anti-p38 MAPK antibody, and p38 MAPK kinase assay was done on the immune complexes. ATF2 was used as substrates for p38 MAPK.

FIGURE 2.

p38 MAPK is involved in Akt activation and cell survival in response to ionizing radiation. A. Regulation of MAPK activity after radiation treatment. HeLa cells were exposed to 10 Gy γ-radiation. After 24, 48, and 72 h, Western blot analysis with anti-p-ERK, anti-ERK, anti-p-p38 MAPK, anti-p38 MAPK, anti-p-JNK, and anti-JNK antibodies were done. B. Effects of MAPK inhibition on radiation-induced cell death. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of the 25 μmol/L PD98059, 30 μmol/L SB203580, or 5 μmol/L SP600125. Cell death was determined by flow cytometric analysis as described in Materials and Methods. Mean ± SE of three independent experiments. *, P < 0.05. C. Effects of MAPK inhibition on radiation-induced Akt phosphorylation. HeLa cells were exposed to 10 Gy γ-radiation in the presence of the 25 μmol/L PD98059, 30 μmol/L SB203580, or 5 μmol/L SP600125. After 48 h, cell lysates were subjected to Western blot analysis with anti-p-ERK, anti-p-JNK, anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-β-actin antibodies. β-Actin was used as a loading control. Cell lysates were also immunoprecipitated with anti-p38 MAPK antibody, and p38 MAPK kinase assay was done on the immune complexes. ATF2 was used as substrates for p38 MAPK.

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Exposure of cells to radiation induced significant phosphorylation of Akt at Thr308 and Ser473 (Fig. 1A). To further determine whether p38 MAPK and/or JNK are involved in phosphorylation of Akt to radiation response, we employed specific inhibitors of these proteins in experiments with human cervical cancer cells. Inhibition of p38 MAPK by pretreatment with SB203580 completely attenuated Ser473 phosphorylation of Akt but did not block Thr308 phosphorylation (Fig. 2C). Inhibition of ERK or JNK did not affect radiation-induced Ser473 phosphorylation of Akt.

p38 MAPK Activation Is Required for Radiation-Induced Akt Activation and Ser473 Phosphorylation

Because p38 MAPK functions in cell survival response of cervical cancer cells to radiation (Fig. 2B and C), we examined involvement of p38 MAPK in phosphorylation of Akt in response to radiation. Inhibition of p38 MAPK by pretreatment with SB203580 completely attenuated Ser473 phosphorylation of Akt but did not block Thr308 phosphorylation in three different human cervical cancer cell lines (Fig. 3A; Supplementary Fig. S3A). Moreover, SB203580 clearly inhibited radiation-induced Akt activity (Fig. 3A). However, inhibition of p38 MAPK failed to block radiation-induced PI3K and PDK1 activations. Consistent with these results, overexpression of dominant-negative form of p38 MAPK also clearly suppressed increase in kinase activity and phosphorylation at Ser473 of Akt in response to radiation but did not at Thr308 (Fig. 3B). Conversely, overexpression of wild-type p38 MAPK enhanced radiation-induced activation and Ser473 phosphorylation of Akt but failed to enhance Thr308 phosphorylation (Fig. 3C). These results indicate that p38 MAPK is required for the Akt activation and phosphorylation at Ser473 in response to radiation.

FIGURE 3.

p38 MAPK activation is required for phosphorylation of Akt at Ser473. A. Effects of inhibition of p38 MAPK on PI3K- and PDK1-mediated phosphorylation of Akt in response to radiation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of 30 μmol/L SB203580. After 48 h, cell lysates were immunoprecipitated with anti-PI3K (anti-p85), PDK1, or Akt antibody. PI3K, PDK1, and Akt kinase assays were done on immune complexes. A representative autoradiogram of PI3K assay is shown, and the position of phosphatidylinositol phosphate is indicated. GST-Akt and GST-GSK3-β fusion proteins were used as substrates for PDK1 and Akt, respectively. Cell lysates were also subjected to Western blot analysis with anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-PTEN antibodies. B. Effect of overexpression of dominant-negative p38 MAPK on radiation-induced Akt phosphorylation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of dominant-negative form of p38 MAPK. After 48 h, cell lysates were immunoprecipitated with anti-p38 MAPK or Ser473-Akt antibody, and p38 MAPK and Akt kinase assays were done on immune complexes. ATF2 and GST-GSK3-β fusion proteins were used as substrates for p38 MAPK and Akt, respectively. Cell lysates were also subjected to Western blot analysis with anti-Flag, anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-β-actin antibodies. β-Actin was used as a loading control. C. Effect of overexpression of p38 MAPK on radiation-induced Akt phosphorylation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of wild-type p38 MAPK. After 48 h, cell lysates were subjected to Western blot analysis with anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, anti-Flag, and anti-β-actin antibodies. β-Actin was used as a loading control. Cell lysates were also immunoprecipitated with anti-Ser473-Akt antibody, and Akt kinase assay was done on immune complexes. GST-GSK3-β fusion protein was used as substrates for Akt.

FIGURE 3.

p38 MAPK activation is required for phosphorylation of Akt at Ser473. A. Effects of inhibition of p38 MAPK on PI3K- and PDK1-mediated phosphorylation of Akt in response to radiation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of 30 μmol/L SB203580. After 48 h, cell lysates were immunoprecipitated with anti-PI3K (anti-p85), PDK1, or Akt antibody. PI3K, PDK1, and Akt kinase assays were done on immune complexes. A representative autoradiogram of PI3K assay is shown, and the position of phosphatidylinositol phosphate is indicated. GST-Akt and GST-GSK3-β fusion proteins were used as substrates for PDK1 and Akt, respectively. Cell lysates were also subjected to Western blot analysis with anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-PTEN antibodies. B. Effect of overexpression of dominant-negative p38 MAPK on radiation-induced Akt phosphorylation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of dominant-negative form of p38 MAPK. After 48 h, cell lysates were immunoprecipitated with anti-p38 MAPK or Ser473-Akt antibody, and p38 MAPK and Akt kinase assays were done on immune complexes. ATF2 and GST-GSK3-β fusion proteins were used as substrates for p38 MAPK and Akt, respectively. Cell lysates were also subjected to Western blot analysis with anti-Flag, anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-β-actin antibodies. β-Actin was used as a loading control. C. Effect of overexpression of p38 MAPK on radiation-induced Akt phosphorylation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of wild-type p38 MAPK. After 48 h, cell lysates were subjected to Western blot analysis with anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, anti-Flag, and anti-β-actin antibodies. β-Actin was used as a loading control. Cell lysates were also immunoprecipitated with anti-Ser473-Akt antibody, and Akt kinase assay was done on immune complexes. GST-GSK3-β fusion protein was used as substrates for Akt.

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Rac1 Acts as an Upstream Regulator of p38 MAPK in Akt Activation and Cell Survival in Response to Radiation

Rac1, an upstream regulator of MAPK, is activated by various types of cellular stress. We further examined whether Rac1 is involved in p38 MAPK activation and cell survival response to radiation. Following irradiation, Rac1-p21-activated kinase binding in cells was dramatically increased, indicative of Rac1 activation (Fig. 4A). Expression of RacN17, a dominant-negative form of Rac1, effectively suppressed p38 MAPK activation and Ser473 phosphorylation of Akt induced by radiation (Fig. 4B) but did not alter the Thr308 phosphorylation. RacN17 also effectively enhanced radiation-induced cell death (Fig. 4C). These results support the theory that Rac1 acts as an upstream regulator of p38 MAPK in activation of Akt and cell survival in response to radiation.

FIGURE 4.

Rac1 acts as an upstream regulator of p38 MAPK in activation of Akt and cell survival in response to radiation. A. Analysis of interaction between Rac1 and Pak in response to radiation. HeLa cells were exposed to 10 Gy γ-radiation. After 24, 48, and 72 h, cell lysates were prepared and interactions between Rac1 and Pak were detected by Western blot analysis with anti-Rac1 antibody after incubation with Pak-conjugated agarose. B. Effect of a dominant-negative form of Rac1 (RacN17) on radiation-induced Akt phosphorylation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of the dominant-negative form of Rac1 (RacN17). After 48 h, cell lysates were subjected to Western blot analysis with anti-Rac1, anti-p-p38 MAPK, anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-β-actin antibodies. β-Actin was used as a loading control. C. Effect of inhibition of Rac1 on radiation-induced cell death. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of dominant-negative form of Rac1 (RacN17). After 48 and 72 h, cell death was determined by flow cytometric analysis as described in Materials and Methods. Mean ± SE of three independent experiments. *, P < 0.05.

FIGURE 4.

Rac1 acts as an upstream regulator of p38 MAPK in activation of Akt and cell survival in response to radiation. A. Analysis of interaction between Rac1 and Pak in response to radiation. HeLa cells were exposed to 10 Gy γ-radiation. After 24, 48, and 72 h, cell lysates were prepared and interactions between Rac1 and Pak were detected by Western blot analysis with anti-Rac1 antibody after incubation with Pak-conjugated agarose. B. Effect of a dominant-negative form of Rac1 (RacN17) on radiation-induced Akt phosphorylation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of the dominant-negative form of Rac1 (RacN17). After 48 h, cell lysates were subjected to Western blot analysis with anti-Rac1, anti-p-p38 MAPK, anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-β-actin antibodies. β-Actin was used as a loading control. C. Effect of inhibition of Rac1 on radiation-induced cell death. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of dominant-negative form of Rac1 (RacN17). After 48 and 72 h, cell death was determined by flow cytometric analysis as described in Materials and Methods. Mean ± SE of three independent experiments. *, P < 0.05.

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c-Src Is Involved in Activation of the Cytoprotective Rac1-p38 MAPK-Akt Signaling Pathway

Recent reports show that Src family kinases are associated with MAPK activation in response to genotoxic and oxidative stress (27-29). Thus, we investigated whether Src family kinases are involved in the radiation response of human cervical cancer cells. Exposure of cell to ionizing radiation induced selective activation of c-Src but not Lyn or Fyn (Fig. 5A). Inhibition of c-Src activation by pretreatment of cells with PP2, a Src family kinase inhibitor, decreased radiation-induced Rac1 and p38 MAPK activations (Fig. 5B). PP2 additionally inhibited Akt phosphorylation at Ser473 but not Thr308 in three different human cervical cancer cell lines (Fig. 5B; Supplementary Fig. S4A). Consistently, small interfering RNA (siRNA) targeting of c-Src clearly revealed that inhibition of c-Src suppresses radiation-induced p38 MAPK activation and Akt phosphorylation at Ser473 (Fig. 5C). Inhibition of c-Src by PP2 or siRNA targeting of c-Src also significantly enhanced radiation-induced cell death in three different human cervical cancer cell lines (Fig. 5D; Supplementary Fig. S4B). These data imply that c-Src is necessary for stimulation of the Rac1-p38 MAPK-Akt signaling pathway and cell survival response against radiation-induced cell death in human cervical cancer cells.

FIGURE 5.

c-Src is involved in activation of the cytoprotective Rac1-p38 MAPK-Akt signaling pathway. A. Activation of Src family kinases in response to radiation. Activities of Src family kinases were detected using the immune complex kinase assay. Cell lysates were immunoprecipitated with anti-c-Src, anti-Fyn, and anti-Lyn antibodies, and kinase assays were done on immune complexes. Enolase was used as substrates for Src family kinases. B. Effect of c-Src inhibition by pretreatment with PP2 on Rac1 activation and phosphorylation of Akt in response to radiation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of the c-Src inhibitor, 10 μmol/L PP2. After 48 h, cell lysates were prepared and interactions between Rac1 and Pak were detected by Western blot analysis with anti-Rac1 antibody after incubation with Pak-conjugated agarose. Cell extracts were also subjected to Western blot analysis with anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-β-actin antibodies. β-Actin was used as a loading control. C. Effect of c-Src inhibition by siRNA targeting on Rac activation and phosphorylation of Akt in response to radiation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of the c-Src siRNA. After 48 h, cell lysates were prepared and interactions between Rac1 and Pak were detected by Western blot analysis with anti-Rac1 antibody after incubation with Pak-conjugated agarose. Cell extracts were also subjected to Western blot analysis with anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-β-actin antibodies. β-Actin was used as a loading control. D. Effect of c-Src inhibition by pretreatment with PP2 or siRNA targeting on radiation-induced cell death. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of 10 μmol/L PP2 or c-Src siRNA. After 48 and 72 h, cell death was determined by flow cytometric analysis. Mean ± SE of three independent experiments. *, P < 0.05.

FIGURE 5.

c-Src is involved in activation of the cytoprotective Rac1-p38 MAPK-Akt signaling pathway. A. Activation of Src family kinases in response to radiation. Activities of Src family kinases were detected using the immune complex kinase assay. Cell lysates were immunoprecipitated with anti-c-Src, anti-Fyn, and anti-Lyn antibodies, and kinase assays were done on immune complexes. Enolase was used as substrates for Src family kinases. B. Effect of c-Src inhibition by pretreatment with PP2 on Rac1 activation and phosphorylation of Akt in response to radiation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of the c-Src inhibitor, 10 μmol/L PP2. After 48 h, cell lysates were prepared and interactions between Rac1 and Pak were detected by Western blot analysis with anti-Rac1 antibody after incubation with Pak-conjugated agarose. Cell extracts were also subjected to Western blot analysis with anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-β-actin antibodies. β-Actin was used as a loading control. C. Effect of c-Src inhibition by siRNA targeting on Rac activation and phosphorylation of Akt in response to radiation. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of the c-Src siRNA. After 48 h, cell lysates were prepared and interactions between Rac1 and Pak were detected by Western blot analysis with anti-Rac1 antibody after incubation with Pak-conjugated agarose. Cell extracts were also subjected to Western blot analysis with anti-Thr308-Akt, anti-Ser473-Akt, anti-Akt, and anti-β-actin antibodies. β-Actin was used as a loading control. D. Effect of c-Src inhibition by pretreatment with PP2 or siRNA targeting on radiation-induced cell death. HeLa cells were exposed to 10 Gy γ-radiation in the presence or absence of 10 μmol/L PP2 or c-Src siRNA. After 48 and 72 h, cell death was determined by flow cytometric analysis. Mean ± SE of three independent experiments. *, P < 0.05.

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Intracellular signaling molecules and survival factors play important roles in determining the radiation response of tumor cells. The Akt signaling and the MAPK pathway have been implicated in tumor cell survival and contribute to radiation resistance (7, 8, 19). In this investigation, we elucidate the molecular basis for link between MAPK and Akt in cell survival response to radiation. Our data show that the c-Src-Rac1-p38 MAPK pathway plays a cytoprotective role in response to radiation through activation of Akt.

It has been known that PI3K activates Akt, which in turn triggers cytoprotective events through Bad phosphorylation and nuclear factor-κB activation (17). Concomitantly, we observed that radiation triggers PI3K-dependent activation of Akt in response to radiation treatment. Moreover, inhibition of Akt enhanced radiation-induced apoptotic cell death, suggesting that activation of Akt plays a cytoprotective role against cell death. Membrane-bound PDK1 phosphorylates Thr308 in the pleckstrin homology domain of Akt, resulting in activation (10, 11). However, full activation of Akt requires additional phosphorylation at Ser473 in the COOH-terminal domain, via either autophosphorylation or an as yet uncharacterized kinase, designated PDK2. In this study, we showed that radiation induces both Thr308 and Ser47 phosphorylation of Akt and that p38 MAPK is required for radiation-induced Ser473 phosphorylation of Akt but not Thr308 phosphorylation as well as activation of Akt. Introduction of p38 MAPK inhibitors completely attenuated radiation-induced Ser473 phosphorylation and activation of Akt. Conversely, overexpression of p38 MAPK markedly enhanced Ser473 phosphorylation and activation of Akt in response to radiation. However, inhibition of p38 MAPK failed to inhibit PI3K and PDK1 activations and Thr308 phosphorylation of Akt in response to radiation. These results indicate that p38 MAPK plays a crucial role in radiation-induced Ser473 phosphorylation and activation of Akt. This is in good agreement with the recent studies showing that the p38 MAPK pathway has an essential role in the Akt activation induced by angiotensin II (30), transforming growth factor-β1 (31), or black tea polyphenols (32). In addition, inhibition of PI3K attenuated not only Thr308 phosphorylation but also Ser473 phosphorylation in response to radiation, suggesting that PI3K-mediated Thr308 phosphorylation of Akt precedes and promotes p38 MAPK-mediated Ser473 phosphorylation.

It is well established that the Rac1/p38 MAPK pathway is activated by various types of membrane-associated cellular signals (33, 34). We provided further evidence that Rac1 is involved in p38 MAPK activation and cell survival in response to radiation. Inhibition of Rac1 activity with RacN17 attenuated p38 MAPK activation and Ser473 phosphorylation of Akt and significantly enhanced cell death, suggesting that Rac1 acts as an upstream regulator of p38 MAPK in cell survival response to ionizing radiation.

We further show that c-Src is essential for the activations of Rac1, p38 MAPK, and Ser473 phosphorylation of Akt in human cervical cancer cells. Our data clearly show that c-Src kinase, but not Lyn or Fyn, is selectively activated in response to radiation and that inhibition of c-Src leads to suppression of Rac1-p38 MAPK signaling and Ser473 phosphorylation of Akt and also leads to enhanced apoptotic cell death. These findings are consistent with recent reports that activation of c-Src in response to genotoxic and oxidative stress is associated with stimulation of protein kinase C and MAPK activity (27-29). However, we failed to find any changes in protein kinase C activities in response to radiation, and inhibition of c-Src did not show any additional effects on protein kinase C activities (data not shown). Based on these findings, we conclude that c-Src kinase is associated with activation of Rac1-p38 MAPK-Akt signaling pathway in cell survival response to radiation.

In summary, we show here that the c-Src-Rac1-p38 MAPK pathway is required for activation of Akt in response to radiation and that the pathway plays an important role in cell survival against radiation-induced cell death. Elucidation of the molecular mechanisms of cell survival response to radiation may ultimately afford novel strategies of intervention in specific signal transduction pathways to favorably alter the therapeutic efficacy of human malignancy treatments.

Materials

Polyclonal antibodies to p-ERK, p38 MAPK (α), Akt, PTEN, Src, Fyn, Lyn, and myc were purchased from Santa Cruz Biotechnology. β-Actin, Flag, l-α-phosphatidylinositol, and enolase were purchased from Sigma. Polyclonal antibodies to ERK, JNK, p-p38 MAPK, p-JNK, p-Akt, poly(ADP-ribose) polymerase, and cleaved caspase-3 were obtained from Cell Signaling Technology. Polyclonal antibodies to p85, glutathione S-transferase (GST)-Akt protein, GST-glycogen synthase kinase 3-β (GSK3-β) fusion protein, and Pak-conjugated agarose were purchased from Upstate Biotechnology. The MEK inhibitor (PD98059), p38 MAPK inhibitor (SB203580), JNK inhibitor (SP600125), Src family kinase inhibitor (PP2), and PI3K inhibitor (LY294002) were obtained from Calbiochem.

Cell Culture and Transfection

Human cervical carcinoma cell lines (HeLa, CaSki, and SiHa) were obtained from the American Type Culture Collection. HeLa and CaSki cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum, whereas SiHa cells were cultured in MEM supplemented with 10% fetal bovine serum and nonessential amino acids. Media were supplemented with 100 units/mL penicillin and 100 μg/mL streptomycin, and all cells were incubated at 37°C in 5% CO2. Cells were transfected with the expression vector of pUSE-dominant-negative Akt, pCMV5-Flag-p38 MAPK, pCMV5-Flag-dominant-negative p38 MAPK (KM), dominant-negative Rac1 (RacN17), or control vector using Lipofectamine PLUS reagent (Invitrogen) according to the manufacturer's recommendations. Cells were analyzed 24 h after transfection.

siRNA Transfection

RNA interference of c-Src was done using 21-bp (including a 2-deoxynucleotide overhang) siRNA duplexes purchased from Ambion. A control siRNA specific for green fluorescent protein (CCACTACCTGAGCACCCAG) was used as the negative control. Cells were plated on 100 mm dishes at 50% confluency, and siRNA duplexes (50 nmol/L) were introduced into cells using Lipofectamine 2000 (Invitrogen) by following the procedure recommended by the manufacturer.

Quantification of Cell Death

Fluorescence-activated cell sorting analysis using propidium iodide staining detects cell death by means of the dye entering the cells along with changes in the target cell membrane and DNA damage. For the cell death assessment, the cells were plated in 60 mm dish with cell density of 2 × 105 per dish and treated with radiation the next day. At indicated time points, cells were harvested by trypsinization, washed in PBS, and then incubated in propidium iodide (2.5 μg/mL) for 5 min at room temperature. Then, cells (10,000 per sample) were analyzed on a FACSscan flow cytometer using Cell Quest software.

Irradiation

Cells were plated in 35, 60, or 100 mm dishes and incubated at 37°C under humidified 5% CO2-95% air in culture medium until 70% to 80% confluent. Cells were then exposed to γ-rays with 137Cs γ-ray source (Atomic Energy of Canada) with a dose rate of 3.81 Gy/min.

Western Blot Analysis

Western blot analysis was done as described previously (35). Briefly, cell lysates were prepared by extracting proteins with lysis buffer [40 mmol/L Tris-HCl (pH 8.0), 120 mmol/L NaCl, 0.1% NP-40] supplemented with protease inhibitors. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in TBS and incubated with primary antibodies for 1 h at room temperature. Blots were developed with a peroxidase-conjugated secondary antibody, and proteins were visualized by enhanced chemiluminescence procedures (Amersham) using the manufacturer's protocol.

Akt Kinase Assay

Akt kinase activity was measured by the ability of immunoprecipitated enzyme to phosphorylate GSK3-β. Briefly, cell lysates were prepared by extracting proteins with lysis in buffer containing 1% (v/v) NP-40, 10% (v/v) glycerol, 137 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.4), 1% (v/v) Triton X-100, and protease inhibitors. Lysates were incubated with anti-Akt antibodies. The immunocomplex was precipitated with protein A Sepharose. Kinase reactions were carried out with 20 mmol/L HEPES (pH 7.4), 5 mmol/L MnCl2, 10 mmol/L MgCl2, 1 mmol/L DTT, 10 μCi γ-[32P]ATP and 1 mg/mL GST-GSK3-β. Proteins were separated on SDS-polyacrylamide gels, and bands were detected by autoradiography.

Src Family Kinase Assay

Cell lysates were prepared by extracting proteins with lysis buffer [40 mmol/L Tris-HCl (pH 8.0), 120 mmol/L NaCl, 0.1% NP-40] supplemented with protease inhibitors and then were incubated with anti-Src, anti-Fyn, and anti-Lyn antibodies, respectively. The immunocomplex was precipitated with protein A Sepharose. Kinase reactions were carried out with 20 mmol/L HEPES (pH 7.4), 3 mmol/L MnCl2, and 10 μCi γ-[32P]ATP. In addition, the enzyme activity of Src family that phosphorylates tyrosine residues of target proteins was monitored by supplementing an exogenous substrate, enolase (0.125 mg/mL; Sigma), to the reaction mixture. Proteins were separated on SDS-polyacrylamide gels, and bands were detected by autoradiography.

PI3K Assay

Cells were lysed in 20 mmol/L Tris-HCl (pH 8.0), 137 mmol/L NaCl, 1 mmol/L MgCl2, 1 mmol/L CaCl2, 1% NP-40, 10% glycerol, 2 mmol/L sodium orthovanadate, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride. Lysates were clarified, and equal amounts of the lysate proteins (400 μg) were immunoprecipitated with an antibody against the p85 subunit of PI3K (Upstate Biotechnology). Immune complexes were washed twice with 1% NP-40, 1 mmol/L sodium orthovanadate, and PBS (pH 7.4), twice with 100 mmol/L Tris-HCl (pH 7.5), 500 mmol/L LiCl, and 1 mmol/L sodium orthovanadate, and twice with 150 mmol/L NaCl and 50 mmol/L Tris-HCl (pH 7.2). Kinase reactions were initiated by adding 5 mg/mL l-α-phosphatidylinositol (Sigma) in 20 mmol/L HEPES (pH 7.4), 5 mmol/L MnCl2, 10 μmol/L ATP, 10 μCi γ-[32P]ATP, and 2.5 mmol/L EGTA. After 20 min incubation, reactions were quenched by adding 1 mol/L HCl. Phospholipids were extracted using a 1:1 mixture of chloroform and methanol and separated by TLC.

Rac1 Activation Assay

Rac1 activation assays were conducted as described previously (36). Cells lysates were prepared by total extracting proteins with lysis buffer [40 mmol/L Tris-HCl (pH 8.0), 120 mmol/L NaCl, 0.1% NP-40] supplemented with protease inhibitors. The cell lysates were used immediately for a pull-down of activated Rac with Pak1-agarose-beads. Pak1-agarose-beads were added to 500 μL cell lysate, and the samples were rotated at 4°C for 60 min. The agarose beads were collected by spinning at 14,000 rpm for 5 s, and the supernatants were removed. The beads were washed three times and suspended in SDS buffer for Rac detection by Western blot.

Statistical Analysis

Statistical analyses were done using Student's t test. The data were expressed as mean ± SE derived from at least three independent experiments. Differences were considered significant at P < 0.05.

No potential conflicts of interest were disclosed.

Grant support: Korea Science and Engineering Foundation and Ministry of Education, Science and Technology, Korean, through its National Nuclear Technology Program.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).

1
Wachsberger P, Burd R, Dicker AP. Tumor response to ionizing radiation combined with antiangiogenesis or vascular targeting agents: exploring mechanisms of interaction.
Clin Cancer Res
2003
;
9
:
1957
–71.
2
Schmidt-Ullrich RK. Molecular targets in radiation oncology.
Oncogene
2003
;
22
:
5730
–3.
3
Kubota Y, Kinoshita K, Suetomi K, et al. Mcl-1 depletion in apoptosis elicited by ionizing radiation in peritoneal resident macrophages of C3H mice.
J Immunol
2007
;
178
:
2923
–31.
4
Wang GJ, Cai L. Induction of cell-proliferation hormesis and cell-survival adaptive response in mouse hematopoietic cells by whole-body low-dose radiation.
Toxicol Sci
2000
;
53
:
369
–76.
5
Vanhaesebroeck B, Waterfield MD. Signaling by distinct classes of phosphoinositide 3-kinases.
Exp Cell Res
1999
;
253
:
239
–54.
6
Li B, Desai SA, MacCorkle-Chosnek RA, et al. A novel conditional Akt ‘survival switch’ reversibly protects cells from apoptosis.
Gene Ther
2002
;
9
:
233
–44.
7
Manning BD. Balancing Akt with S6K: implications for both metabolic diseases and tumorigenesis.
J Cell Biol
2004
;
167
:
399
–403.
8
Testa JR, Bellacosa A. AKT plays a central role in tumorigenesis.
Proc Natl Acad Sci U S A
2001
;
98
:
10983
–5.
9
Wu Y, Zu K, Warren MA, et al. Delineating the mechanism by which selenium deactivates Akt in prostate cancer cells.
Mol Cancer Ther
2006
;
5
:
246
–52.
10
Brognard J, Clark AS, Ni Y, Dennis PA. Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation.
Cancer Res
2001
;
61
:
3986
–97.
11
Li L, Ittmann MM, Ayala G, et al. The emerging role of the PI3-K-Akt pathway in prostate cancer progression.
Prostate Cancer Prostatic Dis
2005
;
8
:
108
–18.
12
Zwang Y, Yarden Y. p38 MAP kinase mediates stress-induced internalization of EGFR: implications for cancer chemotherapy.
EMBO J
2006
;
25
:
4195
–206.
13
Lin A. A five-year itch in TNF-α cytotoxicity: the time factor determines JNK action.
Dev Cell
2006
;
10
:
277
–8.
14
Bowen C, Birrer M, Gelmann EP. Retinoblastoma protein-mediated apoptosis after γ-irradiation.
J Biol Chem
2002
;
277
:
44969
–79.
15
Li T, Dai W, Lu L. Ultraviolet-induced junD activation and apoptosis in myeloblastic leukemia ML-1 cells.
J Biol Chem
2002
;
277
:
32668
–76.
16
Ouwens DM, Gomes de Mesquita DS, Dekker J, et al. Hyperosmotic stress activates the insulin receptor in CHO cells.
Biochim Biophys Acta
2001
;
1540
:
97
–106.
17
Stadheim TA, Kucera GL. c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is required for mitoxantrone- and anisomycin-induced apoptosis in HL-60 cells.
Leuk Res
2002
;
26
:
55
–65.
18
Wang X, McGowan CH, Zhao M, et al. Involvement of the MKK6-38γ cascade in γ-radiation-induced cell cycle arrest.
Mol Cell Biol
2000
;
20
:
4543
–52.
19
Kumar P, Miller AI, Polverini PJ. p38 MAPK mediates γ-irradiation-induced endothelial cell apoptosis, and vascular endothelial growth factor protects endothelial cells through the phosphoinositide 3-kinase-Akt-Bcl-2 pathway.
J Biol Chem
2004
;
279
:
43352
–60.
20
Reinhardt HC, Aslanian AS, Lees JA, et al. p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage.
Cancer Cell
2007
;
11
:
175
–89.
21
Sen P, Chakraborty PK, Raha S. Activation of p38MAPK by repetitive low-grade oxidative stress leads to pro-survival effects.
Biochim Biophys Acta
2007
;
1773
:
367
–74.
22
Yamashita T, Kobayashi Y, Mizoguchi T, et al. MKK6-38 MAPK signaling pathway enhances survival but not bone-resorbing activity of osteoclasts.
Biochem Biophys Res Commun
2008
;
365
:
252
–7.
23
Elenitoba-Johnson KS, Jenson SD, Abbott RT, et al. Involvement of multiple signaling pathways in follicular lymphoma transformation: p38-mitogen-activated protein kinase as a target for therapy.
Proc Natl Acad Sci U S A
2003
;
100
:
7259
–64.
24
Tang J, Qi X, Mercola D, et al. Essential role of p38γ in K-Ras transformation independent of phosphorylation.
J Biol Chem
2005
;
280
:
23910
–7.
25
Crossley LJ. Neutrophil activation by fMLP regulates FOXO (forkhead) transcription factors by multiple pathways, one of which includes the binding of FOXO to the survival factor Mcl-1.
J Leukoc Biol
2003
;
74
:
583
–92.
26
Li F, Malik KU. Angiotensin II-induced Akt activation through the epidermal growth factor receptor in vascular smooth muscle cells is mediated by phospholipid metabolites derived by activation of phospholipase D.
J Pharmacol Exp Ther
2005
;
312
:
1043
–54.
27
Lieskovska J, Ling Y, Badley-Clarke J, et al. The role of Src kinase in insulin-like growth factor-dependent mitogenic signaling in vascular smooth muscle cells.
J Biol Chem
2006
;
281
:
25041
–53.
28
Summy JM, Trevino JG, Baker CH, et al. c-Src regulates constitutive and EGF-mediated VEGF expression in pancreatic tumor cells through activation of phosphatidyl inositol-3 kinase and p38 MAPK.
Pancreas
2005
;
31
:
263
–74.
29
Ishizawar R, Parsons SJ. c-Src and cooperating partners in human cancer.
Cancer Cell
2004
;
6
:
209
–14.
30
Taniyama Y, Ushio-Fukai M, Hitomi H, et al. Role of p38 MAPK and MAPKAPK-2 in angiotensin II-induced Akt activation in vascular smooth muscle cells.
Am J Physiol Cell Physiol
2004
;
287
:
C494
–9.
31
Horowitz JC, Lee DY, Waghray M, et al. Activation of the pro-survival phosphatidylinositol 3-kinase/AKT pathway by transforming growth factor-β1 in mesenchymal cells is mediated by p38 MAPK-dependent induction of an autocrine growth factor.
J Biol Chem
2004
;
279
:
1359
–67.
32
Anter E, Thomas SR, Schulz E, et al. Activation of endothelial nitric-oxide synthase by the p38 MAPK in response to black tea polyphenols.
J Biol Chem
2004
;
279
:
46637
–43.
33
Uddin S, Lekmine F, Sharma N, et al. The Rac1/p38 mitogen-activated protein kinase pathway is required for interferon α-dependent transcriptional activation but not serine phosphorylation of Stat proteins.
J Biol Chem
2000
;
275
:
27634
–40.
34
Turkson J, Bowman T, Adnane J, et al. Requirement for Ras/Rac1-mediated p38 and c-Jun N-terminal kinase signaling in Stat3 transcriptional activity induced by the Src oncoprotein.
Mol Cell Biol
1999
;
19
:
7519
–28.
35
Kim JY, Choi JA, Kim TH, et al. Involvement of p38 mitogen-activated protein kinase in the cell growth inhibition by sodium arsenite.
J Cell Physiol
2002
;
190
:
29
–37.
36
García Arguinzonis MI, Galler AB, Walter U, et al. Increased spreading, Rac/p21-activated kinase (PAK) activity, and compromised cell motility in cells deficient in vasodilator-stimulated phosphoprotein (VASP).
J Biol Chem
2002
;
277
:
45604
–10.

Supplementary data