**4.1 p38 MAPK**

While the cellular response to methylating agent exposure appeared highly dependent on DNA repair processes influenced by the G2 cell cycle checkpoint, the purpose of the cell cycle arrest remains unclear, although it has long been suggested that cell cycle arrest provides cells that have incurred DNA damage time in which to reverse the damage. Despite this suggestion has not been directly tested, it was clear that the prolonged G2 arrest noted in glioma cells exposed to cytotoxic methylating agents alters their response.

It has been appreciated for some time that cells lacking a functional MMR system undergo neither G2 arrest nor cytotoxicity in response to methylating agents, clearly suggesting a link between MMR and G2 arrest.

A potential signaling pathway that might help connect activation of the MMR system to G2 arrest is that controlled by the p38 MAPK family. The p38 family of stress kinases contains four members: , , , and . These MAP kinases are activated by the upstream kinases MKK3 and/or MKK6 in response to diverse stimuli including environmental stress and growth factors. p38 kinases in turn activate a variety of downstream targets including MAPKAP2, the C/EBP family of transcription factors, and various other transcription factors including p53. In this manner p38 is believed to play an important, although not well defined, role in co-ordinating a variety of cellular events including cell growth, cell differentiation, and cell death. At least two recent studies have suggested that the ability of the p38 pathway to co-ordinate cell growth and cell death might also extend to actions on cell cycle progression.

To begin to address the possible role of the p38 pathway in the response of cells to methylating agents, we exposed U87MG cells to TMZ, and the various p38 isoforms were then immunoprecipitated from the nuclear fractions, and equal amounts of the immunoprecipitated p38 isoforms from each time point were incubated with the p38 substrate ATF2 in the presence of ATP. TMZ exposure stimulated p38 kinase activity and p38 kinase activity was also modestly stimulated while the activity of p38 and were not significantly affected (Hirose et al, 2003).

To investigate linkage between MMR and p38 activation, we first exposed MMR-deficient HCT116 human colorectal adenocarcinoma cells and paired MMR-proficient HCT116 cells containing a copy of *Mlh1*-containing chromosome 3 (HCT116 3-6 cells) to TMZ, after which the cells were collected and analyzed for levels of p38 phosphorylation/activation and extent of G2 arrest by Western blot and by FACS analysis, respectively. Neither HCT116 nor HCT116 3-6 cells exhibited p38 induction or G2 arrest in the first two days following TMZ exposure, consistent with previous studies showing that both these cell lines are MGMT-proficient and presumably repair TMZ-induced O6-methylguanine before these lesions mispair with thymine and trigger downstream consequences. Following exposure to the highly specific MGMT depleting agent O6-benzylguanine prior to and after TMZ exposure, however, the MMR-proficient HCT116 3-6 cells exhibited TMZ-induced p38

Stress-activated kinases (SAPKs) could be targeted in an effort to enhance the effect of chemotherapeutic agents because the tumor cells, which grow under various types of cellular stress including hypoxia and genetic instability, might survive in severe environment by modifying on stress-inducing events. We here discuss about two SAPKs,

While the cellular response to methylating agent exposure appeared highly dependent on DNA repair processes influenced by the G2 cell cycle checkpoint, the purpose of the cell cycle arrest remains unclear, although it has long been suggested that cell cycle arrest provides cells that have incurred DNA damage time in which to reverse the damage. Despite this suggestion has not been directly tested, it was clear that the prolonged G2 arrest

It has been appreciated for some time that cells lacking a functional MMR system undergo neither G2 arrest nor cytotoxicity in response to methylating agents, clearly suggesting a

A potential signaling pathway that might help connect activation of the MMR system to G2 arrest is that controlled by the p38 MAPK family. The p38 family of stress kinases contains four members: , , , and . These MAP kinases are activated by the upstream kinases MKK3 and/or MKK6 in response to diverse stimuli including environmental stress and growth factors. p38 kinases in turn activate a variety of downstream targets including MAPKAP2, the C/EBP family of transcription factors, and various other transcription factors including p53. In this manner p38 is believed to play an important, although not well defined, role in co-ordinating a variety of cellular events including cell growth, cell differentiation, and cell death. At least two recent studies have suggested that the ability of the p38 pathway to co-ordinate cell growth and cell death might also extend to actions on

To begin to address the possible role of the p38 pathway in the response of cells to methylating agents, we exposed U87MG cells to TMZ, and the various p38 isoforms were then immunoprecipitated from the nuclear fractions, and equal amounts of the immunoprecipitated p38 isoforms from each time point were incubated with the p38 substrate ATF2 in the presence of ATP. TMZ exposure stimulated p38 kinase activity and p38 kinase activity was also modestly stimulated while the activity of p38 and were not

To investigate linkage between MMR and p38 activation, we first exposed MMR-deficient HCT116 human colorectal adenocarcinoma cells and paired MMR-proficient HCT116 cells containing a copy of *Mlh1*-containing chromosome 3 (HCT116 3-6 cells) to TMZ, after which the cells were collected and analyzed for levels of p38 phosphorylation/activation and extent of G2 arrest by Western blot and by FACS analysis, respectively. Neither HCT116 nor HCT116 3-6 cells exhibited p38 induction or G2 arrest in the first two days following TMZ exposure, consistent with previous studies showing that both these cell lines are MGMT-proficient and presumably repair TMZ-induced O6-methylguanine before these lesions mispair with thymine and trigger downstream consequences. Following exposure to the highly specific MGMT depleting agent O6-benzylguanine prior to and after TMZ exposure, however, the MMR-proficient HCT116 3-6 cells exhibited TMZ-induced p38

noted in glioma cells exposed to cytotoxic methylating agents alters their response.

**4. Stress-activated kinases** 

link between MMR and G2 arrest.

cell cycle progression.

significantly affected (Hirose et al, 2003).

**4.1 p38 MAPK** 

p38MAPK and c-Jun-N-terminal kinase (JNK).

activation in a manner similar to that noted in U87MG glioma cells, and underwent G2 arrest. O6-benzylguanine-exposed, TMZ-treated MMR-deficient HCT116 cells, however, exhibited neither p38 activation nor G2 arrest. These results suggested in a preliminary manner that p38 activation in response to TMZ is dependent on O6-methylguanine lesions and on the recognition and/or processing of these lesions by the DNA MMR system.

While studies in HCT cells suggested an association between the DNA MMR system and TMZ-induced G2 arrest, the MMR-corrected HCT116 3-6 cell line used was derived from a clone that contains a large portion of human chromosome three, and which therefore could differ from the MMR-proficient HCT116 cells in ways more dependent on clonal selection and multiple gene expression that on direct correction of the MMR defect. We therefore also examined TMZ-induced p38 activation and G2 arrest in MGMT-deficient, MMR-proficient human U87MG glioblastoma cells and in paired cells made MMR-deficient by expression of a retrovirally-encoded siRNA targeted to the MMR protein MLH1. Expression of the retrovirally encoded MLH1 siRNA blocked TMZ-induced G2 arrest such that cells expressing the MLH1 siRNA, but not cells expressing the blank vector, avoided TMZinduced G2 arrest in a manner identical to that noted in HCT116 cells in which MLH1 was eliminated by mutation. More importantly, siRNA-mediated reduction of MLH1 levels also blocked the ability of the cells to activate the p38 pathway. These results, in connection with those derived from the studies with HCT cells, suggested that p38 activation was a common response of human cells to the methylating agent TMZ, that this activation is dependent on a functional MMR system, and that p38 activation is associated with methylating agentinduced G2 arrest.

Fig. 5. TMZ-induced p38 activation in MMR-deficient colon carcinoma cells (A) and MMRsilenced glioma cells (B).

Having demonstrated the linkage between DNA MMR and p38 activation, we wished to more clearly define the potential linkage between p38 activation and G2 arrest. To do so we used both pharmacologic and genetic inhibitors of p38, and monitored the effects of these inhibitors on G2 checkpoint proteins and on TMZ-induce G2 arrest. For pharmacologic inhibition studies, MGMT-deficient, MMR-proficient U87MG cells were exposed to the p38/ selective inhibitors SB203580 or SB202190 prior to and following TMZ exposure, after which effects of p38 inhibition on TMZ-induced p38 activation, activation of the G2 checkpoint pathway, and activation of the G2 checkpoint itself were examined. Both

Biological Responses of Glioma Cell to Chemotherapeutic Agents 69

idea that cells incapable of undergoing G2 arrest enter mitosis with damaged DNA and die by mitotic catastrophe. These results suggest that p38 not only links MMR to the G2 checkpoint, but also influences the response of cells to cytotoxic methylating agents. It would be reasonable to suspect that tumors capable of activating the G2 checkpoint via the p38 pathway would be less sensitive than those incapable of doing so. Conversely, because inhibition of p38 sensitizes cells to TMZ, p38 might be a reasonable therapeutic

Since p38, a major component of mitogen-activated protein kinases (MAPKs), had been shown to be involved in TMZ-induced cellular responses, we were interested other MAPKS in terms they could be target for chemosensitization. MAPKs are components of a complex intracellular signaling network that regulates gene expression in response to extracellular stimuli, in turn regulating cell proliferation, differentiation, and cell death. The MAPK family includes components of three major pathways in humans: p38 kinase, c-Jun NH2 terminal kinase (JNK), and extracellular signal-regulated kinase. In the JNK pathway, specific stimuli activate various kinases leading to activation of JNK. Once activated, JNK is translocated to the nucleus, where it phosphorylates and activates transcriptional factors such as components of activator protein, including c-Jun, JunB, and JunD, as well as other factors including ATF-2 and STAT-3. As examples of the importance of JNK on cell signaling, JNK is associated with cell survival, oncogenesis, growth, differentiation, and cell death. The role of the JNK pathway in the mediation of cellular responses (including cellular transformation, cell growth, and cell death) to extracellular stimuli has been studied extensively. Interestingly, JNK can exert completely opposite effects depending on the cell type and stimuli. Since the question of whether JNK is associated with cell death or cell survival appeared to depend on the type of cellular stress, we investigated in the role of JNK

We first investigated whether JNK was activated in response to TMZ and confirmed the activation of JNK in glioma cells treated with TMZ. Previous studies on gliomas had already shown that JNK was activated and played a pro-survival role in response to the DNA crosslinking agent CDDP, and that the inhibition of the JNK pathway sensitized glioma cells to CDDP. Sensitization to chemotherapeutic agents by the inhibition of the JNK pathway has also been reported either in other cancer cells although, on the other hand, the inhibition of JNK reportedly led to the suppression of chemotherapeutic agentinduced apoptosis in several studies. Therefore, we investigated the role of JNK in glioma cells treated with TMZ by utilizing the JNK inhibitor SP600125. As a result, we confirmed that SP600125 potentiated the TMZ-induced cytotoxicity in U87MG cells, and concluded that JNK activation played a cytoprotective role in glioma cells in response to TMZ-

To clarify the mechanism responsible for the JNK inhibitor-induced potentiation of TMZinduced cytotoxicity, the two main downstream proteins of JNK, c-Jun and ATF-2, were investigated, since these proteins are believed to be associated with chemoresistance. In our study, because SP600125 inhibited the phosphorylation of c-Jun but not of ATF-2 at the low concentration at which SP600125 induced chemosensitization to TMZ, c-Jun-related responses were considered to be more important in the JNK-mediated survival of glioma

cells treated with TMZ than ATF-2-related pathways (Figure 7B).

target.

**4.2 c-Jun N-terminal kinase** 

in glioma cells treated with TMZ (Ohba et al., 2009).

induced DNA damage (Figure 7A).

compounds not only blocked TMZ-induced p38 activation but also dramatically inhibited the ability of U87 cells to undergo G2 arrest two days following TMZ exposure. Exposure of cells to SB203580 prior to and following TMZ exposure blocked the inactivation of both cdc25C and cdc2, consistent with the inability of SB compound-treated cells to undergo G2 arrest following TMZ exposure. SB203580 exposure, however, had no significant effect on TMZ-induced Chk1 or Chk2 activation. These results suggest that the p38 pathway, and p38/ specifically, are linked not only to MMR but also to activation of the G2 checkpoint through Chk1/2-independent actions on cdc25C and/or cdc2.

We also employed genetic means to selectively inhibit p38 and to assess the consequences of p38 inhibition on the G2 arrest pathway. To do so, MGMT-deficient, MMR-proficient U87MG cells were transfected with a pool of siRNA targeting p38, after which the cells were exposed to TMZ. Transfection of U87MG cells with p38 siRNA reduced basal levels of p38, and blocked the TMZ-induced ability of immunoprecipitated p38 to phosphorylate the p38 substrate MAPKAP2 *in vitro*. Selective genetic inhibition of p38 blocked TMZ-induced G2 arrest in U87MG cells in manner identical to that mediated by pharmacologic inhibitors of p38/. Furthermore, as was noted in studies using pharmacological inhibitors, p38 siRNA blocked the phosphorylation/inactivation of both cdc25C and cdc2 without affecting TMZ-induced Chk1 or Chk2 activation (Figure 6). These results clearly show that p38 is activated by the DNA MMR system, and that this activation is in turn linked by Chk1- and Chk2-independent means to inactivation of cdc2, cdc25C, and ultimately to G2 arrest.

Fig. 6. p38 inhibition potentiated TMZ-induced cytotoxicity

Although pharmacologic or genetic inhibition of p38 did not alter clonagenicity, extent of senescence-associated beta-galactosidase expression, or extent of mitotic catastrophe, (data not shown), it did enhance the sensitivity of U87MG cells to TMZ-induced cytotoxicity, reducing the colony forming ability of these cells. p38 inhibition increased the percentage of TMZ-treated cells that died by mitotic catastrophe, consistent with the idea that cells incapable of undergoing G2 arrest enter mitosis with damaged DNA and die by mitotic catastrophe. These results suggest that p38 not only links MMR to the G2 checkpoint, but also influences the response of cells to cytotoxic methylating agents. It would be reasonable to suspect that tumors capable of activating the G2 checkpoint via the p38 pathway would be less sensitive than those incapable of doing so. Conversely, because inhibition of p38 sensitizes cells to TMZ, p38 might be a reasonable therapeutic target.

#### **4.2 c-Jun N-terminal kinase**

68 Advances in the Biology, Imaging and Therapies for Glioblastoma

compounds not only blocked TMZ-induced p38 activation but also dramatically inhibited the ability of U87 cells to undergo G2 arrest two days following TMZ exposure. Exposure of cells to SB203580 prior to and following TMZ exposure blocked the inactivation of both cdc25C and cdc2, consistent with the inability of SB compound-treated cells to undergo G2 arrest following TMZ exposure. SB203580 exposure, however, had no significant effect on TMZ-induced Chk1 or Chk2 activation. These results suggest that the p38 pathway, and p38/ specifically, are linked not only to MMR but also to activation of the G2 checkpoint

We also employed genetic means to selectively inhibit p38 and to assess the consequences of p38 inhibition on the G2 arrest pathway. To do so, MGMT-deficient, MMR-proficient U87MG cells were transfected with a pool of siRNA targeting p38, after which the cells were exposed to TMZ. Transfection of U87MG cells with p38 siRNA reduced basal levels of p38, and blocked the TMZ-induced ability of immunoprecipitated p38 to phosphorylate the p38 substrate MAPKAP2 *in vitro*. Selective genetic inhibition of p38 blocked TMZ-induced G2 arrest in U87MG cells in manner identical to that mediated by pharmacologic inhibitors of p38/. Furthermore, as was noted in studies using pharmacological inhibitors, p38 siRNA blocked the phosphorylation/inactivation of both cdc25C and cdc2 without affecting TMZ-induced Chk1 or Chk2 activation (Figure 6). These results clearly show that p38 is activated by the DNA MMR system, and that this activation is in turn linked by Chk1- and Chk2-independent means to inactivation of cdc2,

through Chk1/2-independent actions on cdc25C and/or cdc2.

Fig. 6. p38 inhibition potentiated TMZ-induced cytotoxicity

Although pharmacologic or genetic inhibition of p38 did not alter clonagenicity, extent of senescence-associated beta-galactosidase expression, or extent of mitotic catastrophe, (data not shown), it did enhance the sensitivity of U87MG cells to TMZ-induced cytotoxicity, reducing the colony forming ability of these cells. p38 inhibition increased the percentage of TMZ-treated cells that died by mitotic catastrophe, consistent with the

cdc25C, and ultimately to G2 arrest.

Since p38, a major component of mitogen-activated protein kinases (MAPKs), had been shown to be involved in TMZ-induced cellular responses, we were interested other MAPKS in terms they could be target for chemosensitization. MAPKs are components of a complex intracellular signaling network that regulates gene expression in response to extracellular stimuli, in turn regulating cell proliferation, differentiation, and cell death. The MAPK family includes components of three major pathways in humans: p38 kinase, c-Jun NH2 terminal kinase (JNK), and extracellular signal-regulated kinase. In the JNK pathway, specific stimuli activate various kinases leading to activation of JNK. Once activated, JNK is translocated to the nucleus, where it phosphorylates and activates transcriptional factors such as components of activator protein, including c-Jun, JunB, and JunD, as well as other factors including ATF-2 and STAT-3. As examples of the importance of JNK on cell signaling, JNK is associated with cell survival, oncogenesis, growth, differentiation, and cell death. The role of the JNK pathway in the mediation of cellular responses (including cellular transformation, cell growth, and cell death) to extracellular stimuli has been studied extensively. Interestingly, JNK can exert completely opposite effects depending on the cell type and stimuli. Since the question of whether JNK is associated with cell death or cell survival appeared to depend on the type of cellular stress, we investigated in the role of JNK in glioma cells treated with TMZ (Ohba et al., 2009).

We first investigated whether JNK was activated in response to TMZ and confirmed the activation of JNK in glioma cells treated with TMZ. Previous studies on gliomas had already shown that JNK was activated and played a pro-survival role in response to the DNA crosslinking agent CDDP, and that the inhibition of the JNK pathway sensitized glioma cells to CDDP. Sensitization to chemotherapeutic agents by the inhibition of the JNK pathway has also been reported either in other cancer cells although, on the other hand, the inhibition of JNK reportedly led to the suppression of chemotherapeutic agentinduced apoptosis in several studies. Therefore, we investigated the role of JNK in glioma cells treated with TMZ by utilizing the JNK inhibitor SP600125. As a result, we confirmed that SP600125 potentiated the TMZ-induced cytotoxicity in U87MG cells, and concluded that JNK activation played a cytoprotective role in glioma cells in response to TMZinduced DNA damage (Figure 7A).

To clarify the mechanism responsible for the JNK inhibitor-induced potentiation of TMZinduced cytotoxicity, the two main downstream proteins of JNK, c-Jun and ATF-2, were investigated, since these proteins are believed to be associated with chemoresistance. In our study, because SP600125 inhibited the phosphorylation of c-Jun but not of ATF-2 at the low concentration at which SP600125 induced chemosensitization to TMZ, c-Jun-related responses were considered to be more important in the JNK-mediated survival of glioma cells treated with TMZ than ATF-2-related pathways (Figure 7B).

Biological Responses of Glioma Cell to Chemotherapeutic Agents 71

Whereas an extensive network of proteins are required to work together to initiate G2 arrest in response to DNA damage, a number of additional proteins have been reported to alter activation and maintenance of the G2 checkpoint. One of the most interesting of these is Akt, a member of the phosphatidylinositol-3 kinase family that is recruited to the cell membrane and activated in response to the generation of phospholipids by a variety of signaling pathways. Activated Akt in turn signals to a variety of key downstream molecules including mammalian target of rapamycin (mTOR), glycogen synthetase kinase 3, and S6 kinase, the sum of which is to suppress cell death and to promote cell survival. In addition to effects on apoptosis and cell metabolism, Akt activation has been reported to suppress activation of the G2 checkpoint in human colon carcinoma cells exposed to radiation. Whereas the mechanism by which Akt suppresses G2 arrest has not been defined, the observation that Akt influences the G2 checkpoint is of particular importance to the therapeutic application of TMZ whose cytotoxicity is influenced by the G2 checkpoint and which are frequently used in the treatment of PTEN-deficient, Akt overexpressing gliomas. Furthermore, because exposure of glioma cells to TMZ induces a senescence-like phenomenon and mitotic catastrophe, and because bypass of TMZ–induced G2 arrest by Chk1 inhibitors enhances, rather than suppresses, glioma cell death as described above, the consequences of potential Akt-mediated bypass of methylating agent–induced G2 arrest on cellular outcome remained

To investigate the effect of Akt activation in biological responses of glioma cells to DNAmethylating compound, we created U87MD-derived cells of which Akt activity could be exogeneously controlled. U87MG human glioma cells were infected with a retroviral construct encoding a modified Akt (AktERM+) protein which, by virtue of deletion of the parathyroid hormone domain, fusion to a c-Src myristoylation signal, and fusion to a modified form of the mouse ER hormone binding domain, has been shown rapidly activated in response to 4-hydroxytamoxifen. The levels of AktER rose in response to exposures of 4-

Having created cells with inducible levels of activated Akt, we addressed the consequences of Akt pathway activation on TMZ-induced G2 arrest. In the absence of 4-hydroxytamoxifen, TMZ-treated U87MG-AktERM+ cells showed Cdc2 (Tyr15) phosphorylation/ inactivation, and G2 arrest 3 to 5 days following TMZ exposure. However, U87MG cells expressing the AktERM+ construct exhibited significant inhibition of TMZ-induced phosphorylation/

As described above, inhibition of TMZ-induced G2 arrest by Chk1 inhibitor forced cells into mitosis and enhances cell death by mitotic catastrophe in both p53-proficient and p53 deficient glioma cells. We therefore questioned whether bypass of TMZ-induced G2 arrest by Akt, a protein known to suppress apoptosis in response to radiation-induced DNA damage, would sensitize cells to TMZ or instead protect cells by suppressing pathways linked to senescence and/or mitotic catastrophe. As a result, AktERM+ activation protected rather than sensitized the p53-proficient U87MG cells to TMZ-induced cytotoxicity (Figure 8C). The cytoprotective effects of Akt overexpression were associated with a reduction in the percentage of cells expressing senescence-associated beta-galactosidase activity following TMZ exposure, suggesting that Akt overexpression not only reduced the percentage of cells undergoing TMZ-induced G2 arrest but also reduced the ability of p53-positive glioma cells to undergo a senescence-like phenomenon in response to TMZ. Furthermore, AktERM+

uncertain.

hydroxytamoxifen (Figure 8A).

inactivation of Cdc2, and TMZ-induced G2 arrest (Figure 8B).

(B)

Fig. 7. Effect of JNK inhibitor SB600125 on TMZ-induced cytotoxicity (A) and phosphorylation of associated proteins (B).

As noted above, the inhibition of p38 increased the sensitivity of glioma cells to TMZ in association with the abrogation of G2 arrest, however, in contrast, JNK inhibitor did not affect the cell cycle distribution of TMZ-treated cells nor changed the protein level of phosphorylated cdc2. Thus, the SP600125-induced chemosensitization to TMZ in glioma cells was probably not a consequence of the abrogation of cell cycle arrest. Rather, JNK inhibition increased the percentage of senescence-like cells in U87MG cells and of mitotic catastrophe cells in U87MG-E6 cells after treatment with TMZ. These results suggest that the enhancement of TMZ toxicity by a JNK inhibitor in glioma cells was induced by the potentiation of cell death pathways induced by TMZ alone. These data might be in agreement with previous studies on gliomas which suggested involvement of the JNK pathway in DNA repair.. c-Jun-related responses could be key events in the JNK-mediated cytoprotection of glioma cells treated with TMZ, and further investigations of the involvement of c-Jun in the survival machinery of cells with DNA damage might promote the development of useful chemotherapeutic strategies against malignant gliomas.

#### **5. Survival-promoting protein Akt**

Survival promoting protein such as Akt, which is frequently activated in malignant gliomas, could be a target to enhance the effect of chemotherapeutic agents.

(A)

(B)

As noted above, the inhibition of p38 increased the sensitivity of glioma cells to TMZ in association with the abrogation of G2 arrest, however, in contrast, JNK inhibitor did not affect the cell cycle distribution of TMZ-treated cells nor changed the protein level of phosphorylated cdc2. Thus, the SP600125-induced chemosensitization to TMZ in glioma cells was probably not a consequence of the abrogation of cell cycle arrest. Rather, JNK inhibition increased the percentage of senescence-like cells in U87MG cells and of mitotic catastrophe cells in U87MG-E6 cells after treatment with TMZ. These results suggest that the enhancement of TMZ toxicity by a JNK inhibitor in glioma cells was induced by the potentiation of cell death pathways induced by TMZ alone. These data might be in agreement with previous studies on gliomas which suggested involvement of the JNK pathway in DNA repair.. c-Jun-related responses could be key events in the JNK-mediated cytoprotection of glioma cells treated with TMZ, and further investigations of the involvement of c-Jun in the survival machinery of cells with DNA damage might promote

Fig. 7. Effect of JNK inhibitor SB600125 on TMZ-induced cytotoxicity (A) and

the development of useful chemotherapeutic strategies against malignant gliomas.

could be a target to enhance the effect of chemotherapeutic agents.

Survival promoting protein such as Akt, which is frequently activated in malignant gliomas,

phosphorylation of associated proteins (B).

**5. Survival-promoting protein Akt** 

Whereas an extensive network of proteins are required to work together to initiate G2 arrest in response to DNA damage, a number of additional proteins have been reported to alter activation and maintenance of the G2 checkpoint. One of the most interesting of these is Akt, a member of the phosphatidylinositol-3 kinase family that is recruited to the cell membrane and activated in response to the generation of phospholipids by a variety of signaling pathways. Activated Akt in turn signals to a variety of key downstream molecules including mammalian target of rapamycin (mTOR), glycogen synthetase kinase 3, and S6 kinase, the sum of which is to suppress cell death and to promote cell survival. In addition to effects on apoptosis and cell metabolism, Akt activation has been reported to suppress activation of the G2 checkpoint in human colon carcinoma cells exposed to radiation. Whereas the mechanism by which Akt suppresses G2 arrest has not been defined, the observation that Akt influences the G2 checkpoint is of particular importance to the therapeutic application of TMZ whose cytotoxicity is influenced by the G2 checkpoint and which are frequently used in the treatment of PTEN-deficient, Akt overexpressing gliomas. Furthermore, because exposure of glioma cells to TMZ induces a senescence-like phenomenon and mitotic catastrophe, and because bypass of TMZ–induced G2 arrest by Chk1 inhibitors enhances, rather than suppresses, glioma cell death as described above, the consequences of potential Akt-mediated bypass of methylating agent–induced G2 arrest on cellular outcome remained uncertain.

To investigate the effect of Akt activation in biological responses of glioma cells to DNAmethylating compound, we created U87MD-derived cells of which Akt activity could be exogeneously controlled. U87MG human glioma cells were infected with a retroviral construct encoding a modified Akt (AktERM+) protein which, by virtue of deletion of the parathyroid hormone domain, fusion to a c-Src myristoylation signal, and fusion to a modified form of the mouse ER hormone binding domain, has been shown rapidly activated in response to 4-hydroxytamoxifen. The levels of AktER rose in response to exposures of 4 hydroxytamoxifen (Figure 8A).

Having created cells with inducible levels of activated Akt, we addressed the consequences of Akt pathway activation on TMZ-induced G2 arrest. In the absence of 4-hydroxytamoxifen, TMZ-treated U87MG-AktERM+ cells showed Cdc2 (Tyr15) phosphorylation/ inactivation, and G2 arrest 3 to 5 days following TMZ exposure. However, U87MG cells expressing the AktERM+ construct exhibited significant inhibition of TMZ-induced phosphorylation/ inactivation of Cdc2, and TMZ-induced G2 arrest (Figure 8B).

As described above, inhibition of TMZ-induced G2 arrest by Chk1 inhibitor forced cells into mitosis and enhances cell death by mitotic catastrophe in both p53-proficient and p53 deficient glioma cells. We therefore questioned whether bypass of TMZ-induced G2 arrest by Akt, a protein known to suppress apoptosis in response to radiation-induced DNA damage, would sensitize cells to TMZ or instead protect cells by suppressing pathways linked to senescence and/or mitotic catastrophe. As a result, AktERM+ activation protected rather than sensitized the p53-proficient U87MG cells to TMZ-induced cytotoxicity (Figure 8C). The cytoprotective effects of Akt overexpression were associated with a reduction in the percentage of cells expressing senescence-associated beta-galactosidase activity following TMZ exposure, suggesting that Akt overexpression not only reduced the percentage of cells undergoing TMZ-induced G2 arrest but also reduced the ability of p53-positive glioma cells to undergo a senescence-like phenomenon in response to TMZ. Furthermore, AktERM+

Biological Responses of Glioma Cell to Chemotherapeutic Agents 73

overexpression in U87MG-E6 cells suppressed not only TMZ-induced G2 arrest and loss of clonogenicity but also the percentage of cells undergoing death by mitotic catastrophe following TMZ exposure. These results suggest that Akt overexpression, whereas suppressing TMZ-induced G2 arrest, also protects cells from loss of clonogenicity caused by

In summary, the results showed that Akt activation suppresses the G2 checkpoint by selectively altering activation of the DNA damage signal transducer Chk2 and the downstream effectors of the G2 checkpoint. The overriding effect of Akt activation, however, is suppression of TMZ-induced senescence and mitotic catastrophe in cells that avoid G2 arrest. The Akt pathway may therefore contribute to TMZ resistance in the clinical

It is interesting to note that most high-grade human gliomas have high levels of Akt activation, which are believed to be a consequence of PTEN deletion. Given the role Akt plays in moving cells through the G2 checkpoint and in suppressing TMZ-induced cytotoxicity, it seems likely that an analysis of Akt pathway activation in gliomas before therapy may help identify those individuals for whom TMZ-based therapies are most likely to succeed. Similarly, strategies combining TMZ with inhibitors of the Akt pathway may enhance the likelihood of success. Because Akt overexpression has also been reported to increase the mutagenicity of agents that induce G2 arrest, presumably by promoting cell survival in the absence of genuine DNA repair, strategies designed to suppress Akt may contribute not only to improved tumor cell kill but also to suppression of unwanted

As reviewed above, several ways to potentiate the cytotoxicity of TMZ have been reported, and thus many pathways can be targeted in an effort to sensitize tumor cells to chemotherapeutic agents. A molecular chaperone, 90kD heat shock protein (hsp90) has recently attracted attention as a sensitizing agent because it is expressed at 2–10-fold higher levels in tumor tissue than in normal tissue, and is associated with many proteins (termed client proteins) involved in cell cycle regulation, cell survival and oncogenesis. Taken together, these studies indicated that many hsp90 client proteins are involved in cytoprotective mechanisms against cellular stressors such as DNA damage, suggesting that hsp90 might be important in the survival of tumor cells after exposure to DNA-damaging chemotherapeutic agents. Therefore we hypothesized that hsp90 inhibitors might act as antitumor agents against gliomas and might potentiate the cytotoxicity of DNA-damaging

To investigate this hypothesis, we used 17-AAG, a geldanamycin derivative, as a pharmacological inhibitor of hsp90 and examined whether an hsp90-targeted strategy could be useful for chemosensitizing glioma cells to the DNA-damaging agents BCNU, cisplatin,

The clonogenicity of cells treated with cisplatin, BCNU, or TMZ was depressed with 17- AAG. This 17-AAG–induced potentiation of the effects of these chemotherapeutic agents was recognized at a lower concentration than that needed to induce cytotoxicity with 17- AAG alone, and was more remarkable in the cisplatin- and BCNU-treated cells than in the

mutagenic effects which might otherwise contribute to secondary malignancies.

**6. Molecular chaperone 90kD heat shock protein** 

induction of senescence and mitotic catastrophe.

setting.

agents.

and TMZ (Ohba et al., 2010).

Fig. 8. Akt over-activation in U87MG-AktERM+ cells (A) and its effect on TMZ-induced cell cycle arrest (B) and cytotoxicity (C)

(A)

(B)

(C)

Fig. 8. Akt over-activation in U87MG-AktERM+ cells (A) and its effect on TMZ-induced cell

cycle arrest (B) and cytotoxicity (C)

overexpression in U87MG-E6 cells suppressed not only TMZ-induced G2 arrest and loss of clonogenicity but also the percentage of cells undergoing death by mitotic catastrophe following TMZ exposure. These results suggest that Akt overexpression, whereas suppressing TMZ-induced G2 arrest, also protects cells from loss of clonogenicity caused by induction of senescence and mitotic catastrophe.

In summary, the results showed that Akt activation suppresses the G2 checkpoint by selectively altering activation of the DNA damage signal transducer Chk2 and the downstream effectors of the G2 checkpoint. The overriding effect of Akt activation, however, is suppression of TMZ-induced senescence and mitotic catastrophe in cells that avoid G2 arrest. The Akt pathway may therefore contribute to TMZ resistance in the clinical setting.

It is interesting to note that most high-grade human gliomas have high levels of Akt activation, which are believed to be a consequence of PTEN deletion. Given the role Akt plays in moving cells through the G2 checkpoint and in suppressing TMZ-induced cytotoxicity, it seems likely that an analysis of Akt pathway activation in gliomas before therapy may help identify those individuals for whom TMZ-based therapies are most likely to succeed. Similarly, strategies combining TMZ with inhibitors of the Akt pathway may enhance the likelihood of success. Because Akt overexpression has also been reported to increase the mutagenicity of agents that induce G2 arrest, presumably by promoting cell survival in the absence of genuine DNA repair, strategies designed to suppress Akt may contribute not only to improved tumor cell kill but also to suppression of unwanted mutagenic effects which might otherwise contribute to secondary malignancies.
