**3. DNA checkpoint**

62 Advances in the Biology, Imaging and Therapies for Glioblastoma

Since MGMT has been considered as a key factor in the resistance of gliomas to TMZ, several clinical trials have been conducted. However, TMZ treatment in combination with MGMT-depleting compound did not show remarkable therapeutic affect for malignant gliomas, and recent clinical studies suggest that MGMT is just a prognostic marker for malignant gliomas treated with genotoxic agents including radiation and chemotherapy, and that MGMT is not the only factor that induces TMZ-resistance, and that further biological investigation on glioma cells is needed. In this chapter we review our studies on glioma biology in regard its cellular responses to DNA-damaging compounds, especially TMZ, which could provide a clue to develop safe and effective methods to potentiate anti-

Most of DNA-damaging chemotherapeutic agents induce cell cycle arrest, and so does TMZ (Hirose et al., 2001a). When MGMT-negative and p53 wildtype U87MG human glioblastoma cells were treated with TMZ at the concentration based on the published data of the plasma concentration of the drug in patients involved in its clinical trials (100 microM for 3 hours), FACS analysis revealed no significant difference in the percentage of cells in each phase of the cell cycle between untreated U87MG cells and the cells harvested at 1 day after TMZ treatment. However, cells began to accumulate at the G2/M boundary (4n DNA content) 2 days after TMZ treatment. This G2/M arrest (here defined as a greater percentage of cells in G2/M than G1) was sustained for at least 10 days after TMZ treatment, and was associated with the gradual appearance of hyperploid (>4n DNA content) cells and the gradual loss of cells with 2n DNA content (G1 cells). Although TMZ-treated cells underwent cell cycle arrest, the sub-G1 population, which represents apoptotic cells, was small and did not significantly increase throughout the 10 days following TMZ treatment. Consistent with the G2/M arrest data, p53 and p21Waf1/Cip1 levels were increased approximately 2-4 fold at 2 days after TMZ treatment with the accumulation persisting at least 10 days after the treatment. On the other hand, **g**enetically modified U87MG-E6 cells which have low levels of p53 because of transfection of human papilloma viral oncoprotein E6 mediating ubiquitination and destruction of p53 showed shorter G2/M after TMZ treatment. That is, the proportion of U87MG cells at G2/M began decreasing by 3 days after TMZ treatment and was considerably lessened by 10 days after treatment. The reduction in G2/M arrest in these cells was also associated with a gradual increase in cells with a sub-G1 (less than 2n). Because U87MG and U87MG-E6 cells share a common genetic background except for expression of E6, comparison of the responses of these cells provides more useful information than the comparison of responses of cells with completely different genetic backgrounds. These results support the idea that p53 (and p21Waf1/Cip1), while not important for initiation of TMZ-induced G2/M arrest, do play a role in sustaining the arrest (Figure 2). Colony formation efficiency assay revealed that U87MG cells showed a dose-dependent decrease in clonogenicity, and, on the other hand, U87MG-E6 cells formed far less colonies. As well as being associated with the duration of TMZ-induced arrest in glioma cells, the p53 status of cells was also associated with the ultimate fate of the cells. p53-wildtype cells underwent a prolonged G2/M arrest which left the majority of cells viable yet nonproliferative showing the features of ascent cells. In contrast, p53-deficient cells underwent a more transient arrest, and lost viability in a manner consistent with mitotic catastrophy. Therefore while both p53-wt and p53-deficient cells became non-proliferative in response to

**2. Cell cycle arrest of glioma cells in response to temozolomide** 

tumor activity of the drug.

While both p53-wt and p53-deficient cells initiate G2/M arrest and become non-proliferative in response to TMZ, p53-wt cells which undergo prolonged G2/M arrest are less sensitive than p53-deficient cells to the cytotoxic actions of TMZ. One possible explanation for this effect is that the prolonged G2/M arrest noted in p53-wt cells allows more time for reversal of the cytotoxic effects of the drug prior to entry into mitosis and death by mitotic catastrophe. G2/M arrest in response to TMZ may therefore represent a defense mechanism against the cytotoxic actions of TMZ.

While the linkage between TMZ-induced DNA damage and G2/M arrest has not been fully explored, the linkage between irradiation-induced DNA damage and G2/M arrest has been shown to involve a pathway controlling the cyclin-dependent kinase cdc2. Various types of DNA damage activate Chk1 kinase which phophorylates cdc25C phosphatase at serine-216 which enhances the binding of 14-3-3 proteins and the export of the cdc25C/14-3-3 complex to the cytoplasm. The cytoplasmic sequestration of phosphorylated cdc25C in turn eliminates the potential cdc25C-mediated dephosphorylation of cdc2. Cdc2 therefore remains bound to cyclin B in an inactive, phosphorylated state. The end result of DNA damage-induced Chk1 activation therefore is the phosphorylation of cdc2, and the arrest of cells with damaged DNA at the G2/M boundary. The ability of TMZ to induce DNA damage suggested that, like other DNA damaging agents, TMZ might initiate G2/M arrest via a Chk1-dependent pathway.

Biological Responses of Glioma Cell to Chemotherapeutic Agents 65

a p53-associated prolonged G2/M arrest and senescence in response to TMZ, UCN-01 posttreatment greatly reduced the extent of G2/M arrest, reduced the percentage of cells undergoing TMZ-induced senescence, and increased the percentage of cells undergoing mitotic catastrophe. These results clearly suggested that in p53-proficient glioma cells, the G2 checkpoint serves a protective function, and that elimination of the checkpoint is associated with an increase in the number of cells that die by pre-mature entry into mitosis. UCN-01, however, not only sensitized p53-wt cells but also p53-deficient cells which underwent only a transient G2/M arrest in response to TMZ. This sensitization did not involve changes in TMZ-induced senescence (which was minimal in these cells) but rather was associated exclusively with increases in the number of cells undergoing mitotic

Fig. 4. Enhancement of TMZ-induced mitotic catastrophe by UCN-01.

While increased levels of MGMT and loss of MMR capacity can both confer TMZ resistance, very few gliomas over-express MGMT or are MMR deficient. It appears likely, therefore that at least some of the resistance of gliomas to TMZ involves events downstream of futile MMR activation. The ability of UCN-01 to prevent downstream events which may contribute to TMZ resistance may therefore prove useful in the treatment of TMZ-resistant as well as TMZ-sensitive tumors. As approximately two-thirds of gliomas have defects in the p53 pathway, the ability of UCN-01 to sensitize cells regardless of p53 status also increases the range of tumors for which this approach might be effective. While issues relating to duration of exposure, drug sequencing, and the events that link MMR to Chk1 activation remain to be examined, it has been suggested that the combinations of TMZ with G2 checkpoint inhibitors such as UCN-01 might be useful additions to existing therapies for

catastrophe (Figure 4).

brain tumors.

If Chk1 activation is critical in activation of G2/M arrest in TMZ-treated cells, and if G2/M arrest provides the opportunity for cells to avoid TMZ-induced cytotoxicity, inhibition of cdc2-dependent G2 arrest should sensitize cells to TMZ. A variety of small molecule inhibitors have recently been developed, and we analyzed the effect of UCN-01, a staurosporin derivative Chk1 inhibitor, as a pharmacologic tool to assess the linkage between TMZ exposure and G2/M arrest, to determine if G2/M arrest protects cells from TMZ-induced cytotoxicity, and to determine if Chk1 inhibitors might represent a way to sensitize cells to TMZ (Hirose et al., 2001b).

To better define how TMZ induces G2/M arrest, we analyzed alterations in levels of G2 checkpoint-associated proteins in TMZ-treated cells. The protein levels of Chk1 increased in a transient manner, rising at 1-2 days after TMZ exposure and returning to sub-control levels by 10 days after TMZ exposure. While total levels of cdc2 were unchanged or only slightly increased after TMZ, levels of phosphorylated cdc2 were transiently increased in both U87MG and U87MG-E6 cells in a timeframe and manner similar to that noted for induction of Chk1.

To more directly test the association between TMZ and alterations in G2 checkpoint proteins, U87MG and U87MG-E6 cells were treated with TMZ, and then exposed to UCN-01 for 3 days immediately following TMZ removal. Immunoblot analyses revealed that UCN-01 reduced the level of phosphorylated cdc2 in the cells and significantly inhibited TMZinduced G2/M arrest of both U87MG and U87MG-E6 cells Furthermore, in agreement with the study described above, UCN-01 reduced TMZ-induced senescence-associated betagalactosidase activity and enhances mitotic catastrophe. UCN-01 increases TMZ-induced cytotoxicity in both p53-wt and p53-deficient glioma cells (Figure 3).

Fig. 3. p53-independent sensitization of glioma cells to TMZ by Chk1 inhibitor UCN-01.

Having established an association between TMZ-exposure, Chk1 activation, and TMZinduced G2/M arrest, we examined the hypothesis that G2/M arrest is a protective response of cells to TMZ-induced cytotoxicity, and that elimination of the G2 checkpoint might sensitize cells (and in particular p53-wt cells) to TMZ. In p53 wt cells, which undergo

If Chk1 activation is critical in activation of G2/M arrest in TMZ-treated cells, and if G2/M arrest provides the opportunity for cells to avoid TMZ-induced cytotoxicity, inhibition of cdc2-dependent G2 arrest should sensitize cells to TMZ. A variety of small molecule inhibitors have recently been developed, and we analyzed the effect of UCN-01, a staurosporin derivative Chk1 inhibitor, as a pharmacologic tool to assess the linkage between TMZ exposure and G2/M arrest, to determine if G2/M arrest protects cells from TMZ-induced cytotoxicity, and to determine if Chk1 inhibitors might represent a way to

To better define how TMZ induces G2/M arrest, we analyzed alterations in levels of G2 checkpoint-associated proteins in TMZ-treated cells. The protein levels of Chk1 increased in a transient manner, rising at 1-2 days after TMZ exposure and returning to sub-control levels by 10 days after TMZ exposure. While total levels of cdc2 were unchanged or only slightly increased after TMZ, levels of phosphorylated cdc2 were transiently increased in both U87MG and U87MG-E6 cells in a timeframe and manner similar to that noted for

To more directly test the association between TMZ and alterations in G2 checkpoint proteins, U87MG and U87MG-E6 cells were treated with TMZ, and then exposed to UCN-01 for 3 days immediately following TMZ removal. Immunoblot analyses revealed that UCN-01 reduced the level of phosphorylated cdc2 in the cells and significantly inhibited TMZinduced G2/M arrest of both U87MG and U87MG-E6 cells Furthermore, in agreement with the study described above, UCN-01 reduced TMZ-induced senescence-associated betagalactosidase activity and enhances mitotic catastrophe. UCN-01 increases TMZ-induced

Fig. 3. p53-independent sensitization of glioma cells to TMZ by Chk1 inhibitor UCN-01.

Having established an association between TMZ-exposure, Chk1 activation, and TMZinduced G2/M arrest, we examined the hypothesis that G2/M arrest is a protective response of cells to TMZ-induced cytotoxicity, and that elimination of the G2 checkpoint might sensitize cells (and in particular p53-wt cells) to TMZ. In p53 wt cells, which undergo

cytotoxicity in both p53-wt and p53-deficient glioma cells (Figure 3).

sensitize cells to TMZ (Hirose et al., 2001b).

induction of Chk1.

a p53-associated prolonged G2/M arrest and senescence in response to TMZ, UCN-01 posttreatment greatly reduced the extent of G2/M arrest, reduced the percentage of cells undergoing TMZ-induced senescence, and increased the percentage of cells undergoing mitotic catastrophe. These results clearly suggested that in p53-proficient glioma cells, the G2 checkpoint serves a protective function, and that elimination of the checkpoint is associated with an increase in the number of cells that die by pre-mature entry into mitosis. UCN-01, however, not only sensitized p53-wt cells but also p53-deficient cells which underwent only a transient G2/M arrest in response to TMZ. This sensitization did not involve changes in TMZ-induced senescence (which was minimal in these cells) but rather was associated exclusively with increases in the number of cells undergoing mitotic catastrophe (Figure 4).

Fig. 4. Enhancement of TMZ-induced mitotic catastrophe by UCN-01.

While increased levels of MGMT and loss of MMR capacity can both confer TMZ resistance, very few gliomas over-express MGMT or are MMR deficient. It appears likely, therefore that at least some of the resistance of gliomas to TMZ involves events downstream of futile MMR activation. The ability of UCN-01 to prevent downstream events which may contribute to TMZ resistance may therefore prove useful in the treatment of TMZ-resistant as well as TMZ-sensitive tumors. As approximately two-thirds of gliomas have defects in the p53 pathway, the ability of UCN-01 to sensitize cells regardless of p53 status also increases the range of tumors for which this approach might be effective. While issues relating to duration of exposure, drug sequencing, and the events that link MMR to Chk1 activation remain to be examined, it has been suggested that the combinations of TMZ with G2 checkpoint inhibitors such as UCN-01 might be useful additions to existing therapies for brain tumors.

Biological Responses of Glioma Cell to Chemotherapeutic Agents 67

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

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 agent-

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

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

and on the recognition and/or processing of these lesions by the DNA MMR system.

induced G2 arrest.

silenced glioma cells (B).
