**Biological Responses of Glioma Cell to Chemotherapeutic Agents**

Yuichi Hirose and Shigeo Ohba

*Department of Neurosurgery, Fujita Health University, Toyoake Department of Neurosurgery, Ashikaga Red Cross Hospital, Ashikaga Japan* 

#### **1. Introduction**

60 Advances in the Biology, Imaging and Therapies for Glioblastoma

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Because gliomas are not curable surgically, development of effective adjuvant therapies is warranted. A chemotherapeutic agent temozolimide (TMZ) has been widely used not only because it is well tolerated and easily administrated orally but because various clinical trials had revealed that high grade gliomas could show objective response or stable disease to this compound (Stupp et al., 2005). The action of TMZ had been extensively studied primarily in leukemia and lymphoma cells. TMZ spontaneously decomposes in aqueous solution to form the cytotoxic methylating agent, and the cytotoxicity of TMZ appears to be mediated mainly through adduction of a methyl group to O6 position of guanine (G) in genomic DNA. The methyl group can be removed from O6-methylguanine by O6-methylguanine-DNA methyltransferase (MGMT). If MGMT is deficient in the cell, however, O6-methylguanine is not repaired, and incorporation of a thymine (T) rather than a cytosine opposite the O6 methylguanine during the next cycle of DNA replication leads to the formation of GT mismatches in DNA. This triggers the DNA mismatch repair (MMR) system which removes the T, only to have the T reinserted during repair synthesis. Futile cycles of MMR triggered by GT mismatches can lead to a variety of outcomes in TMZ-treated cells (Figure 1).

Fig. 1. Mechanism of temozolomide-induced DNA damage creation

Biological Responses of Glioma Cell to Chemotherapeutic Agents 63

TMZ, the means by which this was accomplished differed in a manner consistent with p53

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

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

Fig. 2. TMZ-induced cell cycle arrest and the fate of the cells.

**3. DNA checkpoint** 

against the cytotoxic actions of TMZ.

via a Chk1-dependent pathway.

status.

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 antitumor activity of the drug.

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

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 TMZ, the means by which this was accomplished differed in a manner consistent with p53 status.

Fig. 2. TMZ-induced cell cycle arrest and the fate of the cells.
