Are There Thresholds in Glioblastoma Cell Death Responses Triggered by Temozolomide?

Yang He,Bernd Kaina

doi:10.3390/ijms20071562

Abstract

Temozolomide (TMZ) is an alkylating agent used in the treatment of high-grade malignant glioma, notably glioblastoma multiforme, the most aggressive form of brain cancer. The drug induces a dozen DNA methylation adducts, including O6-methylguanine (O6MeG), which is the most toxic primary DNA lesion as it causes the formation of DNA double-strand breaks (DSBs) that trigger apoptosis. In p53 wild-type cells, TMZ activates p-p53ser15 and p-p53ser46, which have opposing dual functions regulating survival and death, respectively. Since the use of TMZ in a therapeutic setting is limited because of its side effects, the question arises as to the existence of threshold doses that activate the death pathway and start apoptosis. To determine whether there is a threshold for the TMZ-induced DNA damage response and exploring the factors regulating the switch between p53 dependent survival and death, the glioblastoma lines LN-229 (deficient in MGMT) and LN-229MGMT (stably transfected with MGMT) were exposed to different doses of TMZ. p53 protein expression and phosphorylation levels of p-p53ser15 and p-p53ser46 were determined by Western blotting. Also, apoptosis, senescence and autophagy levels were checked after different doses of TMZ. The results show that pro-survival p-p53ser15 and pro-death p-p53ser46 were induced by O6MeG in a specific dose- and time-dependent manner. p-p53ser15 was an early response while p-p53ser46 was activated at later times following treatment. Unexpectedly, the dose-response curves for total p53, p-p53ser15 and p-p53ser46 were linear, without an obvious threshold. O6MeG induces apoptosis late after treatment as a linear function of TMZ dose. This was observed for both p53 proficient LN-229 and p53 lacking LN-308 cells. A linear dose-response after TMZ was also observed for senescence and autophagy as well as γH2AX, an indicator of DSBs that are considered to be the downstream trigger of apoptosis, senescence and autophagy. LN-229MGMT cells were highly resistant to all measured endpoints because of repair of the critical primary lesion. Although LN-308 were less responsive than LN-229 to TMZ, they displayed the same TMZ-induced DSB level. The observed linear dose-responses are not compatible with the view that low DNA damage level evokes survival while high damage level activates death functions. The data bear important therapeutic implications as they indicate that even low doses of TMZ may elicit a cytotoxic response. However, since O6MeG triggers apoptosis, senescence and autophagy in the same dose range, it is likely that the accumulation of senescent cells in the population counteracts the killing effect of the anticancer drug.

Highlights

In cancer therapy, chemotherapeutics with genotoxic activity are being used routinely
We further used the isogenic line LN-229MGMT, which was stably transfected with human methylguanine-DNA methyltransferase (MGMT) cDNA and expresses MGMT [18], in order to find out whether the responses observed are triggered by the specific TMZ-induced DNA damage O6MeG
In LN-229MGMT, we did not observe any significant increase in p53, p-p53ser15 and p-p53ser46 (Figure 1A, right panel), demonstrating that the effects observed were triggered by the O6MeG lesion, which is repaired by MGMT

Summary

Introduction

Chemotherapeutics with genotoxic activity are being used routinely. It is well known that these anticancer drugs induce DNA damage that triggers complex cellular DNA damage responses (DDR), which determine the fate of the cell, making the decision between survival and death [1]. It is generally believed that low DNA damage levels activate pro-survival and high damage levels activate pro-death genes and cellular functions [4,5,6]. At high dose levels p53 turns into a “killer” through activation of pro-death functions such as the proapoptotic genes Bax, Bak and Fas [2]. This concept derived mostly from work with ionizing radiation is reasonable, there is not much experimental proof of it for chemical genotoxins, notably anticancer drugs. The concept implicates that there are threshold doses for cell death, i.e., low doses do not elicit activation of apoptosis pathways while high doses do

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