Repair gene O6 -methylguanine-DNA methyltransferase is controlled by SP1 and up-regulated by glucocorticoids, but not by temozolomide and radiation.

Therapy of malignant glioma relies on treatment with the O6 -methylating agent temozolomide (TMZ) concomitant with ionizing radiation followed by adjuvant TMZ. For the treatment of recurrences, DNA chloroethylating drugs are also used. The main killing lesion induced by these drugs is O6 -alkylguanine. Since this damage is repaired by O6 -methylguanine-DNA methyltransferase (MGMT), the repair enzyme represents a most important factor of drug resistance, limiting the therapy of malignant high-grade gliomas. Although MGMT has been shown to be transcriptionally up-regulated in rodents following genotoxic stress, it is still unclear whether human MGMT is subject to up-regulation. Here, we addressed the question whether MGMT in glioma cells is enhanced following alkylating drugs or ionizing radiation, using promoter assays. We also checked the response of glioma cell lines to dexamethasone. In a series of experiments, we found no evidence that the human MGMT promoter is significantly up-regulated following treatment with TMZ, the chloroethylating agent nimustine or radiation. It was activated, however, by dexamethasone. Using deletion constructs, we further show that the basal level of MGMT is mainly determined by the transcription factor SP1. The high amount of SP1 sites in the MGMT promoter likely prevents transcriptional up-regulation following genotoxic stress by neutralizing inducible signals. The regulation of MGMT by miRNAs plays only a minor role, as shown by DICER knockdown experiments. Since high dose dexamethasone concomitant with temozolomide is frequently used in glioblastoma therapy, induction of the MGMT gene through glucocorticoids in MGMT promoter unmethylated cases might cause further elevation of drug resistance, while radiation and alkylating drugs seem not to induce MGMT at transcriptional level.

Site-directed mutagenesis of the ATM promoter: consequences for response to proliferation and ionizing radiation.

Although ATM, the protein defective in ataxia-telangiectasia (A-T), is activated primarily by radiation, there is also evidence that expression of the protein can be regulated by both radiation and growth factors. Computer analysis of the ATM promoter proximal 700-bp sequence reveals a number of potentially important cis-regulatory sequences. Using nucleotide substitutions to delete putative functional elements in the promoter of ATM, we examined the importance of some of these sites for both the basal and the radiation-induced activity of the promoter. In lymphoblastoid cells, most of the mutations in transcription factor consensus sequences [Sp1(1), Sp1(2), Cre, Ets, Xre, gammaIre(2), a modified AP1 site (Fse), and GCF] reduced basal activity to various extents, whereas others [gammaIre(1), NF1, Myb] left basal activity unaffected. In human skin fibroblasts, results were generally the same, but the basal activity varied up to 8-fold in these and other cell lines. Radiation activated the promoter approximately 2.5-fold in serum-starved lymphoblastoid cells, reaching a maximum by 3 hr, and all mutated elements equally blocked this activation. Reduction in Sp1 and AP1 DNA binding activity by serum starvation was rapidly reversed by exposure of cells to radiation. This reduction was not evident in A-T cells, and the response to radiation was less marked. Data provided for interaction between ATM and Sp1 by protein binding and co-immunoprecipitation could explain the altered regulation of Sp1 in A-T cells. The data described here provide additional evidence that basal and radiation-induced regulation of the ATM promoter is under multifactorial control.

Ionizing radiation stimulates octamer factor DNA binding activity in human carcinoma cells.

In mammalian cells, the octamer motif (ATGCAAAT) binding proteins, Oct-1 and Oct-2, play an important role in the transcriptional transactivation of several ubiquitously expressed genes as well as cell-specifically expressed genes. To date, a role of the octamer binding proteins in damage-stimulated response is not known. In this report, we demonstrate that DNA-binding activity of Oct-1, as demonstrated by the electrophoretic mobility shift assay, is significantly induced in a dose-dependent manner upon treatment of human head and neck squamous carcinoma cells (PCI-04A) with ionizing radiation (5 Gy: 5-fold; 15 Gy: 11-fold). By comparison, activities of other transcription factors were modestly increased (15 Gy: AP-1, 2.5-fold; NF-kappaB, 2.6-fold; SP-1, 5-fold). Radiation stimulation of Oct-1 activity was also noted in two other human cancer cell lines, albeit to a lesser extent (MDA-MB231 breast carcinoma cells and PC-3 prostate carcinoma cells (5 Gy: approximately 2-fold). These data represent the first report of the activation of an octamer factor DNA binding activity in response to environmental cues and suggest a novel role of Oct-1 in the radiation signaling cascade in these cancer cells.