O6-methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells

Temozolomide (TMZ) is a methylating agent which prolongs survival when administered during and after radiotherapy in the first-line treatment of glioblastoma and which also has significant activity in recurrent disease. O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme attributed a role in cancer cell resistance to O6-alkylating agent-based chemotherapy. Using a panel of 12 human glioma cell lines, we here defined the sensitivity to TMZ in acute cytotoxicity and clonogenic survival assays in relation to MGMT, mismatch repair and p53 status and its modulation by dexamethasone, irradiation and BCL-X(L). We found that the levels of MGMT expression were a major predictor of TMZ sensitivity in human glioma cells. MGMT activity and clonogenic survival after TMZ exposure are highly correlated (p < 0.0001, r2 = 0.92). In contrast, clonogenic survival after TMZ exposure does not correlate with the expression levels of the mismatch repair proteins mutS homologue 2, mutS homologue 6 or post-meiotic segregation increased 2. The MGMT inhibitor O6-benzylguanine sensitizes MGMT-positive glioma cells to TMZ whereas MGMT gene transfer into MGMT-negative cells confers protection. The antiapoptotic BCL-X(L) protein attenuates TMZ cytotoxicity in MGMT-negative LNT-229 but not in MGMT-positive LN-18 cells. Neither ionizing radiation (4 Gy) nor clinically relevant concentrations of dexamethasone modulate MGMT activity or TMZ sensitivity. Abrogation of p53 wild-type function strongly attenuates TMZ cytotoxicity. Conversely, p53 mimetic agents designed to stabilize the wild-type conformation of p53 sensitize glioma cells for TMZ cytotoxicity. Collectively, these results suggest that the determination of MGMT expression and p53 status will help to identify glioma patients who will or will not respond to TMZ.     

Adenosine 3',5'-cyclic monophosphate dependent and independent protein kinase activities in 1,2-dimethylhydrazine induced rat colon cancer

The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent and cAMP-independent kinase activities were measured in the 1,2-dimethylhydrazine (DMH) induced rat colon cancer and in untreated colon. Previous studies had shown that intestinal tumors induced by chronic exposure to DMH contained 2-fold less intracellular cAMP. The present findings indicate that reduction in cAMP-dependent protein kinase activities also occur in colon cancer cells. Similar hydrogen ion dependence (pH 6-7) and approximate association constants (Ka approximately 0.1 microM) were observed for the enzymes existing in both normal and tumor tissues, while the cAMP-dependent tumor protein kinase was found to phosphorylate phosvitin and casein to a greater degree. These recent findings are consistent with the concept that the concentrations of cAMP and activities of its associated enzyme system are inversely related to the cell proliferation state.     

Co-expression of MGMT(P140K) and alpha-L-iduronidase in primary hepatocytes from mucopolysaccharidosis type I mice enables efficient selection with metabolic correction

BACKGROUND: Systemic in vivo gene therapy has resulted in widespread correction in animal models when treated at birth. However, limited improvement was observed in postnatally treated animals with mainly targeting to the liver and bone marrow. It has been shown that an O(6)-methylguanine-DNA-methyltransferase variant (MGMT(P140K)) mediated in vivo selection of transduced hematopoietic stem cells (HSC) in animals. METHODS: We investigated the feasibility of MGMT(P140K)-mediated selection in primary hepatocytes from a mouse model of mucopolysaccharidosis type I (MPS I) in vitro using lentiviral vectors. RESULTS: We found that multiple cycles of O(6)-benzylguanine (BG)/1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment at a dosage effective for ex vivo HSC selection led to a two-fold increase of MGMT-expressing primary hepatocytes under culture conditions with minimum cell expansion. This enrichment level was comparable to that obtained after selection at a hepatic maximal tolerated dose of BCNU. Similar levels of increase were observed regardless of initial transduction frequency, or the position of MGMT (upstream or downstream of internal ribosome entry site) in the vector constructs. In addition, we found that elongation factor 1alpha promoter was superior to the long-terminal repeat promoter from spleen focus-forming virus with regard to transgene expression in primary hepatocytes. Moreover, the levels of therapeutic transgene expression in transduced, enzyme-deficient hepatocytes directly correlated with the doses of BCNU, leading to metabolic correction in transduced hepatocytes and metabolic cross-correction in neighbouring non-transduced MPS I cells. CONCLUSIONS: These results demonstrate that MGMT(P140K) expression confers successful protection/selection in primary hepatocytes, and provide 'proof of concept' to the prospect of MGMT(P140K)-mediated co-selection for hepatocytes and HSC using BG/BCNU treatment.     

Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamster and virus-transformed hamster cells. In vivo and in vitro methylation

Information has been lacking as to whether mitochondrial DNA of animal cells is methylated. The methylation patterns of mitochondrial and nuclear DNAs of several mammalian cell lines have therefore been compared by four methods: (1) in vivo transfer of the methyl group from [methyl-³H]methionine; (2) in vivo incorporation of [³²P]orthophosphate and a combination of (1) and (2); (3) in vivo incorporation of [³H]deoxycytidine; (4) in vitro methylation of DNAs with ³H-labeled S-adenosylmethionine as methyl donor and DNA methylase preparations from L cell nuclei. The cell lines were mouse L cells, $\text{BHK21}\text{C13}$, $\text{C13}\text{B4}$ (baby hamster kidney cells transformed by the Bryan strain of Rouse sarcoma virus), and PyY (BHK cells transformed by polyoma virus). DNA bases were separated chromatographically, using 5-methylcytosine, 6-methylaminopurine and, in some cases, 7-methylguanine as markers.Mitochondrial DNA was found to be significantly less methylated than nuclear DNA with respect to 5-methylcytosine in all cell types studied and by all methods used. The relative advantages and disadvantages of each method have been discussed. The level of 5-methylcytosine in mitochondrial DNA as compared with that in nuclear DNA was estimated as one-fourth to one-fourteenth in various cell lines. The estimated 5-methylcytosine content per circular mitochondrial DNA molecule (mol. wt 10 × 10⁶) was about 12 methylcytosine residues for L cells and 24, 30 and 36 methylcytosine residues for BHK, B4 and PyY cells, respectively. Relative to cytosine residues, the estimate was one 5-methylcytosine per 500 cytosine residues of mitochondrial DNA and one 5-methylcytosine per 36 cytosine residues of nuclear DNA from L-cells. The values for methylcytosine of mitochondrial DNA are presumed to be maximal. PyY cells as compared with other cells had the highest methylcytosine content of both mitochondrial and nuclear DNA as estimated by method (3). No methylation of nuclear DNA was observed in confluent L cells.Evidence for the presence of DNA methylase activity associated with mitochondrial fractions was obtained. This activity could be distinguished from other cellular DNA methylase activity by differential response to mercaptoethanol. Radioactivity from ³H-labeled S-adenosylmethionine was found only in 5-methyl-cytosine of DNA.     

反义RNA调节肿瘤细胞O~6-甲基鸟嘌呤-DNA甲基转移酶活性

报道了逆转录病毒载体介导的反义ＲＮＡ对肿瘤细胞Ｏ６－甲基鸟嘌呤－ＤＮＡ甲基转移酶（ＭＧＭＴ）活性的调节作用．构建了三个表达ＭＧＭＴ反义ＲＮＡ的逆转录病毒载体并用它们转染ＨｅＬａＳ３肿瘤细胞,观察细胞在转染前后ＭＧＭＴｍＲＮＡ水平、ＭＧＭＴ活性及其对ＡＣＮＵ抗药性的变化．发现针对ＭＧＭＴｍＲＮＡ５’端的反义ＲＮＡ能够有效地降低ＭＧＭＴｍＲＮＡ水平和ＭＧＭＴ活性并使细胞对ＡＣＮＵ的敏感性提高４．６倍;针对ＭＧＭＴｍＲＮＡ全长的反义ＲＮＡ也能在一定程度上调节细胞的ＭＧＭＴｍＲＮＡ水平和ＭＧＭＴ活性并增加细胞对ＡＣＮＵ的敏感性,而针对３’端序列的反义ＲＮＡ对ＭＧＭＴ活性没有调节作用．     

The mismatch repair-dependent DNA damage response: Mechanisms and implications

An important role for the DNA mismatch repair (MMR) pathway in maintaining genomic stability is embodied in its conservation through evolution and the link between loss of MMR function and tumorigenesis. The latter is evident as inheritance of mutations within the major MMR genes give rise to the cancer predisposition condition, Lynch syndrome. Nonetheless, how MMR loss contributes to tumorigenesis is not completely understood. In addition to preventing the accumulation of mutations, MMR also directs cellular responses, such as cell cycle checkpoint or apoptosis activation, to different forms of DNA damage. Understanding this MMR-dependent DNA damage response may provide insight into the full tumor suppressing capabilities of the MMR pathway. Here, we delve into the proposed mechanisms for the MMR-dependent response to DNA damaging agents. We discuss how these pre-clinical findings extend to the clinical treatment of cancers, emphasizing MMR status as a crucial variable in selection of chemotherapeutic regimens. Also, we discuss how loss of the MMR-dependent damage response could promote tumorigenesis via the establishment of a survival advantage to endogenous levels of stress in MMR-deficient cells.     

Animal models of brain maldevelopment induced by cycad plant genotoxins

Cycads are long‐lived tropical and subtropical plants that contain azoxyglycosides (e.g., cycasin, macrozamin) and neurotoxic amino acids (notably β‐N‐methylamino‐l ‐alanine l ‐BMAA), toxins that have been implicated in the etiology of a disappearing neurodegenerative disease, amyotrophic lateral sclerosis and parkinsonism‐dementia complex that has been present in high incidence among three genetically distinct populations in the western Pacific. The neuropathology of amyotrophic lateral sclerosis/parkinsonism‐dementia complex includes features suggestive of brain maldevelopment, an experimentally proven property of cycasin attributable to the genotoxic action of its aglycone methylazoxymethanol (MAM). This property of MAM has been exploited by neurobiologists as a tool to study perturbations of brain development. Depending on the neurodevelopmental stage, MAM can induce features in laboratory animals that model certain characteristics of epilepsy, schizophrenia, or ataxia. Studies in DNA repair‐deficient mice show that MAM perturbs brain development through a DNA damage‐mediated mechanism. The brain DNA lesions produced by systemic MAM appear to modulate the expression of genes that regulate neurodevelopment and contribute to neurodegeneration. Epigenetic changes (histone lysine methylation) have also been detected in the underdeveloped brain after MAM administration. The DNA damage and epigenetic changes produced by MAM and, perhaps by chemically related substances (e.g., nitrosamines, nitrosoureas, hydrazines), might be an important mechanism by which early‐life exposure to genotoxicants can induce long‐term brain dysfunction. Birth Defects Research (Part C) 99:247–255, 2013. © 2013 Wiley Periodicals, Inc.