Quantification of O 6 -methylguanine-DNA methyltransferase mRNA in human brain tumors

Mineura, Katsuyoshi; Watanabe, Katsuo; Yanagisawa, Toshiharu; Kowada, Masayoshi

doi:10.1016/0304-4165(95)00123-9

Cited by 20 publications

(11 citation statements)

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“…One cycle consisted of a 60-sec incubation at 94"C, a 90-sec incubation at 5 6 T , then a 60-sec incubation at 72°C. PCR products of brain tumor MGMT mRNA were quantified using kinetic analysis as reported previously (Mineura et a/., 1996). The level of MGMT mRNA expression was represented as a percentage relativc to the MGMT mRNA in U138MG-positive control brain-tumor cells, which have the highest level of MGMT activity (1,316 fmol/mg) and MGMT mRNA expression among brain-tumor cells maintained in our laboratory (Mineura et a/., 1994).…”

Section: Total Rna Extraction and First-strand Cdna Librarymentioning

confidence: 99%

“…Nonradioisotopic and kinctic analyses have since supplemented the PCR method in molecular research. We previously tested the relationship between the level of MGMT mRNA, determined by a quantificative RT-PCR method based on kinetics analysis, and actual MGMT activity, determined by a conventional assay with [3H]-methylnitrosourea-containing substrate DNA, in human glioma cell lines (Mineura et al, 1996). The level of MGMT mRNA correlated positively with the MGMT activity.…”

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confidence: 99%

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Human brain tumorO6-methylguanine-DNA methyltransferase mRNA and its significance as an indicator of selective chloroethylnitrosourea chemotherapy

et al. 1996

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Section: Total Rna Extraction and First-strand Cdna Librarymentioning

confidence: 99%

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Human brain tumorO6-methylguanine-DNA methyltransferase mRNA and its significance as an indicator of selective chloroethylnitrosourea chemotherapy

et al. 1996

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“…2 μg total RNA was reverse transcribed with ReverTra Ace 40°C for 1 h, followed by 95°C for 5 min to inactive the reverse transcriptase. The condition for PCR was 30 cycles of 94°C 40 s, 52°C 40 s, 72°C 40 s to amplify hMGMT [3] and β-actin [4] using primer set PM1-2 and PB1-2 respectively, the latter as inner control. The products were electrophoresized on 1% agarose gel, stained with ethidium bromide, and visualized under UV light (Tab.…”

Section: Rna Extraction and Rt-pcrmentioning

confidence: 99%

Expression silence of DNA repair gene hMGMT induced by RNA interference

Lai

2007

Chin. J. Cancer Res.

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Objective: MGMT protein expression has been associated with tumor resistance to alkylating agents. The objective of this paper is to construct the RNA interference vector which can specifically induce the expression silence of human DNA repair gene hMGMT. Methods: The hMGMT specific siRNA expression cassette was made by two steps PCR, linked with pUC19 to get pU6-MGMTi, co-transfected with pEGFP-C1 into 16HBE and screened by G418. The MGMT mRNA and protein levels were detected by RT-PCR and Western Blot respectively. Results: hMGMT specific RNA interfere vector pU6-MGMTi was constructed successfully. In transfected 16HBE cells MGMT mRNA level could hardly be detected and the protein level was only 10% of control. Conclusion: MGMT specific RNAi expression cassette can effectively inhibit MGMT expression. MGMT silence cell line was built by co-transfection technology, which offered condition for studying the gene function of MGMT.

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“…[7] The characterization of the MGMT gene has made it possible to determine MGMT expression by RNA and DNA blot analyses and the reverse transcription-polymerase chain reaction techniques. [9,23,32] However, determining MGMT expression with cellular extracts may be misleading because they may contain normal tissues that influence the results. Immunofluorescence assays have the advantage of allowing quantitation of MGMT activity in only the malignant cells.…”

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“…[6,28,29] Clinical trials are currently underway to determine its efficacy; [18] however, there have been only a few clinical investigations of MGMT expression in brain tumor samples in relation to patient survival and/or clinical response to chemotherapy. [5,23,25] Traditionally, MGMT levels have been determined by bioenzymatic assay in which fresh tissue is used. [7] The characterization of the MGMT gene has made it possible to determine MGMT expression by RNA and DNA blot analyses and the reverse transcription-polymerase chain reaction techniques.…”

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Clinical response of O6-methylguanine-DNA methyltransferase levels to 1,3-(2-chloroethyl)-1-nitrosourea chemotherapy in glioma patients

Chen¹,

Yarosh²,

Garcia³

et al. 1998

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Adjuvant nitrosourea chemotherapy fails to prolong patient survival significantly as many tumors demonstrate resistance to these drugs. It has been documented in cell lines that O6-methylguanine-DNA methyltransferase (MGMT) plays an important role in chloroethylnitrosourea (CENU) drug resistance. The authors evaluated MGMT expression in 22 glioma specimens by using an immunofluorescence assay and compared the results with clinical response of the patients to CENU-based chemotherapy. The patients were treated with CENU after evidence of progressive disease following surgery and radiotherapy. Eight tumor samples had no detectable MGMT, whereas other samples had from 9989 to 982,401 molecules/nucleus. In one group (12 patients), the tumor decreased in size or was stable (effective group), whereas in the other group (10 patients), the tumor demonstrated continuous growth during chemotherapy (progressive group). The median time to progression (TTP) was 6.7 months with a median survival of 13 months. The Mer− patients (MGMT < 60,000 molecules/nucleus) appeared to have more chance of stable disease or response to CENU therapy than the Mer+ patients (MGMT > 60,000 molecules/nucleus) (chi-square = 4.791, p = 0.0286). In patients with glioblastomas multiforme (GBMs), the TTP of Mer+ patients was shorter than that of Mer− patients (t = 2.04, p = 0.049). As a corollary, the MGMT levels were significantly higher in GBM tumors from the progressive group than those from the effective group (t = -2.26, p = 0.029). The TTP and survival time in the effective GBM group were also longer than those in the progressive GBM group. However, there was no significant correlation between MGMT levels and either the survival time (r = 0.04, p = 0.8595) or TTP (r = 0.107, p = 0.6444). Results from this study suggested that MGMT positivity is indicative of more aggressive disease that progresses more rapidly when exposed to CENU therapy. However, MGMT-negative tumors are not always sensitive to CENU agents, suggesting that other factors may also be important.

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Quantification of O 6 -methylguanine-DNA methyltransferase mRNA in human brain tumors

Cited by 20 publications

References 20 publications

Human brain tumorO6-methylguanine-DNA methyltransferase mRNA and its significance as an indicator of selective chloroethylnitrosourea chemotherapy

Human brain tumorO6-methylguanine-DNA methyltransferase mRNA and its significance as an indicator of selective chloroethylnitrosourea chemotherapy

Expression silence of DNA repair gene hMGMT induced by RNA interference

Clinical response of O6-methylguanine-DNA methyltransferase levels to 1,3-(2-chloroethyl)-1-nitrosourea chemotherapy in glioma patients

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