Katsuo Watanabe

Induction of mitochondrial heat shock protein 60 and 10 mRNAs following transient focal cerebral ischemia in the rat

Heat shock proteins (HSPs) 60 and 10 are stress-inducible mitochondrial matrix proteins that form a chaperonin complex that is important for mitochondrial protein folding and function. The effect of cerebral ischemia on mitochondrial HSPs is unclear. The topographical and chronological patterns of HSP60 and HSP10 messenger ribonucleic acid (mRNA) expression and induction were investigated in the rat focal cerebral ischemia model. Focal cerebral ischemia was produced by transient middle cerebral artery occlusion for 30 or 90 min. Expression of mRNAs was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. RT-PCR analysis showed that both HSP60 and HSP10 mRNA levels increased significantly in the ischemic cortex from 4 to 24 h of reperfusion after 30 min of occlusion. In situ hybridization analysis demonstrated significant induction of both mRNAs in the whole ischemic cortex after 30 min of occlusion and in the dorsomedial border (penumbra) of the ischemic cortex and ipsilateral hippocampus after 90 min of occlusion. Expression patterns and the timing of the induction of both HSP60 and HSP10 mRNAs were identical throughout the experiments. Simultaneous induction of the mRNAs for the mitochondrial chaperonins, HSP60 and HSP10, in various regions in focal cerebral ischemia demonstrates that mitochondrial stress conditions persist concomitantly with cytosolic stress conditions in focal cerebral ischemia.

Human brain tumor O6-methylguanine-DNA methyltransferase mRNA and its significance as an indicator of selective chloroethylnitrosourea chemotherapy

O6‐methylguanine‐DNA methyltransferase (MGMT) removes and repairs chloroethylnitrosourea (CENU)‐induced O6‐methylguanine‐DNA by accepting the alkyl group at a cysteine moiety. MGMT activity is, therefore, predictive of resistance or sensitivity to CENU chemotherapy. We measured the levels of MGMT mRNA expression in human brain tumors using a reverse transcription‐polymerase chain reaction (RT‐PCR) method, and studied the significance of MGMT mRNA levels in CENU chemotherapy. The level of MGMT mRNA was represented as a percentage relative to the MGMT mRNA in UI38MG brain tumor cells. Forty‐three patients with brain tumors were entered into the study. High‐grade gliomas had significantly lower levels of MGMT mRNA than did low‐grade gliomas and non‐glial tumors (p < 0.05 determined by analysis of co‐variance). Out of 14 high‐grade gliomas, 4 had a level of MGMT mRNA below 10%, indicating chemosensitivity to CENU. Out of 11 patients who received CENU chemotherapy, 3 had a partial response. All 3 responders had a low level of MGMT mRNA. The time to tumor progression (TTP) for 6 patients with a level lower than the median was short, but significantly longer than the TTP for 5 patients with a higher level (p < 0.05 determined by Gehans Wilcoxon test). These results indicate that a fraction of brain tumors have a low expression of MGMT mRNA, and that the level of MGMT mRNA is a useful indicator of effectiveness in selective CENU chemotherapy.

QUANTIFICATION OF O6-METHYLGUANINE-DNA METHYLTRANSFERASE MRNA IN HUMAN BRAIN TUMORS

O6-Methylguanine-DNA methyltransferase (MGMT) is strongly involved in drug resistance mechanism of tumor cells to chloroethylnitrosoureas (CENUs), because it removes and repairs CENU-induced O6-alkylguanine-DNA by accepting the alkyl group at a cysteine moiety. MGMT activity and MGMT mRNA expression are good indicators for detection of sensitive cells or resistant cells to CENUs. In the present study, we applied a non-radioactive reverse transcription-polymerase chain reaction (RT-PCR) method on quantitative measurement of MGMT mRNA expression. Estimated levels of MGMT mRNA expression determined by this RT-PCR method were consistent with the actual doses of MGMT mRNA. This relationship was noted at a wide range from 10 fg to 10 pg. The relative expression levels of MGMT mRNA estimated from kinetic analysis correlated well with MGMT activity determined using 3H-methyl-nitrosourea-treated DNA substrate in brain tumor cells (P<0.001 with a correlation coefficient of 0.997). The RT-PCR method facilitated quantitative measurements in even a small amount of biopsy specimens obtained by stereotactic brain surgery.

Characterization and chemosensitivity of two cell lines derived from human glioblastomas

SummaryWe have characterized two human glioblastoma cell lines, which were designated as YH cells and AM cells. The two cell lines maintained morphological appearance observed in the primary culture and immunohis-tochemically expressed glial fibrillary acidic protein (GFAP) and S-100 protein. Population doubling time for YH cells and AM cells indicated 30 hours and 25 hours, respectively, in an exponential phase of culture. Inoculation of AM cells into athymic nude mice formed large tumors at a high incidence. As with chemo-sensitivity to chloroethylnitrosourea, O6-methylguanine-DNA methyltransferase (MGMT) activity was measured inin vitro cultured cells as well as tumor specimens obtained at surgery. YH cells showed a high MGMT activity of 1196 fmol/mg and drug resistance to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-cloroethyl)-3-nitrosourea hydrochloride (ACNU) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. YH tumor specimens indicated an MGMT activity of 301 fmol/mg, which reflected poor effectiveness of ACNU chemotherapy in the clinical evaluation. AM cells had an extremely low MGMT activity of 16 fmol/mg and were vulnerable to ACNU. Original tumor specimens of AM cells however expressed a high value of 628 fmol/mg. Considering that ACNU chemotherapy was not effective in the both patients, an MGMT activity of original tumors related with responsiveness to ACNU. Discrepancy in an MGMT activity between thein vitro cell lines and the respective tumor specimens comes from selection of ACNU-sensitive cells or alteration in biological characteristics during long term culture. These results suggest that cell lines derived human brain tumors are useful targets for understanding the chemosensitivity of human malignant gliomas and for establishing a pertinent chemosensitivity test.

Modulation of BUdR labeling index in rat brain tumors following intracarotid ACNU administration

SummaryChloroethy1nitrosourea (CENU) chemotherapy has yielded limited benefit on survival of malignant brain tumors. Intracarotid administration of CENU is expected to have the advantage of increasing drug concentration reaching tumors. To understand basic knowledge of intracarotid chemotherapy, we monitor changes of proliferating rate after intracarotid injection of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2chloroethyl)-3-nitrosourea hydrochloride (ACNU), using a bromodeoxyuridine (BUdR) labeling index (LI) in transplanted brain tumors of three cell strains.C6-2 tumor cells were in vitro sensitive to ACNU, and C6-2/ACNU and C6-1 cells were resistant. The drug sensitivity to ACNU was as follows: 11.9 μtM in the C6-2 cells, 46.0 μM in the C6-2/ACNU cells, and 49.7 μM in the C6-1 cells at SD10, which gives 10% survival of clonogenic cells. The intracarotid ACNU at a dose of 30 mg/kg abruptly decreased the LI to 11% (mean) from 36% in C6-2 transplanted tumors. The LI remained low between 12 and 48 hours after, and then increased to the pretreatment level by 96 hours. In contrast, the LI of C6-1 tumors transiently fell to 15% from 42% at 12 hours after the injection, and subsequently increased to 36% at 24 hours and 37% at 48 hours.These results indicate that intracarotid ACNU administration shortly suppresses proliferating activity of tumors and that combined and alternating chemotherapy are mandatory for enhancing effectiveness of brain tumor chemotherapy.

O6-Methylguanine-DNA Methyltransferase (O6-MT) Activity as an Index of Drug Responsiveness to Antitumor Chloroethylnitrosoureas in Xenografted Brain Tumors

The synthesized chloroethylnitrosoureas (CENUs), 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), and methyl-6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-α-D-glucopyranoside (MCNU), have been among the antitumor agents widely used for the treatment of malignant brain tumors [1]. Methods for monitoring responsiveness of brain tumors to CENUs on the basis of a cell-killing mechanism have been needed to enhance therapeutic effectiveness. It is well documented that a DNA repair enzyme O6-methylguanine-DNA methyltransferase (O6-MT), identical to the term O6-alkylguanine-DNA alkyltransferase, eliminates the O6-methylguanine induced by CENUs and reflects the cellular resistance to CENUs in cultured cell strains and xenografted tumors [2–5]. Further in vivo work was warranted to establish a chemoserisitivity index of O6-MT activity. In the present paper, we test the relationship of O6-MT activity and responsiveness to CENUs in xenografted brain tumors.

Enhancement of ACNU cytotoxicity by pretreatment with O6-methylguanine in ACNU-resistant brain tumors

SummaryO6-Methylguanine is a substrate of the DNA repair enzymeO6-methylguanine-DNA methyltransferase, which is involved in the repair mechanism of DNA damage induced by chloroethylnitrosoureas such as 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU). We tested the enhancement effect ofO6-methylguanine pretreatment on ACNU cytotoxicity in ACNU-resistant brain tumors. Exposure toO6-methylguanine at various times ranging from 2 to 48 hours increased the cytotoxic effects of ACNU on C6-1 cells, and this effect was highest at higher concentrations 500 and 1,000 μM. Colorimetric cytotoxicity assay revealed at least a two-fold increase in ACNU cytotoxicity relative to controls withoutO6-methylguanine. Intraarterial ACNU after treatment withO6-methylguanine (two intravenous bolus injections of 80 and 40 mg/kg) significantly (P < 0.05 or P < 0.01) reduced the proliferation activity of transplanted C6-1 tumors for 96 hours after injection, whereas intravenous ACNU together withO6-methylguanine significantly (P < 0.05) reduced C6-1 activity for only 48 hours. Thus, pretreatment withO6-methylguanine prolonged the suppression effect of ACNU. The C6-1 tumors treated only with intravenous or intraarterial ACNU showed transient inhibition and rapid regrowth for 24 hours after treatment. These results indicate thatO6-methylguanine increases ACNU cytotoxicity in anin vitro andin vivo brain tumor model.

Potential of O6-methylguanine or O6-benzylguanine in the enhancement of chloroethylnitrosourea cytotoxicity on brain tumours

SummaryThe purine analogues O6-methylguanine and O6-benzylguanine are well-known as a chemical modulator of the DNA repair enzyme O6-methylguanine-DNA methyltransferase. Inactivation of the enzyme by O6-methylguanine or O6-benzylguanine is expected to enhance sensitivity of tumours to chloroethylnitrosoureas.We studied the effect of O6-methylguanine or O6-benzylguanine pretreatment on cytotoxity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in brain tumour cells and transplanted brain tumours. Two-hour exposure of O6-methylguanine at higher concentrations (500 μM, 1,000 μM) increased ACNU cytotoxicity by only 2 times in ACNU-resistant C6-1 brain tumour cells. O6-Benzylguanine at concentrations between 10 and 100 μM markedly enhanced the cytotoxic effecct. The ACNU sensitivity of the tumour cels pretreated with O6-benzylguanine was 5–40 times that of the cells without O6-benzylguanine. Neither O6-methylguanine nor O6-benzylguamne appreciably enhanced ACNU cytotoxicity of 9 L cells, which were origininally sensitive to ACNU. Intracarotid ACNU with O6-methylguanine or O6-benzylguanine decreased proliferating activity of transplanted C6-1 brain tumours significantly during 48 hours. O6-Benzylguanine pretreatment resulted in a greater degree of suppression for a long time. The C6-1 tumours treated only with intracarotid ACNU showed a transient inhibition and a rapid regrowth during 24 hours after the treatment.These results indicate that O6-methylguanine or O6-benzylguanine increases ACNU cytotoxicity and may be feasible for effective combination therapy with chloroethylnitrosourea in the chemotherapy of malignant brain tumours.

O6-(fluorobenzyl)guanine and chloroethylnitrosourea in xenografted rat brain tumor in vivo.

O6-methylguanine-DNA methyltransferase (MGMT), one of the DNA repair enzymes, potently repairs DNA damage induced by chloroethylnitrosoureas (CENUs). Depletion of MGMT activity after treatment with MGMT inhibitors increases the sensitivity of tumor cells to CENUs. We tested the effect of O6-(4-, 3- and 2-fluorobenzyl)guanines (4F, 3F and 2F, respectively), three newly synthesized MGMT inhibitors, on 1-(4-amino-2-methyl-5-pyrimidinyl)methy-3-(2-chloroethyl)-3-nitrosoureahydrochloride (ACNU) therapy in C6 tumor xenografts. Treatment with 4F+ACNU and 3F+ACNU significantly decreased tumor volume and extended the delay of growth in comparison to untreated mice (control group, p<0.05). Both groups showed significantly lower proliferating indices than the control group (p<0.05) 12 h after treatment. In contrast, 2F did not enhance the ACNU anti-tumor effect. These results indicate that O6-(4- and 3-fluorobenzyl)guanines as well as O6-benzylguanines enhance the effect of ACNU on the growth of C6 tumor xenografts in vivo.

CT and MRI of multicentric oligoastrocytoma

We report a rare biofrontal multicentric oligoastrocytoma shown by CT and MRI in a 37-year-old man hospitalised for generalised convulsions.