The standard chemotherapy for malignant gliomas is BCNU which produces only a 35% partial response. In a search for more active agents we have identified SarCNU, a new chloroethylnitrosourea (CNU) with a methylglycinamide carrier. SarCNU, as compared to BCNU, has enhanced in vitro and in vivo cytotoxicity against gliomas with less in vitro myelotoxicity. SarCNU is accumulated to a greater extent in the sensitive SK-MG-1 glioma cells as compared to BCNU. In addition, there is less accumulation of SarCNU in resistant SKI-1 glioma cells as compared to SK-MG-1 cells. The increased accumulation of SarCNU in SK- MG-1 cells is associated with a more rapid initial uptake via the catecholamine (uptake 2) carrier that is not detectable in SKI-1 cells. CNU drug resistance is responsible for the failure of BCNU/CCNU drug therapy in gliomas. While SarCNU may have increased activity against gliomas, it will almost certainly share some of the CNU resistance mechanisms. In glioma cell lines at least 2 mechanisms of CNU resistance have been recognized: (1) O6-methylguanine-DNA methyltransferase (MGMT) mediated removal of CNU monoadducts and (2) elevated glutathione-S- transferase (GST) possibly related to increased CNU metabolism. Furthermore, the lack of effective nucleotide excision repair (NER) increases CNU cytotoxicity and results in diminished removal of O6- alkylguanine lesions in spite of the presence of MGMT activity. This suggests that NER is an important factor in CNU cytotoxicity and possibly CNU resistance. In order to investigate these mechanisms of CNU resistance and examine other possible mechanisms we will utilize 10 human glioma cell lines: (1) the CNU sensitive MGMT-negative SK-MG-1; (2) the CNU resistant MGMT-negative SKI-1; (3) the CNU resistant MGMT- positive T98G and (4) 7 glioma cell lines of varying sensitivity to BCNU. We will: (1) examine the expression of several genes associated with drug resistance in the glioma cell lines, plus 30 samples of primary malignant gliomas, grade III-IV (initial results suggest that increased MGMT, ERCC-2 and metallothionein expression are associated with CNU resistance in gliomas); (2) correlate response to BCNU in glioma patients with the level of mRNA expression of the above-mentioned genes in their tumor specimens; (3) transfect genes, which are overexpressed in the resistant glioma cell lines and some of the clinical samples, via expression vectors into the sensitive SK-MG-1 cell line in order to determine the effects on CNU cytotoxicity in these cells; and (4) examine potential differences in DNA repair in 3 glioma cell lines utilizing a host cell reactivation assay.
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