We previously found that 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU), a new chloroethylnitrosoureaanalogue presently in phase I clinical trials, is aselective cytotoxin that enters cells via the extraneuronaltransporter for monoamine transmitters (EMT). In thisstudy, we assessed whether EMT expression correlates withSarCNU cytotoxicity by determining EMT expression in 23human tumor cell lines with reverse-transcription PCR.Western blot analysis was used to measure protein levels ofthe DNA repair genes, O6-methylguanine-DNA methyltransferase(MGMT), and excision repair cross-complementing rodentrepair deficiency gene 2 (ERCC2). SarCNU cytotoxicitywas determined by the sulforhodamine B colorimetric anticancer-drug screening assay and correlated with gene expression.Almost all of the cell lines screened were positivefor EMT expression. However, seven cell lines (MGR-1,MGR-2, T98-G, SKI-1, SKNSH, 297, and GBM) expressedlow levels of EMT. Although there was no linear correlationbetween SarCNU cytotoxicity and EMT expression,SarCNU cytotoxicity significantly correlated with ERCC2protein levels, and MGMT-rich (Mer1) cell lines (MGMTprotein level >0.1) were more resistant to SarCNU thanMGMT-poor (Mer2) cell lines (MGMT protein level <0.1). Moreover, multiple regression analysis indicated that thebest correlation with SarCNU cytotoxicity was attainablewith EMT plus MGMT and ERCC2 expression. This studysuggests that in human tumor cell lines both EMT and DNArepair factors, specifically, MGMT and ERCC2, are importantdeterminants of SarCNU activity. Because EMT is expressedin a wide variety of human tumors, SarCNU shouldbe a more widely effective alternative chemotherapeuticagent.

We examined the O6-methylguanine-DNA methyltransferase(MGMT) protein as well as MGMT activity levels and the excisionrepair cross-complementing rodent repair deficiency gene,ERCC2 (XPD), protein levels in 14 human tumor cell lines notselected for chloroethylnitrosourea (CENU) resistance. Theseresults were compared with 1,3-bis-(2-chloroethyl)-1-nitrosourea(BCNU) cytotoxicity and UV light sensitivity. MGMTprotein correlated significantly with MGMT activity (r=0.9497,p=0.0001). There was no significant linear correlation betweenBCNU cytotoxicity and MGMT content as determined by bothWestern analysis (r=0.139, p=0.6348) and activity assay (r=0.131, p=0.6515). However, MGMT-rich cell lines were foundto be more resistant than MGMT-poor cell lines to BCNU (t=2.2375, p=0.0225) but not to UV (t=1.1734, p=0.1317).Furthermore, the most BCNU-sensitive cell lines were allMGMT-poor. UV sensitivity was significantly correlated toBCNU cytotoxicity (r=0.858, p=0.0001). Significant correlationswere found between ERCC2 protein levels and BCNUcytotoxicity (r=0.786, p=0.0009) or UV sensitivity (r=5 0.874, p=5 0.0001). Our results confirm that MGMT plays an importantrole in CENU resistance, but not in UV resistance. The correlationof UV sensitivity with BCNU cytotoxicity suggests thatnucleotide excision repair is an important modifying factor ofMGMT-mediated innate CENU resistance in human tumor celllines, especially in highly resistant cell lines. ERCC2 may beimplicated in this process.

Objective To confirm the enhancing effect of excision repair cross complementing rodent repairdeficiency gene 2 (ERCC2) on alkylating agents resistance. Methods The authors constructed a pcDNA3-ERCC2 plasmid. The pcDNA3-ERCC2 was transfected into aselected ERCC2 negative human glioma cell line, SKMG-4, using liposome-mediated transfection. AfterG418 selection, a stable transfected cell line was obtained and tested for cytotoxicity of several alkylatingagents.Results The stable transfectant was obtained and confirmed by RT-PCR as well as Western blot analysisto be strongly expressing ERCC2 at both mRNA and protein levels. The 1O90 (pmol/L) of two alkylatingagents, cisplatin and melphalan, increased from 1.0 to 1.75 (75%) and 5.6 to 9.0 (61%), respectively,compared with control cell line.Conclusion The present data provided evidences and confirmed the authors'previous results thatERCC2 contributes, at least partially, to alkylating agent resistance in human glioma cell line.

Vasculogenic mimicry (VM) has been observed in melanoma and in some nonmelanomatumor types. It is unknown whether a similar VM phenomenon exists in astrocytoma.The present study was to examine 45 astrocytomas (including World Health Organizationgrade II 15 cases, grade III 15 cases, and grade IV 15 cases) by CD34 endothelialmarker periodic acid–Schiff (PAS) dual staining to see if VM existing in these tumors. Theresults demonstrated that endothelium-lined vessels dominated the tumor microvasculatureand stained positively for PAS, laminin, and endothelial marker. PAS-positive patternof VM was found in two grade IV astrocytomas. Channels stained positively for PAS, laminin,and negatively for CD34 of the VM entrapped in the tumor tissue. Erythrocytes couldbe observed in some of these channels. In these networks of PAS-positive pattern, spots ofweak reaction for CD34 were observed, suggesting the incorporation of VM channel andnormal vessel. Furthermore, in astrocytoma, especially glioblastoma, focus of anaplastic tumorcells appeared with CD34 expression, whereas some tumor cells lost glial fibrillary acidprotein expression. It is assumed that genetically deregulated tumor cells in astrocytomacould lose the astrocyte-specific protein and express inappropriate markers not expected incells of astrocyte lineage. The present results suggest that VM phenomenon exists in somemalignant astrocytoma.

Nucleotide excision repair (NER) is a multi-enzyme DNA repairpathway in eukaryotes. Several NER genes in this pathway includingXPB,XPD,XPAand ERCC-1have been implicated in anticancerdrug resistance in human tumor cells. In this study, we assessedthe levels of the above-mentioned proteins in the NCI panel of 60human tumor cell lines in relation to the cytotoxicity patterns of 170compounds that constitute the standard agent (SA) database.Thedatabase consists of drugs used in the clinic for which a mechanismofaction has been at least partially de