1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (VNP40101M): II. Role of O6-alkylguanine-DNA alkyltransferase in cytotoxicity.
OBJECTIVE
VNP40101M (1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine) is a sulfonylhydrazine prodrug that possesses broad spectrum antitumor efficacy in murine models. VNP40101M activation generates chloroethylating species that alkylate DNA at the O(6)-position of guanine, and a carbamoylating agent, methyl isocyanate, which inhibits O(6)-alkylguanine-DNA alkyltransferase (AGT) in model systems. We determined whether expression of AGT in Chinese hamster ovary (CHO) cells decreased sensitivity to VNP40101M and explored the mechanism of VNP40101M cytotoxicity by employing analogs of VNP40101M that generate reactive intermediates with either carbamoylating or chloroethylating activity.


METHODS
AGT was overexpressed in CHO cells by transfection with an expression vector containing the human AGT gene. Cell lines expressing AGT were employed in clonogenic assays to determine the cytotoxicity of VNP40101M and its analogs.


RESULTS
VNP40101M was more active against AGT-expressing CHO cells than 90CE (1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine), a chloroethylating generator devoid of carbamoylating activity. Furthermore, the greater the degree of AGT expression the more resistance to VNP40101M cytotoxicity. Combination chemotherapy experiments support the conclusions that methyl isocyanate and the chloroethylating species generated from the activation of VNP40101M function synergistically to kill cells.


CONCLUSIONS
The findings support the concept that alkylation of the O(6)-position of guanine residues in DNA is the predominant lesion created by VNP40101M, and that methyl isocyanate resulting from the base-catalyzed activation of VNP40101M inhibits AGT and presumably other enzymes involved in DNA repair, thereby enhancing the yield of the DNA G-C interstrand crosslinks responsible for the antitumor activity of this agent.
Baumann
R P
RP
Department of Pharmacology and Cancer Center, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520, USA.
Shyam
K
K
Penketh
P G
PG
Remack
J S
JS
Brent
T P
TP
Sartorelli
A C
AC
D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology*; D017953:Isocyanates / Q000494:pharmacology; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000096:biosynthesis* / Q000235:genetics; D013329:Structure-Activity Relationship; D014162:Transfection
C424963:1,2-bis(methylsulfonyl),1-(2-chloroethyl)-2-(methylamino)carbonylhydrazine; D018906:Antineoplastic Agents, Alkylating; D006834:Hydrazines; D017953:Isocyanates; C008461:methyl isocyanate; D019853:O(6)-Methylguanine-DNA Methyltransferase