Philip G. Penketh

Comparison of DNA lesions produced by tumor-inhibitory 1,2-bis(sulfonyl)hydrazines and chloroethylnitrosoureas.

1,2-Bis(sulfonyl)hydrazine derivatives, designed to generate several of the electrophilic species classically believed to be responsible for the alkylating (chloroethylating) and/or carbamoylating activities of the chloroethylnitrosoureas (CNUs), were compared with respect to the cross-linking and nicking of T7 DNA to that caused by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and 1-(2-chloroethyl)-3-(4-trans-methylcyclohexyl)-1-nitrosourea (MeCCNU). In the case of BCNU, a large proportion of T7 DNA strand nicking was found to be due to the generation of 2-chloroethylamine, produced from the hydrolysis of 2-chloroethylisocyanate, in turn formed during the decomposition of the parental nitrosourea. 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (compound 1) gave a greater yield of DNA cross-links than the CNUs. Compound 1, as well as its derivatives that were incapable of generating 2-chloroethylisocyanate, did not produce detectable levels of strand nicking, indicating that N7-alkylation of guanine did not occur to a significant extent with these agents. Since compound 1 and its derivatives are believed to generate chloronium and chloroethyldiazonium ions, it would appear that these species could not be significantly involved in the N7-alkylation of guanine caused by the CNUs. The relatively low level of N7-alkylation of guanine residues and the relatively high yield of cross-links generated by some of the 1,2-bis(sulfonyl)-1-(2-chloroethyl)hydrazine derivatives implies that they are more exclusive O6-guanine chloroethylating agents than the CNUs. O6-Guanine chloroethylation is believed to be the therapeutically relevant event produced by the CNUs; therefore, compound 1 derivatives represent promising new cancer chemotherapeutic agents, since they appear to generate lower quantities of therapeutically unimportant, yet carcinogenic lesions, and more of the therapeutically relevant O6-guanine chloroethylation than the CNUs.

Trypanosomatid hydrogen peroxidase metabolism

The rate of whole cell H2O2 metabolism in several salivarian and stercorarian trypanosomes and Leishmania species was measured. These cells metabolized H2O2 at rates between 2.3 and 48.2 nmol/108 cells per min depending upon the species employed. H2O2 metabolism was largely insensitive to NaN3, implying that typical catalase and peroxidase haemoproteins are not important in H2O2 metabolism. The metabolism of H2O2, however, was almost completely inhibited by N‐ethylmaleimide. In representative species, H2O2 metabolism was shown to occur through a trypanothione‐dependent mechanism.

The antineoplastic efficacy of the prodrug Cloretazine is produced by the synergistic interaction of carbamoylating and alkylating products of its activation

Cloretazine {1,2-bis(methylsulfonyl)-1-[(2-chloroethyl)-2-(methylamino)carbonyl]hydrazine; VNP40101M; 101M} is a sulfonylhydrazine prodrug that possesses broad spectrum antitumor efficacy against transplanted murine and human tumor models and has shown activity in clinical trials against relapsed or refractory acute myeloid leukemia. Base catalyzed activation of this prodrug generates two different reactive intermediates: chloroethylating species that covalently interact with DNA at the O6-position of guanine residues that progress to a G-C interstrand cross-link, and a carbamoylating agent, methyl isocyanate. Previous findings from this laboratory have provided initial evidence that methyl isocyanate can contribute to the efficacy of Cloretazine by enhancing the cytotoxicity of the generated chloroethylating species. This action may be due in part to inhibition of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT); however, activity in cells devoid of AGT indicates that other actions are involved in the synergistic cytotoxicity. Herein we demonstrate that O6-benzylguanine can also produce synergistic cell kill with the alkylating component of Cloretazine but differs from methyl isocyanate in that the enhancement occurs in AGT-containing cells, but not in cells devoid of AGT. Methyl isocyanate generated by the decomposition of 1,2-bis(methylsulfonyl)-1-[methylaminocarbonyl]hydrazine also acts to enhance the activity of a variety of DNA cross-linking agents, while only producing additive cytotoxicity with methylating agents. Flow cytometric studies using annexin as a marker for apoptosis indicate that in Chinese hamster ovary cells and in human leukemia cells Cloretazine-induced apoptosis is primarily caused by the generated methyl isocyanate. Comet assays designed to detect DNA cross-links in intact cells indicate that the chloroethylating species generated by the activation of Cloretazine produce DNA cross-links, with the co-generated methyl isocyanate increasing the degree of cross-linking produced by the reactive chloroethylating species. These findings provide further evidence that the methyl isocyanate produced by the activation of Cloretazine can be a major contributor to the cytotoxicity produced by this antineoplastic agent.

Generation of Oxygen Deficiency in Cell Culture Using a Two- Enzyme System to Evaluate Agents Targeting Hypoxic Tumor Cells

Abstract Baumann, R. P., Penketh, P. G., Seow, H. A., Shyam, K. and Sartorelli, A. C. Generation of Oxygen Deficiency in Cell Culture Using a Two-Enzyme System to Evaluate Agents Targeting Hypoxic Tumor Cells. Radiat. Res. 170, 651–660 (2008). The poor and aberrant vascularization of solid tumors makes them susceptible to localized areas of oxygen deficiency that can be considered sites of tumor vulnerability to prodrugs that are preferentially activated to cytotoxic species under conditions of low oxygenation. To readily facilitate the selection of agents targeted to oxygen-deficient cells in solid tumors, we have developed a simple and convenient two-enzyme system to generate oxygen deficiency in cell cultures. Glucose oxidase is employed to deplete oxygen from the medium by selectively oxidizing glucose and reducing molecular oxygen to hydrogen peroxide; an excess of catalase is also used to scavenge the peroxide molecules. Rapid and sustained depletion of oxygen occurs in medium or buffer, even in the presence of oxygen at the liquid/air interface. Studies using CHO/AA8 Chinese hamster cells, EMT6 murine mammary carcinoma cells, and U251 human glioma cells indicate that this system generates an oxygen deficiency that produces activation of the hypoxia-targeted prodrug KS119. This method of generating oxygen deficiency in cell culture is inexpensive, does not require cumbersome equipment, permits longer incubation times to be used without the loss of sample volume, and should be adaptable for high-throughput screening in 96-well plates.

Mode of action of the chloroethylating and carbamoylating moieties of the prodrug cloretazine

Cloretazine is an antitumor sulfonylhydrazine prodrug that generates both chloroethylating and carbamoylating species. The cytotoxic potency of these species was analyzed in L1210 leukemia cells using analogues with chloroethylating or carbamoylating function only. Clonogenic assays showed that the chloroethylating-only agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) produced marked differential cytotoxicity against wild-type and O6-alkylguanine-DNA alkyltransferase–transfected L1210 cells (LC10, 1.4 versus 31 μmol/L), indicating that a large portion of the cytotoxicity was due to alkylation of DNA at the O-6 position of guanine. Consistent with the concept that O-6 chloroethylation of DNA guanine progresses to interstrand cross-links, the comet assay, in which DNA cross-links were measured by a reduction in DNA migration induced by strand breaks, showed that cloretazine and 90CE, but not the carbamoylating-only agent 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]hydrazine (101MDCE), produced DNA cross-links and that cloretazine caused more DNA cross-links than 90CE at equimolar concentrations. Cell cycle analyses showed that 90CE and 101MDCE at concentrations of 5 and 80 μmol/L, respectively, produced similar degrees of G2-M arrest. 90CE produced selective inhibition of DNA synthesis after overnight incubation, whereas 101MDCE caused rapid and nonselective inhibition of RNA, DNA, and protein syntheses. Both 90CE and 101MDCE induced phosphorylation of histone H2AX, albeit with distinct kinetics. These results indicate that (a) differential expression of O6-alkylguanine-DNA alkyltransferase in tumor and host cells seems to be responsible for tumor selectivity exerted by cloretazine; (b) 101MDCE enhances DNA cross-linking activity; and (c) 90CE induces cell death at concentrations lower than those causing alterations in the cell cycle and macromolecular syntheses. [Mol Cancer Ther 2006;5(4):969–76]

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (VNP40101M): II. Role of O6-alkylguanine-DNA alkyltransferase in cytotoxicity

PurposeVNP40101M (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 O6-position of guanine, and a carbamoylating agent, methyl isocyanate, which inhibits O6-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.MethodsAGT 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.ResultsVNP40101M 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.ConclusionsThe findings support the concept that alkylation of the O6-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.

Bioactivation and Resistance to Mitomycin C

Publisher Summary This chapter analyzes the bioactivation and resistance of tumor cells to mitomycin C (MC). MC is a naturally occurring antibiotic that was isolated originally from the microorganism Streptomyces caspitosus . MC exerts its antitumor activity primarily by damaging DNA through both monofunctional and bifunctional alkylations (cross-links). Numerous studies have employed HPLC separation methods to identify specific MC-DNA lesions associated with MC treatment. Monoalkylations initially occur through the C1 position of MC to the N2 position of aguanine base in DNA and may proceed to form a DNA cross-link through the C10 position of MC to an adjacent DNA guanine at its N2 position. MC-induced cross-links are believed to be primarily responsible for cell death. A single cross-link per genome has been reported to be sufficient to cause the death of a bacterial cell. In this chapter, properties of MC as a prototypic bioreductive agent are described, and mechanism of the reductive activation of MC is elaborated. The chapter presents an overview of MC resistance protein A (MCRA). Mammalian MCRA functional homolog is discussed and rapid screening for DTD activity by using a microtiter assay is discussed. Methodology for the indirect determination of MC activation is also described in the chapter.

Role of O6-alkylguanine-DNA alkyltransferase in the cytotoxic activity of cloretazine

Cloretazine (VNP40101M; 101M; 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine) is a sulfonylhydrazine prodrug that generates both chloroethylating and carbamoylating species on activation. To explore the molecular mechanisms underlying the broad anticancer activity observed in preclinical studies, cloretazine and chloroethylating-only [i.e., 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine] and carbamoylating-only (i.e., 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]hydrazine) analogues were evaluated in five murine hematopoietic cell lines. These cell lines were separable into two groups by virtue of their sensitivity to 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine; the sensitive group included L1210, P388, and F-MEL leukemias (IC50s, 6–8 μmol/L) and the resistant group consisted of Ba/F3 bone marrow and WEHI-3B leukemia cells (IC50s, 50–70 μmol/L). Resistant cells expressed O6-alkylguanine-DNA alkyltransferase (AGT), whereas sensitive cells did not. A correlation existed between AGT expression and the functional status of p53; AGT− cells possessed defective p53, whereas AGT+ cells contained wild-type p53. Based on recent findings on regulation of AGT gene expression by others, we suspect that silencing of the AGT gene by promoter hypermethylation frequently occurs during tumor progression involving p53 inactivation. O6-Chloroethylguanine is the initial DNA lesion that progresses to lethal interstrand DNA cross-links. Cloretazine exhibited a much higher preference toward the O6-chloroethylation of guanine, as measured by the difference in IC50s to wild-type and AGT-transfected L1210 cells, than 1,3-bis(2-chloroethyl)-1-nitrosourea, which targets the same site in DNA. Preferential toxicity of cloretazine against AGT− tumor cells coupled with decreased toxicity to AGT+ cells in host tissues constitute the therapeutic basis for cloretazine.

Development of an O6-alkylguanine—DNA alkyltransferase assay based on covalent transfer of the benzyl moiety from [benzene-3H]O6-benzylguanine to the protein

Although it is known that (i) O(6)-alkylguanine-DNA alkyltransferase (AGT) confers tumor cell resistance to guanine O(6)-targeting drugs such as cloretazine, carmustine, and temozolomide and that (ii) AGT levels in tumors are highly variable, measurement of AGT activity in tumors before treatment is not a routine clinical practice. This derives in part from the lack of a reliable clinical AGT assay; therefore, a simple AGT assay was devised based on transfer of radioactive benzyl residues from [benzene-3H]O(6)-benzylguanine ([3H]BG) to AGT. The assay involves incubation of intact cells or cell homogenates with [3H]BG and measurement of radioactivity in a 70% methanol precipitable fraction. Approximately 85% of AGT in intact cells was recovered in cell homogenates. Accuracy of the AGT assay was confirmed by examination of AGT levels by Western blot analysis with the exception of false-positive results in melanin-containing cells due to [3H]BG binding to melanin. Second-order kinetic constants for human and murine AGT were 1100 and 380 M(-1)s(-1), respectively. AGT levels in various human cell lines ranged from less than 500 molecules/cell (detection limit) to 45,000 molecules/cell. Rodent cell lines frequently lacked AGT expression, and AGT levels in rodent cells were much lower than in human cells.

Lethality to leukemia cell lines of DNA interstrand cross-links generated by Cloretazine derived alkylating species.

Cloretazine [1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine; VNP40101M; 101M] is a relatively new prodrug with activity in elderly acute myelogenous leukemia (AML) patients. Its therapeutic action is due largely to the production of 1-(3-cytosinyl),2-(1-guanyl)ethane cross-links (G-C ethane cross-links) in DNA. The numbers of cross-links produced in three experimental leukemia lines (L1210, U937 and HL-60) were fewer than 10 per genome at their respective LC50 concentrations. Only 1 in approximately 20,000 90CE molecules produces a cross-link in the AGT (O6-alkylguanine-DNA alkyltransferase) negative L1210 and U937 cell lines and 1 in 400,000 in the AGT positive HL-60 cell line.