Triapine potentiates platinum-based combination therapy by disruption of homologous recombination repair.

BACKGROUND: Platinum resistance may be attributable to inherent or acquired proficiency in homologous recombination repair (HRR) in epithelial ovarian cancer (EOC). The objective of this study was to evaluate the efficacy of the small molecule inhibitor triapine to disrupt HRR and sensitise BRCA wild-type EOC cells to platinum-based combination therapy in vitro and in vivo. METHODS: The sensitivity of BRCA wild-type cancer cells to olaparib, cisplatin, carboplatin, doxorubicin, or etoposide in combination with triapine was evaluated by clonogenic survival assays. The effects of triapine on HRR activity in cells were measured with a DR-GFP reporter assay. The ability of triapine to enhance the effects of the carboplatin-doxil combination on EOC tumour growth delay was determined using a xenograft tumour mouse model. RESULTS: Platinum resistance is associated with wild-type BRCA status. Triapine inhibits HRR activity and enhances the sensitivity of BRCA wild-type cancer cells to cisplatin, olaparib, and doxorubicin. However, sequential combination of triapine and cisplatin is necessary to achieve synergism. Moreover, triapine potentiates platinum-based combination therapy against BRCA wild-type EOC cells and produces significant delay of EOC tumour growth. CONCLUSIONS: Triapine promises to augment the clinical efficacy of platinum-based combination regimens for treatment of platinum-resistant EOC with wild-type BRCA and proficient HRR activity.

MK-2206 sensitizes BRCA-deficient epithelial ovarian adenocarcinoma to cisplatin and olaparib.

BACKGROUND: Platinum resistance is a major obstacle in the treatment of epithelial ovarian cancer (EOC). Activation of the AKT pathway promotes platinum resistance while inhibition of AKT sensitizes chemoresistant cells. Patients with BRCA mutant EOC, and thus a defect in the homologous recombination (HR) repair pathway, demonstrate greater clinical response to platinum and olaparib therapy than patients with BRCA wild-type EOC. MK-2206, an allosteric inhibitor of AKT phosphorylation, sensitizes a variety of cell types to various anticancer agents and is currently undergoing phase II trials as monotherapy for platinum-resistant ovarian, fallopian tube, and peritoneal cancer. This study examines the differential effects of AKT inhibition with cisplatin and olaparib therapy in BRCA1/2-deficient versus wild-type EOC. METHODS: PEO1, a chemosensitive BRCA2-mutant serous ovarian adenocarcinoma, and PEO4, a reverted BRCA2-proficient line from the same patient after the development of chemotherapeutic resistance, were primarily used for the study. In PEO1, MK-2206 demonstrated moderate to strong synergism with cisplatin and olaparib at all doses, while demonstrating antagonism at all doses in PEO4. RESULTS: Baseline phospho-AKT activity in untreated cells was upregulated in both BRCA1- and 2-deficient cell lines. MK-2206 prevented cisplatin- and olaparib-induced AKT activation in the BRCA2-deficient PEO1 cells. We propose that BRCA-deficient EOC cells upregulate baseline AKT activity to enhance survival in the absence of HR. Higher AKT activity is also required to withstand cytotoxic agent-induced DNA damage, leading to strong synergism between MK-2206 and cisplatin or olaparib therapy in BRCA-deficient cells. CONCLUSIONS: MK-2206 shows promise as a chemosensitization agent in BRCA-deficient EOC and merits clinical investigation in this patient population.

When alcohol is the answer: Trapping, identifying and quantifying simple alkylating species in aqueous environments.

Alkylating agents are a significant class of environmental carcinogens as well as commonly used anticancer therapeutics. Traditional alkylating activity assays have utilized the colorimetric reagent 4-(4-nitrobenzyl)pyridine (4NBP). However, 4NBP based assays have a relatively low sensitivity towards harder, more oxophilic alkylating species and are not well suited for the identification of the trapped alkyl moiety due to adduct instability. Herein we describe a method using water as the trapping agent which permits the trapping of simple alkylating electrophiles with a comparatively wide range of softness/hardness and permits the identification of donated simple alkyl moieties.

Tumor-associated mutations in O6 -methylguanine DNA-methyltransferase (MGMT) reduce DNA repair functionality.

MGMT is the primary vehicle for cellular removal of alkyl lesions from the O-6 position of guanine and the O-4 position of thymine. While key to the maintenance of genomic integrity, MGMT also removes damage induced by alkylating chemotherapies, inhibiting the efficacy of cancer treatment. Germline variants of human MGMT are well-characterized, but somatic variants found in tumors were, prior to this work, uncharacterized. We found that MGMT G132R, from a human esophageal tumor, and MGMT G156C, from a human colorectal cancer cell line, are unable to rescue methyltransferase-deficient Escherichia coli as well as wild type (WT) human MGMT after treatment with a methylating agent. Using pre-steady state kinetics, we biochemically characterized these variants as having a reduced rate constant. G132R binds DNA containing an O6 -methylguanine lesion half as tightly as WT MGMT, while G156C has a 40-fold decrease in binding affinity for the same damaged DNA versus WT. Mammalian cells expressing either G132R or G156C are more sensitive to methylating agents than mammalian cells expressing WT MGMT. G132R is slightly resistant to O6 -benzylguanine, an inhibitor of MGMT in clinical trials, while G156C is almost completely resistant to this inhibitor. The impared functionality of expressed variants G132R and G156C suggests that the presence of somatic variants of MGMT in a tumor could impact chemotherapeutic outcomes.

Influence of phosphate and phosphoesters on the decomposition pathway of 1,2-bis(methylsulfonyl)-1-(2-chloroethyhydrazine (90CE), the active anticancer moiety generated by Laromustine, KS119, and KS119W.

Prodrugs of the short-lived chloroethylating agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) and its methylating analogue 1,2-bis(methylsulfonyl)-1-(methyl)hydrazine (KS90) are potentially useful anticancer agents. This class of agents frequently yields higher ratios of therapeutically active oxophilic electrophiles responsible for DNA O(6)-guanine alkylations to other electrophiles with lower therapeutic relevance than the nitrosoureas. This results in improved selectivity toward tumors with diminished levels of O(6)-alkylguanine-DNA alkyltransferase (MGMT), the resistance protein responsible for O(6)-alkylguanine repair. The formation of O(6)-(2-chloroethyl)guanine, which leads to the formation of a DNA-DNA interstrand cross-link, accounts for the bulk of the anticancer activity of 90CE prodrugs. Herein, we describe a new decomposition pathway that is available to 90CE but not to its methylating counterpart. This pathway appears to be subject to general/acid base catalysis with phosphate (Pi), phosphomonoesters, and phosphodiesters, being particularly effective. This pathway does not yield a chloroethylating species and results in a major change in nucleophile preference since thiophilic rather than oxophilic electrophiles are produced. Thus, a Pi concentration dependent decrease in DNA-DNA interstand cross-link formation was observed. Changes in 90CE decomposition products but not alkylation kinetics occurred in the presence of Pi since the prebranch point elimination of the N-1 methanesulfinate moiety remained the rate-limiting step. The Pi catalyzed route is expected to dominate at Pi and phosphoester concentrations totaling >25-35 mM. In view of the abundance of Pi and phosphoesters in cells, this pathway may have important effects on agent toxicity, tumor selectivity, and resistance to prodrugs of 90CE. Furthermore, it may be possible to design analogues that diminish this thiophile-generating pathway, which is likely superfluous at best and potentially detrimental to the targeting of hypoxic regions where Pi concentrations can be significantly elevated.

Influence of glutathione and glutathione S-transferases on DNA interstrand cross-link formation by 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine, the active anticancer moiety generated by laromustine.

Prodrugs of 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) are promising anticancer agents. The 90CE moiety is a readily latentiated, short-lived (t1/2 ∼ 30 s) chloroethylating agent that can generate high yields of oxophilic electrophiles responsible for the chloroethylation of the O-6 position of guanine in DNA. These guanine O-6 alkylations are believed to be responsible for the therapeutic effects of 90CE and its prodrugs. Thus, 90CE demonstrates high selectivity toward tumors with diminished levels of O(6)-alkylguanine-DNA alkyltransferase (MGMT), the resistance protein responsible for O(6)-alkylguanine repair. The formation of O(6)-(2-chloroethyl)guanine lesions ultimately leads to the generation of highly cytotoxic 1-(N(3)-cytosinyl),-2-(N(1)-guaninyl)ethane DNA interstrand cross-links via N(1),O(6)-ethanoguanine intermediates. The anticancer activity arising from this sequence of reactions is thus identical to this component of the anticancer activity of the clinically used chloroethylnitrosoureas. Herein, we evaluate the ability of glutathione (GSH) and other low molecular weight thiols, as well as GSH coupled with various glutathione S-transferase enzymes (GSTs) to attenuate the final yields of cross-links generated by 90CE when added prior to or immediately following the initial chloroethylation step to determine the major point(s) of interaction. In contrast to studies utilizing BCNU as a chloroethylating agent by others, GSH (or GSH/GST) did not appreciably quench DNA interstrand cross-link precursors. While thiols alone offered little protection at either alkylation step, the GSH/GST couple was able to diminish the initial yields of cross-link precursors. 90CE exhibited a very different GST isoenzyme susceptibility to that reported for BCNU, this could have important implications in the relative resistance of tumor cells to these agents. The protection afforded by GSH/GST was compared to that produced by MGMT.

Chloroethylating and methylating dual function antineoplastic agents display superior cytotoxicity against repair proficient tumor cells.

Two new agents based upon the structure of the clinically active prodrug laromustine were synthesized. These agents, 2-(2-chloroethyl)-N-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (1) and N-(2-chloroethyl)-2-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (2), were designed to retain the potent chloroethylating and DNA cross-linking functions of laromustine, and gain the ability to methylate DNA at the O-6 position of guanine, while lacking the carbamoylating activity of laromustine. The methylating arm was introduced with the intent of depleting the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT). Compound 1 is markedly more cytotoxic than laromustine in both AGT minus EMT6 mouse mammary carcinoma cells and high AGT expressing DU145 human prostate carcinoma cells. DNA cross-linking studies indicated that its cross-linking efficiency is nearly identical to its predicted active decomposition product, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which is also produced by laromustine. AGT ablation studies in DU145 cells demonstrated that 1 can efficiently deplete AGT. Studies assaying methanol and 2-chloroethanol production as a consequence of the methylation and chloroethylation of water by 1 and 2 confirmed their ability to function as methylating and chloroethylating agents and provided insights into the superior activity of 1.

Poly (ADP-ribose) polymerase inhibitors: on the horizon of tailored and personalized therapies for epithelial ovarian cancer.

PURPOSE OF REVIEW: Management of the epithelial ovarian cancer (EOC) remains a therapeutic challenge, with continued poor overall survival (OS). Given low chemotherapy response rates for recurrent disease and short survival times, new treatment options with improved therapeutic indices for targeting cancer's vulnerability are urgently needed in this patient population. RECENT FINDINGS: In this review, we summarize the recent development and clinical evaluations of inhibitors of poly (ADP-ribose) polymerase (PARP) as novel targeting agents for EOC. PARP inhibitors exploit synthetic lethality to target DNA repair defects in hereditary breast and ovarian cancer.In recent clinical trials, EOC patients with BRCA mutations exhibited favorable responses to the PARP inhibitor olaparib compared with patients without BRCA mutations. Additionally, olaparib has been reported to augment the effects of cisplatin and carboplatin on recurrence-free survival and OS in mice bearing BRCA1/2-deficient tumors.Given that hereditary EOC with deleterious BRCA1/2 mutations and BRCAness sporadic EOC are profoundly susceptible to synthetic lethality with PARP inhibition, it is imperative to identify a population of EOC patients that is likely to respond to PARP inhibitors. Recent studies have identified the gene expression profiles of DNA repair defects and BRCAness that predict clinical outcomes and response to platinum-based chemotherapy in EOC patients. SUMMARY: Ovarian cancer continues to carry the highest mortality among gynecologic cancers in the western world. Clinical development of PARP inhibitors that target DNA repair defects in cancer is a novel and imperative stride in individualized identification of molecular characteristics in management of ovarian cancer.

Design of a hypoxia-activated prodrug inhibitor of O6-alkylguanine-DNA alkyltransferase.

The efficacy of agents that alkylate the O-6 position of guanine is inhibited by O(6)-alkylguanine-DNA alkyltransferase (AGT) which removes these lesions from the tumor DNA. To increase differential toxicity, inhibitors must selectively deplete AGT in tumors, while sparing normal tissues where this protein serves a protective function. A newly synthesized prodrug of the AGT inhibitor O(6)-benzylguanine (O(6)-BG) with an α,α-dimethyl-4-nitrobenzyloxycarbonyl moiety masking the essential 2-amino group has demonstrated the feasibility of targeting hypoxic regions that are unique to solid tumors, for drug delivery. However, these modifications resulted in greatly decreased solubility. Recently, new potent global AGT inhibitors with improved formulatability such as O(6)-[(3-aminomethyl)benzylguanine (1) have been developed. However, acetylamino (N-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)acetamide) (2) exhibits a pronounced decrease in activity. Thus, 1 would be inactivated by N-acetylation and probably N-glucuronidation. To combat potential conjugational inactivation while retaining favorable solubility, we synthesized 6-((3-((dimethylamino)methyl)benzyl)oxy)-9H-purin-2-amine (3) in which the 3-aminomethyl moiety is protected by methylation; and to impart tumor selectivity we synthesized 2-(4-nitrophenyl)propan-2-yl(6-((3-((dimethylamino)methyl)benzyl)oxy)-9H-purin-2-yl)carbamate (7), a hypoxia targeted prodrug of 3 utilizing an α,α-dimethyl-4-nitrobenzyloxycarbonyl moiety. Consistent with this design, 7 demonstrates both hypoxia selective conversion by EMT6 cells of 7 to 3 and hypoxic sensitization of AGT containing DU145 cells to the cytotoxic actions of laromustine, while exhibiting improved solubility.

7-Nitro-4-(phenylthio)benzofurazan is a potent generator of superoxide and hydrogen peroxide.

Here, we report on 7-nitro-4-(phenylthio)benzofurazan (NBF-SPh), the most potent derivative among a set of patented anticancer 7-nitrobenzofurazans (NBFs), which have been suggested to function by perturbing protein-protein interactions. We demonstrate that NBF-SPh participates in toxic redox-cycling, rapidly generating reactive oxygen species (ROS) in the presence of molecular oxygen, and this is the first report to detail ROS production for any of the anticancer NBFs. Oxygraph studies showed that NBF-SPh consumes molecular oxygen at a substantial rate, rivaling even plumbagin, menadione, and juglone. Biochemical and enzymatic assays identified superoxide and hydrogen peroxide as products of its redox-cycling activity, and the rapid rate of ROS production appears to be sufficient to account for some of the toxicity of NBF-SPh (LC(50) = 12.1 µM), possibly explaining why tumor cells exhibit a sharp threshold for tolerating the compound. In cell cultures, lipid peroxidation was enhanced after treatment with NBF-SPh, as measured by 2-thiobarbituric acid-reactive substances, indicating a significant accumulation of ROS. Thioglycerol rescued cell death and increased survival by 15-fold to 20-fold, but pyruvate and uric acid were ineffective protectants. We also observed that the redox-cycling activity of NBF-SPh became exhausted after an average of approximately 19 cycles per NBF-SPh molecule. Electrochemical and computational analyses suggest that partial reduction of NBF-SPh enhances electrophilicity, which appears to encourage scavenging activity and contribute to electrophilic toxicity.

Reduced level of ribonucleotide reductase R2 subunits increases dependence on homologous recombination repair of cisplatin-induced DNA damage.

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the production of deoxyribonucleoside triphosphates (dNTPs) required for replicative and repair DNA synthesis. Mammalian RNR is a heteromeric enzyme consisting primarily of R1 and R2 subunits during the S phase of the cell cycle. We have shown previously that the presence of excess R2 subunits protects p53-deficient human colon cancer cells from cisplatin-induced DNA damage and replication stress. However, the mode of DNA repair influenced by changes in the level of the R2 subunit remained to be defined. In the present study, we demonstrated that depletion of BRCA1, an important factor of homologous recombination repair (HRR), preferentially sensitized stable R2-knockdown p53(-/-) HCT116 cells to the cytotoxicity of cisplatin and γ-H2AX induction. In accord with this finding, these R2-knockdown cells exhibited increased dependence on HRR, as evidenced by elevated levels of cisplatin-induced Rad51 foci and sister chromatid exchange frequency. Furthermore, stable knockdown of the R2 subunit also led to decreased cisplatin-induced gap-filling synthesis in nucleotide excision repair (NER) and a reduced dATP level in the G(2)/M phase of the cell cycle. These results suggest that an increased level of the R2 subunit extends the availability of dATP in the G(2)/M phase to promote the repair of NER-mediated single-strand gaps that are otherwise converted into double-strand breaks in the subsequent S phase. We propose that HRR becomes important for recovery from cisplatin-DNA lesions when the postexcision process of NER is restrained by reduced levels of the R2 subunit and dATP in p53-deficient cancer cells.

Ataxia-telangiectasia mutated kinase regulates ribonucleotide reductase and mitochondrial homeostasis.

Ataxia-telangiectasia mutated (ATM) kinase orchestrates nuclear DNA damage responses but is proposed to be involved in other important and clinically relevant functions. Here, we provide evidence for what we believe are 2 novel and intertwined roles for ATM: the regulation of ribonucleotide reductase (RR), the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates, and control of mitochondrial homeostasis. Ataxia-telangiectasia (A-T) patient fibroblasts, wild-type fibroblasts treated with the ATM inhibitor KU-55933, and cells in which RR is inhibited pharmacologically or by RNA interference (RNAi) each lead to mitochondrial DNA (mtDNA) depletion under normal growth conditions. Disruption of ATM signaling in primary A-T fibroblasts also leads to global dysregulation of the R1, R2, and p53R2 subunits of RR, abrogation of RR-dependent upregulation of mtDNA in response to ionizing radiation, high mitochondrial transcription factor A (mtTFA)/mtDNA ratios, and increased resistance to inhibitors of mitochondrial respiration and translation. Finally, there are reduced expression of the R1 subunit of RR and tissue-specific alterations of mtDNA copy number in ATM null mouse tissues, the latter being recapitulated in tissues from human A-T patients. Based on these results, we propose that disruption of RR and mitochondrial homeostasis contributes to the complex pathology of A-T and that RR genes are candidate disease loci in mtDNA-depletion syndromes.

Disruption of cAMP and prostaglandin E2 transport by multidrug resistance protein 4 deficiency alters cAMP-mediated signaling and nociceptive response.

Multidrug resistance protein 4 (MRP4; ABCC4) is a member of the MRP/ATP-binding cassette family serving as a transmembrane transporter involved in energy-dependent efflux of anticancer/antiviral nucleotide agents and of physiological substrates, including cyclic nucleotides and prostaglandins (PGs). Phenotypic consequences of mrp4 deficiency were investigated using mrp4-knockout mice and derived immortalized mouse embryonic fibroblast (MEF) cells. Mrp4 deficiency caused decreased extracellular and increased intracellular levels of cAMP in MEF cells under normal and forskolin-stimulated conditions. Mrp4 deficiency and RNA interference-mediated mrp4 knockdown led to a pronounced reduction in extracellular PGE(2) but with no accumulation of intracellular PGE(2) in MEF cells. This result was consistent with attenuated cAMP-dependent protein kinase activity and reduced cyclooxygenase-2 (Cox-2) expression in mrp4-deficient MEF cells, suggesting that PG synthesis is restrained along with a lack of PG transport caused by mrp4 deficiency. Mice lacking mrp4 exhibited no outward phenotypes but had a decrease in plasma PGE metabolites and an increase in inflammatory pain threshold compared with wild-type mice. Collectively, these findings imply that mrp4 mediates the efflux of PGE(2) and concomitantly modulates cAMP mediated signaling for balanced PG synthesis in MEF cells. Abrogation of mrp4 affects the regulation of peripheral PG levels and consequently alters inflammatory nociceptive responses in vivo.

Relationship between the induction of leukemia cell differentiation and the enhancement of reporter gene expression in 3T3 Swiss cells.

The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and hexamethylene bisacetamide (HMBA), which lacks HDAC inhibitory activity, both possess the capacity to induce leukemia cell differentiation and to enhance the expression of a wide range of transiently transfected reporter genes in 3T3 Swiss cells. In addition, known inducers of leukemia cell differentiation, including hypoxanthine, diazepam, 6-thioguanine and phorbol 12-myristate 13-acetate, also exhibited the ability to enhance reporter gene expression, while randomly chosen compounds that did not induce leukemia cell differentiation did not enhance reporter gene expression. The activity of TSA in the transfection system was modified by co-expression of histone acetyltransferase p300 and HDAC1; whereas, that of HMBA was enhanced by co-expression of the TATA-binding protein TBP. The stimulatory effects of diverse chemical inducers on transiently transfected genes suggest the existence of multiple exploitable targets for the selection of novel inducers of differentiation that function as modulators of gene activity.

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.

Development of an O(6)-alkylguanine-DNA alkyltransferase assay based on covalent transfer of the benzyl moiety from [benzene-3H]O(6)-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.

Generation of oxygen deficiency in cell culture using a two-enzyme system to evaluate agents targeting hypoxic tumor cells.

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.

Hyperinduction of Wnt activity: a new paradigm for the treatment of colorectal cancer?

Constitutive canonical Wnt signaling, resulting from mutations in the adenomatous polyposis coli (APC), beta-catenin, or axin genes, has been implicated in the initiation of most human colorectal cancers (CRCs). Some of the proposed approaches for CRC prevention and treatment involve the downregulation of canonical Wnt activity in an attempt to inhibit proliferation and promote apoptosis of the neoplastic cells. However, a number of studies have shown an association between high levels of canonical Wnt transcriptional activity and apoptosis. This relationship is also supported by the "just right hypothesis" for CRC formation where, in CRC patients, a selection for APC mutations occurs that results in a moderate level of canonical Wnt signaling and mutations leading to high levels of Wnt signaling are selected against, presumably due to apoptosis. In comparative studies of 10 human CRC cell lines, we found that inhibitors of histone deacetylases (HDACis), one of which is used clinically, promote apoptosis of CRC cells, at least partially by hyperinduction of canonical Wnt signaling. Based upon these findings, we propose a new paradigm for the activity of HDACis in the prevention and treatment of CRCs and other Wnt signaling-positive cancers. Herein, we review the evidence for the relationship between hyper-induced canonical Wnt activity and enhanced apoptosis in HDACi-treated CRC cells, discussing the implications of this relationship for cancer prevention and treatment, and pointing out the possible caveats of treating these tumors with HDACis.

Excess ribonucleotide reductase R2 subunits coordinate the S phase checkpoint to facilitate DNA damage repair and recovery from replication stress.

Ribonucleotide reductase (RNR), which consists of R1 and R2 subunits, catalyzes a key step of deoxyribonucleoside triphosphate (dNTP) synthesis for DNA replication and repair. The R2 subunit is controlled in a cell cycle-specific manner for timely DNA synthesis and is negatively regulated by p53 in response to DNA damage. Herein we demonstrate that the presence of excess R2 subunits in p53(-/-) HCT-116 human colon cancer cells protects against DNA damage and replication stress. siRNA-mediated stable knockdown (>80%) of excess R2 subunits has no effect on proliferative growth but results in enhanced accumulation of gamma-H2Ax and delayed recovery from DNA lesions inflicted by exposure to cisplatin and Triapine. This accentuated induction of gamma-H2Ax in R2-knockdown cells is attributed to reduced ability to repair damaged DNA and overcome replication blockage. The lack of excess R2 subunits consequently augments chk1 activation and cdc25A degradation, causing impeded cell progression through the S phase and enhanced apoptosis in response to DNA damage and replication stress. In contrast, the level of R1 subunits appears to be limiting, since depletion of the R1 subunit directly activates the S phase checkpoint due to replication stress associated with impaired RNR activity. These findings suggest that excess R2 subunits facilitate DNA damage repair and recovery from replication stress through coordination with the S phase checkpoint in the absence of functional p53. Thus, the level of the R2 subunit constitutes an important determinant of the chemosensitivity of cancer cells and serves as a potential target for enhancement of DNA-damage based therapy.

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 micromol/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 micromol/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.