Design Strategy for the EPR Tumor-Targeting of 1,2-Bis(sulfonyl)-1-alkylhydrazines.

A design strategy for macromolecular prodrugs is described, that are expected to exhibit robust activity against most solid tumor types while resulting in minimal toxicities to normal tissues. This approach exploits the enhanced permeability, and retention (EPR) effect, and utilizes carefully engineered rate constants to selectively target tumor tissue with short-lived cytotoxic moieties. EPR based tumor accumulation (half-life ~ 15 h) is dependent upon the ubiquitous abnormal solid tumor capillary morphology and is expected to be independent of individual tumor cell genetic variability that leads to resistance to molecularly targeted agents. The macromolecular sulfonylhydrazine-based prodrugs hydrolyze spontaneously with long half-life values (~10 h to >300 h dependent upon their structure) resulting in the majority of the 1,2-bis(sulfonyl)-1-alkylhydrazines (BSHs) cytotoxic warhead being released only after tumor sequestration. The very short half-life (seconds) of the finally liberated BSHs localizes the cytotoxic stress to the tumor target site by allowing insufficient time for escape. Thus, short lifespan anticancer species are liberated, and exhibit their activity largely within the tumor target. The abnormal tumor cell membrane pH gradients favor the uptake of BSHs compared to that of normal cells, further enhancing their selectivity. The reliance on physicochemical/chemical kinetic parameters and the EPR effect is expected to reduce response variability, and the acquisition of resistance.

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.

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.

Digital spatial proteomic profiling reveals immune checkpoints as biomarkers in lymphoid aggregates and tumor microenvironment of desmoplastic melanoma.

BACKGROUND: Desmoplastic melanoma (DM) is a rare melanoma subtype characterized by dense fibrous stroma, a propensity for local recurrence, and a high response rate to programmed cell death protein 1 (PD-1) blockade. Occult sentinel lymph node positivity is significantly lower in both pure and mixed DM than in conventional melanoma, underscoring the need for better prognostic biomarkers to inform therapeutic strategies. METHODS: We assembled a tissue microarray comprising various cores of tumor, stroma, and lymphoid aggregates from 45 patients with histologically confirmed DM diagnosed between 1989 and 2018. Using a panel of 62 validated immune-oncology markers, we performed digital spatial profiling using the NanoString GeoMx platform and quantified expression in three tissue compartments defined by fluorescence colocalization (tumor (S100+/PMEL+/SYTO+), leukocytes (CD45+/SYTO+), and non-immune stroma (S100-/PMEL-/CD45-/SYTO+)). RESULTS: We observed higher expression of immune checkpoints (lymphocyte-activation gene 3 [LAG-3] and cytotoxic T-lymphocyte associated protein-4 [CTLA-4]) and cancer-associated fibroblast (CAF) markers (smooth muscle actin (SMA)) in the tumor compartments of pure DMs than mixed DMs. When comparing lymphoid aggregates (LA) to non-LA tumor cores, LAs were more enriched with CD20+B cells, but non-LA intratumoral leukocytes were more enriched with macrophage/monocytic markers (CD163, CD68, CD14) and had higher LAG-3 and CTLA-4 expression levels. Higher intratumoral PD-1 and LA-based LAG-3 expression appear to be associated with worse survival. CONCLUSIONS: Our proteomic analysis reveals an intra-tumoral population of SMA+CAFs enriched in pure DM. Additionally, increased expressions of immune checkpoints (LAG-3 and PD-1) in LA and within tumor were associated with poorer prognosis. These findings might have therapeutic implications and help guide treatment selection in addition to informing potential prognostic significance.

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.

Case report: Metastatic Merkel cell carcinoma presenting seven years after loco-regional disease resection of primary tumor with interval in-transit and nodal metastases.

Merkel cell carcinoma (MCC) is a rare tumor with a high risk of recurrence after definitive therapy; however, the optimal duration of surveillance is unclear. First recurrences typically occur within 3 years. National guidelines recommend that patients undergo physical examination and imaging for surveillance during this time period. However, the duration of surveillance beyond this is not defined. Here, we describe a case of a patient developing a recurrence of MCC 7 years after the primary diagnosis with interval in-transit and regional lymph node metastases 15 months following the treatment of the primary MCC. Such late recurrences are rare, largely not reported, and the risk factors contributing to late recurrences are not well described. This case highlights the possibility of late recurrences of MCC after an initial in-transit and nodal recurrence and underscores the importance of identifying predictors of recurrence that may better guide the duration of surveillance.

Melanoma in pregnancy.

Melanoma is one of the most common types of cancer diagnosed during pregnancy. Patients with advanced disease require frequent staging examinations (e.g., CT, PET, MRI, ultrasound), which, during pregnancy must be modified from routine protocol to minimize risk to the fetus. We will review the diagnostic and treatment approach to pregnant patients with melanoma, with a discussion and pictorial examples of imaging protocol modifications, and the appearance of metastatic melanoma on radiology exams using modified protocols due to pregnancy.

The role of imaging and sentinel lymph node biopsy in patients with T3b-T4b melanoma with clinically negative disease.

Background: Previous studies demonstrate minimal utility of pre-operative imaging for low-risk melanoma; however, imaging may be more critical for patients with high-risk disease. Our study evaluates the impact of peri-operative cross-sectional imaging in patients with T3b-T4b melanoma. Methods: Patients with T3b-T4b melanoma who underwent wide local excision were identified from a single institution (1/1/2005 - 12/31/2020). Cross-sectional imaging was defined as body CT, PET and/or MRI in the perioperative period, with the following findings: in-transit or nodal disease, metastatic disease, incidental cancer, or other. Propensity scores were created for the odds of undergoing pre-operative imaging. Recurrence free survival was analyzed using the Kaplan-Meier method and log-rank test. Results: A total of 209 patients were identified with a median age of 65 (IQR 54-76), of which the majority were male (65.1%), with nodular melanoma (39.7%) and T4b disease (47.9%). Overall, 55.0% underwent pre-operative imaging. There were no differences in imaging findings between the pre- and post-operative cohorts. After propensity-score matching, there was no difference in recurrence free survival. Sentinel node biopsy was performed in 77.5% patients, with 47.5% resulting in a positive result. Conclusion: Pre-operative cross-sectional imaging does not impact the management of patients with high-risk melanoma. Careful consideration of imaging use is critical in the management of these patients and highlights the importance of sentinel node biopsy for stratification and decision making.

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.

Methods of Sentinel Lymph Node Identification in Auricular Melanoma.

Sentinel lymph node biopsy is used to evaluate for micrometastasis in auricular melanoma. However, lymphatic drainage patterns of the ear are not well defined and predicting the location of sentinel nodes can be difficult. The goal of this study was to define the lymphatic drainage patterns of the ear and to compare multiple modalities of sentinel node identification. METHODS: A retrospective review of a prospectively maintained database evaluated 80 patients with auricular melanoma who underwent sentinel lymph node biopsy by comparing preoperative imaging with intraoperative identification of sentinel nodes. Patients were placed into two cohorts, based on the modality of preoperative imaging: (1) planar lymphoscintigraphy only (n = 63) and (2) single-photon emission computerized tomography combined with computerized tomography (SPECT-CT) only (n = 17). Sites of preoperative mapping and sites of intraoperative identification were recorded as parotid/preauricular, mastoid/postauricular, and/or cervical. RESULTS: In patients that underwent planar lymphoscintigraphy preoperatively (n = 63), significantly more sentinel nodes were identified intraoperatively than were mapped preoperatively in both the parotid/preauricular (P = 0.0017) and mastoid/postauricular (P = 0.0047) regions. Thirty-two nodes were identified intraoperatively that were not mapped preoperatively in the planar lymphoscintigraphy group (n = 63), two of which were positive for micrometastatic disease. In contrast, there were no discrepancies between preoperative mapping and intraoperative identification of sentinel nodes in the SPECT-CT group (n = 17). CONCLUSIONS: SPECT-CT is more accurate than planar lymphoscintigraphy for the preoperative identification of draining sentinel lymph nodes in auricular melanoma. If SPECT-CT is not available, planar lymphoscintigraphy can also be used safely, but careful intraoperative evaluation, even in basins not mapped by lymphoscintigraphy, must be performed to avoid missed sentinel nodes.

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.

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.

pH-dependent general base catalyzed activation rather than isocyanate liberation may explain the superior anticancer efficacy of laromustine compared to related 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine prodrugs.

Laromustine (also known as cloretazine, onrigin, VNP40101M, 101M) is a prodrug of 90CE, a short-lived chloroethylating agent with anticancer activity. The short half-life of 90CE necessitates the use of latentiated prodrug forms for in vivo treatments. Alkylaminocarbonyl-based prodrugs such as laromustine exhibit significantly superior in vivo activity in several murine tumor models compared to analogs utilizing acyl, and alkoxycarbonyl latentiating groups. The alkylaminocarbonyl prodrugs possess two exclusive characteristics: (i) They are primarily unmasked by spontaneous base catalyzed elimination; and (ii) they liberate a reactive carbamoylating species. Previous speculations as to the therapeutic superiority of laromustine have focused upon the inhibition of enzymes by carbamoylation. We have investigated the therapeutic interactions of analogs with segregated chloroethylating and carbamoylating activities (singly and in combination) in the in vivo murine L1210 leukemia model. The combined treatment with chloroethylating and carbamoylating prodrugs failed to result in any synergism and produced a reduction in the therapeutic efficacy compared to the chloroethylating prodrug alone. Evidence supporting an alternative explanation for the superior tumor selectivity of laromustine is presented that is centered upon the high pH sensitivity of its base catalyzed activation, and the more alkaline intracellular pH values commonly found within tumor cells.

Cataloging antineoplastic agents according to their effectiveness against platinum-resistant and platinum-sensitive ovarian carcinoma cell lines.

Although epithelial ovarian cancers (EOCs) are initially treated with platinum-based chemotherapy, EOCs vary in platinum responsiveness. Cataloging antineoplastic agents according to their effectiveness against platinum-resistant and platinum-sensitive EOC cell lines is valuable for development of therapeutic strategies to avoid platinum inefficacy and to exploit platinum sensitivity. TOV-21G devoid of FANCF expression, OV-90 and SKOV-3 were employed as examples of platinum-sensitive, platinum-intermediate and platinum-resistant cell lines, respectively. Antineoplastic agents examined included mitomycin C, doxorubicin, etoposide, gemcitabine, chlorambucil, paclitaxel, triapine and X-rays. Their effectiveness against cell lines was analyzed by clonogenic assays. Cytotoxic profiles of mitomycin C and carboplatin were similar, with mitomycin C exhibiting greater potency and selectivity against TOV-21G than carboplatin. Cytotoxic profiles of doxorubicin, etoposide and X-rays overlapped with that of carboplatin, while OV-90 overexpressing Rad51 was more resistant to chlorambucil than SKOV-3. The efficacy of paclitaxel and triapine was independent of platinum sensitivity or resistance. Consistent with these cytotoxic profiles, cisplatin/mitomycin C, triapine, and paclitaxel differed in the capacity to induce phosphorylation of H2AX, and produced unique inhibitory patterns of DNA/RNA syntheses in HL-60 human leukemia cells. Paclitaxel and triapine in combination produced additive antitumor effects in M109 murine lung carcinoma. In conclusion, mitomycin C is potentially more effective against Fanconi anemia pathway-deficient EOCs than carboplatin. Doxorubicin and etoposide, because of their overlapping cytotoxic properties with carboplatin, are unlikely to be efficacious against platinum-refractory EOCs. Paclitaxel and triapine are effective regardless of platinum sensitivity status, and promising in combination for both platinum-sensitive and platinum-refractory EOCs.

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.

Antitumor sulfonylhydrazines: design, structure-activity relationships, resistance mechanisms, and strategies for improving therapeutic utility.

1,2-Bis(sulfonyl)-1-alkylhydrazines (BSHs) were conceived as more specific DNA guanine O-6 methylating and chloroethylating agents lacking many of the undesirable toxicophores contained in antitumor nitrosoureas. O(6)-Alkylguanine-DNA alkyltransferase (MGMT) is the sole repair protein for O(6)-alkylguanine lesions in DNA and has been reported to be absent in 5-20% of most tumor types. Many BSHs exhibit highly selective cytotoxicity toward cells deficient in MGMT activity. The development of clinically useful MGMT assays should permit the identification of tumors with this vulnerability and allow for the preselection of patient subpopulations with a high probability of responding. The BSH system is highly versatile, permitting the synthesis of many prodrug types with the ability to incorporate an additional level of tumor-targeting due to preferential activation by tumor cells. Furthermore, it may be possible to expand the spectrum of activity of these agents to include tumors with MGMT activity by combining them with tumor-targeted MGMT inhibitors.

Novel selection marker for mammalian cell transfection.

The availability of selectable markers suitable for use in mammalian cells has permitted the analysis of the influence of the stable overexpression of single or multiple genes on specific cell properties. This powerful technique has led directly to many fundamental advances in molecular biology and increased our overall understanding of cell growth and regulatory events. Although a variety of selectable markers are currently available, some cell lines continue to be naturally resistant to certain markers, making direct selection difficult or not feasible. Thus, the characterization of additional cell selectable markers continues to be of interest. We have developed a novel selectable marker based on mitomycin C resistance that is suitable for stable transfection of mammalian cells. This system is based on the ability of the mcrA gene, isolatedfrom Streptomyces lavendulae, to confer mitomycin C resistance to both bacterial and mammalian cells by expression of the MCRA protein. Here we demonstrate that mcrA can be used as a selectable gene marker in Chinese hamster ovary cells when cells transfected with the mcrA gene are either pulsed or cultured continuously with mitomycin C This unique selection system may be of use for transfection of cells that are resistant to currently available selectable markers.

Distinct mechanisms of cell-kill by triapine and its terminally dimethylated derivative Dp44mT due to a loss or gain of activity of their copper(II) complexes.

Triapine, currently being evaluated as an antitumor agent in phase II clinical trials, and its terminally dimethylated derivative Dp44mT share the α-pyridyl thiosemicarbazone backbone that functions as ligands for transition metal ions. Yet, Dp44mT is approximately 100-fold more potent than triapine in cytotoxicity assays. The aims of this study were to elucidate the mechanisms underlying their potency disparity and to determine their kinetics of cell-kill in culture to aid in the formulation of their clinical dosing schedules. The addition of Cu(2+) inactivated triapine in a 1:1 stoichiometric fashion, while it potentiated the cytotoxicity of Dp44mT. Clonogenic assays after finite-time drug-exposure revealed that triapine produced cell-kill in two phases, one completed within 20 min that caused limited cell-kill, and the other occurring after 16 h of exposure that produced extensive cell-kill. The ribonucleotide reductase inhibitor triapine at 0.4 µM caused immediate complete arrest of DNA synthesis, whereas Dp44mT at this concentration did not appreciably inhibit DNA synthesis. The inhibition of DNA synthesis by triapine was reversible upon its removal from the medium. Cell death after 16 h exposure to triapine paralleled the appearance of phospho-(γ)H2AX, a marker of DNA double-strand breaks induced by collapse of DNA replication forks after prolonged replication arrest. In contrast to triapine, Dp44mT produced robust cell-kill within 1h in a concentration-dependent manner. The short-term action of both agents was prevented by thiols, indicative of the involvement of reactive oxygen species. The time dependency in the production of cell-kill by triapine should be considered in treatment regimens.