Mycoplasma infection of cancer cells enhances anti-tumor effect of oxidized methylcytidines.

Oxidized methylcytidines 5-hydroxymethyl-2'deoxycytidine (5hmdC) and 5-formy-2'deoxycytidine (5fdC) are deaminated by cytidine deaminase (CDA) into genome-toxic variants of uridine, triggering DNA damage and cell death. These compounds are promising chemotherapeutic agents for cancer cells that are resistant to pyrimidine derivative drugs, such as decitabine and cytarabine, which are inactivated by CDA. In our study, we found that cancer cells infected with mycoplasma exhibited a markedly increased sensitivity to 5hmdC and 5fdC, which was independent of CDA expression of cancer cells. In vitro biochemical assay showed that the homologous CDA protein from mycoplasma was capable of deaminating 5hmdC and 5fdC into their uridine form. Moreover, mycoplasma infection increased the sensitivity of cancer cells to 5hmdC and 5fdC, whereas administration of Tetrahydrouridine (THU) attenuated this effect, suggesting that mycoplasma CDA confers a similar effect as human CDA. As mycoplasma infection occurs in many primary tumors, our findings suggest that intratumoral microbes could enhance the tumor-killing effect and expand the utility of oxidized methylcytidines in cancer treatment.

Combined inhibition of histone deacetylase and cytidine deaminase improves epigenetic potency of decitabine in colorectal adenocarcinomas.

BACKGROUND: Targeting the epigenome of cancerous diseases represents an innovative approach, and the DNA methylation inhibitor decitabine is recommended for the treatment of hematological malignancies. Although epigenetic alterations are also common to solid tumors, the therapeutic efficacy of decitabine in colorectal adenocarcinomas (COAD) is unfavorable. Current research focuses on an identification of combination therapies either with chemotherapeutics or checkpoint inhibitors in modulating the tumor microenvironment. Here we report a series of molecular investigations to evaluate potency of decitabine, the histone deacetylase inhibitor PBA and the cytidine deaminase (CDA) inhibitor tetrahydrouridine (THU) in patient derived functional and p53 null colon cancer cell lines (CCCL). We focused on the inhibition of cell proliferation, the recovery of tumor suppressors and programmed cell death, and established clinical relevance by evaluating drug responsive genes among 270 COAD patients. Furthermore, we evaluated treatment responses based on CpG island density. RESULTS: Decitabine caused marked repression of the DNMT1 protein. Conversely, PBA treatment of CCCL recovered acetylation of histone 3 lysine residues, and this enabled an open chromatin state. Unlike single decitabine treatment, the combined decitabine/PBA treatment caused > 95% inhibition of cell proliferation, prevented cell cycle progression especially in the S and G2-phase and induced programmed cell death. Decitabine and PBA differed in their ability to facilitate re-expression of genes localized on different chromosomes, and the combined decitabine/PBA treatment was most effective in the re-expression of 40 tumor suppressors and 13 genes typically silenced in cancer-associated genomic regions of COAD patients. Furthermore, this treatment repressed expression of 11 survival (anti-apoptotic) genes and augmented expression of X-chromosome inactivated genes, especially the lncRNA Xist to facilitate p53-mediated apoptosis. Pharmacological inhibition of CDA by THU or its gene knockdown prevented decitabine inactivation. Strikingly, PBA treatment recovered the expression of the decitabine drug-uptake transporter SLC15A1, thus enabling high tumor drug-loads. Finally, for 26 drug responsive genes we demonstrated improved survival in COAD patients. CONCLUSION: The combined decitabine/PBA/THU drug treatment improved drug potency considerably, and given their existing regulatory approval, our findings merit prospective clinical trials for the triple combination in COAD patients.

Upregulation of cytidine deaminase in NAT1 knockout breast cancer cells.

PURPOSE: Arylamine N-acetyltransferase 1 (NAT1), a phase II metabolic enzyme, is frequently upregulated in breast cancer. Inhibition or depletion of NAT1 leads to growth retardation in breast cancer cells in vitro and in vivo. A previous metabolomics study of MDA-MB-231 breast cancer cells suggests that NAT1 deletion leads to a defect in de novo pyrimidine biosynthesis. In the present study, we observed that NAT1 deletion results in upregulation of cytidine deaminase (CDA), which is involved in the pyrimidine salvage pathway, in multiple breast cancer cell lines (MDA-MB-231, MCF-7 and ZR-75-1). We hypothesized that NAT1 KO MDA-MB-231 cells show differential sensitivity to drugs that either inhibit cellular pyrimidine homeostasis or are metabolized by CDA. METHODS: The cells were treated with (1) inhibitors of dihydroorotate dehydrogenase or CDA (e.g., teriflunomide and tetrahydrouridine); (2) pyrimidine/nucleoside analogs (e.g., gemcitabine and 5-azacytidine); and (3) naturally occurring, modified cytidines (e.g., 5-formyl-2'-deoxycytidine; 5fdC). RESULTS: Although NAT1 KO cells failed to show differential sensitivity to nucleoside analogs that are metabolized by CDA, they were markedly more sensitive to 5fdC which induces DNA damage in the presence of high CDA activity. Co-treatment with 5fdC and a CDA inhibitor, tetrahydrouridine, abrogated the increase in 5fdC cytotoxicity in NAT1 KO cells, suggesting that the increased sensitivity of NAT1 KO cells to 5fdC is dependent on their increased CDA activity. CONCLUSIONS: The present findings suggest a novel therapeutic strategy to treat breast cancer with elevated NAT1 expression. For instance, NAT1 inhibition may be combined with cytotoxic nucleosides (e.g., 5fdC) for breast cancer treatment.

A pilot clinical trial of oral tetrahydrouridine/decitabine for noncytotoxic epigenetic therapy of chemoresistant lymphoid malignancies.

One mechanism by which lymphoid malignancies resist standard apoptosis-intending (cytotoxic) treatments is genetic attenuation of the p53/p16-CDKN2A apoptosis axis. Depletion of the epigenetic protein DNA methyltransferase 1 (DNMT1) using the deoxycytidine analog decitabine is a validated approach to cytoreduce malignancy independent of p53/p16. In vivo decitabine activity, however, is restricted by rapid catabolism by cytidine deaminase (CDA). We, therefore, combined decitabine with the CDA-inhibitor tetrahydrouridine and conducted a pilot clinical trial in patients with relapsed lymphoid malignancies: the doses of tetrahydrouridine/decitabine used (∼10/0.2 mg/kg orally (PO) 2×/week) were selected for the molecular pharmacodynamic objective of non-cytotoxic, S-phase dependent, DNMT1-depletion, guided by previous Phase 1 studies. Patients with relapsed/refractory B- or T-cell malignancies (n = 7) were treated for up to 18 weeks. Neutropenia without concurrent thrombocytopenia is an expected toxicity of DNMT1-depletion and occurred in all patients (Grade 3/4). Subjective and objective clinical improvements occurred in 4 of 7 patients, but these responses were lost upon treatment interruptions and reductions to manage neutropenia. We thus performed parallel experiments in a preclinical in vivo model of lymphoma to identify regimen refinements that might sustain DNMT1-targeting in malignant cells but limit neutropenia. We found that timed-alternation of decitabine with the related molecule 5-azacytidine, and combination with inhibitors of CDA and de novo pyrimidine synthesis could leverage feedback responses of pyrimidine metabolism to substantially increase lymphoma cytoreduction but with less neutropenia. In sum, regimen innovations beyond incorporation of a CDA-inhibitor are needed to sustain decitabine DNMT1-targeting and efficacy against chemo-resistant lymphoid malignancy. Such potential solutions were explored in preclinical in vivo studies.

Nucleoside-catabolizing enzymes in mycoplasma-infected tumor cell cultures compromise the cytostatic activity of the anticancer drug gemcitabine.

The intracellular metabolism and cytostatic activity of the anticancer drug gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdC) was severely compromised in Mycoplasma hyorhinis-infected tumor cell cultures. Pronounced deamination of dFdC to its less cytostatic metabolite 2',2'-difluoro-2'-deoxyuridine was observed, both in cell extracts and spent culture medium (i.e. tumor cell-free but mycoplasma-containing) of mycoplasma-infected tumor cells. This indicates that the decreased antiproliferative activity of dFdC in such cells is attributed to a mycoplasma cytidine deaminase causing rapid drug catabolism. Indeed, the cytostatic activity of gemcitabine could be restored by the co-administration of tetrahydrouridine (a potent cytidine deaminase inhibitor). Additionally, mycoplasma-derived pyrimidine nucleoside phosphorylase (PyNP) activity indirectly potentiated deamination of dFdC: the natural pyrimidine nucleosides uridine, 2'-deoxyuridine and thymidine inhibited mycoplasma-associated dFdC deamination but were efficiently catabolized (removed) by mycoplasma PyNP. The markedly lower anabolism and related cytostatic activity of dFdC in mycoplasma-infected tumor cells was therefore also (partially) restored by a specific TP/PyNP inhibitor (TPI), or by exogenous thymidine. Consequently, no effect on the cytostatic activity of dFdC was observed in tumor cell cultures infected with a PyNP-deficient Mycoplasma pneumoniae strain. Because it has been reported that some commensal mycoplasma species (including M. hyorhinis) preferentially colonize tumor tissue in cancer patients, our findings suggest that the presence of mycoplasmas in the tumor microenvironment could be a limiting factor for the anticancer efficiency of dFdC-based chemotherapy. Accordingly, a significantly decreased antitumor effect of dFdC was observed in mice bearing M. hyorhinis-infected murine mammary FM3A tumors compared with uninfected tumors.

Tetrahydrouridine inhibits cell proliferation through cell cycle regulation regardless of cytidine deaminase expression levels.

Tetrahydrouridine (THU) is a well characterized and potent inhibitor of cytidine deaminase (CDA). Highly expressed CDA catalyzes and inactivates cytidine analogues, ultimately contributing to increased gemcitabine resistance. Therefore, a combination therapy of THU and gemcitabine is considered to be a potential and promising treatment for tumors with highly expressed CDA. In this study, we found that THU has an alternative mechanism for inhibiting cell growth which is independent of CDA expression. Three different carcinoma cell lines (MIAPaCa-2, H441, and H1299) exhibited decreased cell proliferation after sole administration of THU, while being unaffected by knocking down CDA. To investigate the mechanism of THU-induced cell growth inhibition, cell cycle analysis using flow cytometry was performed. This analysis revealed that THU caused an increased rate of G1-phase occurrence while S-phase occurrence was diminished. Similarly, Ki-67 staining further supported that THU reduces cell proliferation. We also found that THU regulates cell cycle progression at the G1/S checkpoint by suppressing E2F1. As a result, a combination regimen of THU and gemcitabine might be a more effective therapy than previously believed for pancreatic carcinoma since THU works as a CDA inhibitor, as well as an inhibitor of cell growth in some types of pancreatic carcinoma cells.

Epigenetic regulation by decitabine of melanoma differentiation in vitro and in vivo.

Apoptosis genes, such as TP53 and p16/CDKN2A, that mediate responses to cytotoxic chemotherapy, are frequently nonfunctional in melanoma. Differentiation may be an alternative to apoptosis for inducing melanoma cell cycle exit. Epigenetic mechanisms regulate differentiation, and DNA methylation alterations are associated with the abnormal differentiation of melanoma cells. The effects of the deoxycytidine analogue decitabine (5-aza-2'-deoxycytidine), which depletes DNA methyl transferase 1 (DNMT1), on melanoma differentiation were examined. Treatment of human and murine melanoma cells in vitro with concentrations of decitabine that did not cause apoptosis inhibited proliferation accompanied by cellular differentiation. A decrease in promoter methylation, and increase in expression of the melanocyte late-differentiation driver SOX9, was followed by increases in cyclin-dependent kinase inhibitors (CDKN) p27/CDKN1B and p21/CDKN1A that mediate cell cycle exit with differentiation. Effects were independent of the TP53, p16/CDKN2A and also the BRAF status of the melanoma cells. Resistance, when observed, was pharmacologic, characterized by diminished ability of decitabine to deplete DNMT1. Treatment of murine melanoma models in vivo with intermittent, low-dose decitabine, administered sub-cutaneously to limit high peak drug levels that cause cytotoxicity and increase exposure time for DNMT1 depletion, and with tetrahydrouridine to decrease decitabine metabolism and further increase exposure time, inhibited tumor growth and increased molecular and tumor stromal factors implicated in melanocyte differentiation. Modification of decitabine dose, schedule and formulation for differentiation rather than cytotoxic objectives inhibits the growth of melanoma cells in vitro and in vivo.

Effects of tetrahydrouridine on pharmacokinetics and pharmacodynamics of oral decitabine.

The deoxycytidine analog decitabine (DAC) can deplete DNA methyl-transferase 1 (DNMT1) and thereby modify cellular epigenetics, gene expression, and differentiation. However, a barrier to efficacious and accessible DNMT1-targeted therapy is cytidine deaminase, an enzyme highly expressed in the intestine and liver that rapidly metabolizes DAC into inactive uridine counterparts, severely limiting exposure time and oral bioavailability. In the present study, the effects of tetrahydrouridine (THU), a competitive inhibitor of cytidine deaminase, on the pharmacokinetics and pharmacodynamics of oral DAC were evaluated in mice and nonhuman primates. Oral administration of THU before oral DAC extended DAC absorption time and widened the concentration-time profile, increasing the exposure time for S-phase-specific depletion of DNMT1 without the high peak DAC levels that can cause DNA damage and cytotoxicity. THU also decreased interindividual variability in pharmacokinetics seen with DAC alone. One potential clinical application of DNMT1-targeted therapy is to increase fetal hemoglobin and treat hemoglobinopathy. Oral THU-DAC at a dose that would produce peak DAC concentrations of less than 0.2µM administered 2×/wk for 8 weeks to nonhuman primates was not myelotoxic, hypomethylated DNA in the γ-globin gene promoter, and produced large cumulative increases in fetal hemoglobin. Combining oral THU with oral DAC changes DAC pharmacology in a manner that may facilitate accessible noncytotoxic DNMT1-targeted therapy.

Plasma pharmacokinetics and oral bioavailability of the 3,4,5,6-tetrahydrouridine (THU) prodrug, triacetyl-THU (taTHU), in mice.

PURPOSE: Cytidine drugs, such as gemcitabine, undergo rapid catabolism and inactivation by cytidine deaminase (CD). 3,4,5,6-tetrahydrouridine (THU), a potent CD inhibitor, has been applied preclinically and clinically as a modulator of cytidine analogue metabolism. However, THU is only 20% orally bioavailable, which limits its preclinical evaluation and clinical use. Therefore, we characterized THU pharmacokinetics after the administration to mice of the more lipophilic pro-drug triacetyl-THU (taTHU). METHODS: Mice were dosed with 150 mg/kg taTHU i.v. or p.o. Plasma and urine THU concentrations were quantitated with a validated LC-MS/MS assay. Plasma and urine pharmacokinetic parameters were calculated non-compartmentally and compartmentally. RESULTS: taTHU did not inhibit CD. THU, after 150 mg/kg taTHU i.v., had a 235-min terminal half-life and produced plasma THU concentrations >1 µg/mL, the concentration shown to inhibit CD, for 10 h. Renal excretion accounted for 40-55% of the i.v. taTHU dose, 6-12% of the p.o. taTHU dose. A two-compartment model of taTHU generating THU fitted the i.v. taTHU data best. taTHU, at 150 mg/kg p.o., produced a concentration versus time profile with a plateau of approximately 10 µg/mL from 0.5-2 h, followed by a decline with a 122-min half-life. Approximately 68% of i.v. taTHU is converted to THU. Approximately 30% of p.o. taTHU reaches the systemic circulation as THU. CONCLUSIONS: The availability of THU after p.o. taTHU is 30%, when compared to the 20% achieved with p.o. THU. These data will support the clinical studies of taTHU.

Stability of 5-fluoro-2'-deoxycytidine and tetrahydrouridine in combination.

In vivo, the DNA methyltransferase inhibitor, 5-fluoro-2'-deoxycytidine (FdCyd, NSC-48006), is rapidly converted to its unwanted metabolites. Tetrahydrouridine (THU, NSC-112907), a cytidine deaminase inhibitor can block the first metabolic step in FdCyd catabolism. Clinical studies have shown that co-administration with THU can inhibit the metabolism of FdCyd. The National Cancer Institute is particularly interested in a 1:5 FdCyd/THU formulation. The purpose of this study was to investigate the in vitro pH stability of FdCyd and THU individually and in combination. A stability-indicating high-performance liquid chromatography method for the quantification of both compounds and their degradants was developed using a ZIC(R)-HILIC column. The effect of THU and FdCyd on the in vitro degradation of each other was studied as a function of pH from 1.0 to 7.4 in aqueous solutions at 37 degrees C. The degradation of FdCyd appears to be first-order and acid-catalyzed. THU equilibrates with at least one of its degradants. The combination of FdCyd and THU in solution does not affect the stability of either compound. The stability and compatibility of FdCyd and THU in the solid state at increased relative humidity and at various temperatures are also evaluated.

Is the resistance of gemcitabine for pancreatic cancer settled only by overexpression of deoxycytidine kinase?

The prognosis of pancreatic cancer remains poor, and the standard first-line chemotherapy with gemcitabine (GEM) has a response rate of less than 20%. Since expression of deoxycytidine kinase (dCK) seems important for improvement of GEM sensitivity, overexpression of dCK was investigated using pancreatic cancer cell lines (Panc-1, MIAPaCa-2 and BxPC-3). dCK gene was introduced into the cell lines by retrovirus and changes in IC50 were examined. Sensitivity of two pancreatic cancer cell lines to GEM elevated dramatically in comparison with control cells, but change of sensitivity remained at 1.8 times in BxPC-3. Since addition of tetrahydro uridine (THU), an inhibitor of deoxycytidine deaminase (CDA), increased the sensitivity 54-fold, overexpression of CDA seems to be the mechanism for improvement of the sensitivity. In conclusion, dCK is a key enzyme of GEM, but resistance of GEM is not improved in all pancreatic cancer cells by overexpression of dCK. Combination treatment based on expression of GEM metabolism-related gene may become an effective therapy in the future.

Optimized blood sampling with cytidine deaminase inhibitor for improved analysis of capecitabine metabolites.

The 5FU prodrug capecitabine undergoes a 3-step enzymatic conversion, including the conversion of 5'DFRC into 5'DFUR by cytidine deaminase (CDA). The presence of CDA activity in blood led us to analyze the possible ex vivo conversion of 5'DFCR into 5'DFUR in blood samples. We thus examined the impact of the addition of a CDA inhibitor (tetrahydrouridine (THU) 1 microM final) in blood. Blood samples from 3 healthy volunteers were taken on tubes containing or not THU. Blood was spiked with 5'DFCR (20 microM final) (T0) and was maintained at room temperature for 2 h. Plasma concentrations of 5'DFRC and 5'DFUR were analyzed with an optimized HPLC assay. In the absence of THU, 5'DFUR was detectable as early as T0. The percent of 5'DFUR produced relative to 5'DFCR increased over time, up to 7.7 % at 2h. In contrast, the presence of THU totally prevents the formation of 5'DFUR. The impact of THU for preventing the conversion of 5'DFCR was confirmed by the analysis of blood samples from 2 capecitabine-treated patients. Addition of THU in the sampling-tube before the introduction of blood is thus strongly recommended in order to guarantee accurate conditions for reliable measurement of capecitabine metabolites in plasma, and thus faithful pharmacokinetic data.

Plasma pharmacokinetics and oral bioavailability of 3,4,5,6-tetrahydrouridine, a cytidine deaminase inhibitor, in mice.

Cytidine analogues such as cytosine arabinoside, gemcitabine, decitabine, 5-azacytidine, 5-fluoro-2'-deoxycytidine and 5-chloro-2'-deoxycytidine undergo rapid catabolism by cytidine deaminase (CD). 3,4,5,6-tetrahydrouridine (THU) is a potent CD inhibitor that has been applied preclinically and clinically as a modulator of cytidine analogue metabolism. However, THU pharmacokinetics has not been fully characterized, which has impaired the optimal preclinical evaluation and clinical use of THU. Therefore, we characterized the THU pharmacokinetics and bioavailability in mice. Mice were dosed with THU iv (100 mg/kg) or po (30, 100, or 300 mg/kg). Plasma and urine THU concentrations were quantitated with a validated LC-MS/MS assay. Plasma pharmacokinetic parameters were calculated compartmentally and non-compartmentally. THU, at 100 mg/kg iv had a 73 min terminal half-life and produced plasma THU concentrations >1 microg/ml, the concentration shown to effectively block deamination, for 4 h. Clearance was 9.1 ml/min/kg, and the distribution volume was 0.95 l/kg. Renal excretion accounted for 36-55% of the THU dose. A three-compartment model fit the iv THU data best. THU, at 100 mg/kg po, produced a concentration versus time profile with a plateau of approximately 10 mug/ml from 0.5-3 h, followed by a decline with an 85 min half-life. The oral bioavailability of THU was approximately 20%. The 20% oral bioavailability of THU is sufficient to produce and sustain, for several hours, plasma concentrations that inhibit CD. This suggests the feasibility of using THU to decrease elimination and first-pass metabolism of cytidine analogues by CD. THU pharmacokinetics are now being evaluated in humans.

The 1.48 A resolution crystal structure of the homotetrameric cytidine deaminase from mouse.

Cytidine deaminase (CDA) is a zinc-dependent enzyme that catalyzes the deamination of cytidine or deoxycytidine to form uridine or deoxyuridine. Here we present the crystal structure of mouse CDA (MmCDA), complexed with either tetrahydrouridine (THU), 3-deazauridine (DAU), or cytidine. In the MmCDA-DAU complex, it clearly demonstrates that cytidine is distinguished from uridine by its 4-NH(2) group that acts as a hydrogen bond donor. In the MmCDA-cytidine complex, cytidine, unexpectedly, binds as the substrate instead of the deaminated product in three of the four subunits, and in the remaining subunit it binds as the product uridine. Furthermore, the charge-neutralizing Arg68 of MmCDA has also exhibited two alternate conformations, I and II. In conformation I, the only conformation observed in the other structurally known homotetrameric CDAs, Arg68 hydrogen bonds Cys65 and Cys102 to modulate part of their negative charges. However, in conformation II the side chain of Arg68 rotates about 130 degrees around the Cgamma-Cdelta bond and abolishes these hydrogen bonds. The lack of hydrogen bonding may indirectly weaken the zinc-product interaction by increased electron donation from cysteine to the zinc ion, suggesting a novel product-expelling mechanism. On the basis of known structures, structural analysis further reveals two subclasses of homotetrameric CDAs that can be identified according to the position of the charge-neutralizing arginine residue. Implications for CDA-RNA interaction have also been considered.

Relatively small increases in the steady-state levels of nucleobase deamination products in DNA from human TK6 cells exposed to toxic levels of nitric oxide.

Nitric oxide (NO) is a physiologically important molecule that has been implicated in the pathophysiology of diseases associated with chronic inflammation, such as cancer. While the complicated chemistry of NO-mediated genotoxicity has been extensively study in vitro, neither the spectrum of DNA lesions nor their consequences in vivo have been rigorously defined. We have approached this problem by exposing human TK6 lymphoblastoid cells to controlled steady-state concentrations of 1.75 or 0.65 microM NO along with 186 microM O2 in a recently developed reactor that avoids the anomalous gas-phase chemistry of NO and approximates the conditions at sites of inflammation in tissues. The resulting spectrum of nucleobase deamination products was defined using a recently developed liquid chromatography/mass spectrometry (LC/MS) method, and the results were correlated with cytotoxicity and apoptosis. A series of control experiments revealed the necessity of using dC and dA deaminase inhibitors to avoid adventitious formation of 2'-deoxyuridine (dU) and 2'-deoxyinosine (dI), respectively, during DNA isolation and processing. Exposure of TK6 cells to 1.75 microM NO and 186 microM O2 for 12 h (1260 microM x min dose) resulted in 32% loss of cell viability measured immediately after exposure and 87% cytotoxicity after a 24 h recovery period. The same exposure resulted in 3.5-, 3.8-, and 4.1-fold increases in dX, dI, and dU, respectively, to reach the following levels: dX, 7 (+/- 1) per 10(6) nt; dI, 25 (+/- 2.1) per 10(6) nt; and dU, 40 (+/- 3.8) per 10(6) nt. dO was not detected above the limit of detection of 6 lesions per 10(7) nt in 50 microg of DNA. A 12 h exposure to 0.65 microM NO and 190 microM O2 (468 microM x min dose) caused 1.7-, 1.8-, and 2.0-fold increases in dX, dI, and dU, respectively, accompanied by a approximately 15% (+/- 3.6) reduction in cell viability immediately after exposure. Again, dO was not detected. These results reveal modest increases in the steady-state levels of DNA deamination products in cells exposed to relatively cytotoxic levels of NO. This could result from limited nitrosative chemistry in nuclear DNA in cells exposed to NO or high levels of formation balanced by rapid repair of nucleobase deamination lesions in DNA.

Epimer interconversion, isomerization, and hydrolysis of tetrahydrouridine: implications for cytidine deaminase inhibition.

Tetrahydrouridine (THU) is an inhibitor of cytidine deaminase (CDA), the enzyme responsible for the deactivation of ara-C and other cytidine analogues in vivo, and therefore is capable of improving the therapeutic efficacy of these antitumor agents. In aqueous solution formulations, THU exists as a mixture of epimers differing in stereochemistry of the 4-OH substituent. The aims of this study were to investigate the interconversion kinetics of the epimers of THU, the CDA inhibitory effects of these epimers, and the stability and degradation mechanisms of THU epimer mixtures in aqueous solution with the ultimate goal of developing optimal conditions for a parenteral formulation of THU. A stability indicating HPLC assay utilizing a derivatized beta-cyclodextrin column was developed to separate the two epimers of THU and to monitor their reversible isomerization to their beta-ribopyranosyl counterparts and their hydrolysis to form N-glycosidic bond cleavage products. MS and one- and two-dimensional (1)H- and (13)C-NMR measurements were conducted to identify THU epimers and degradation products and to quantitatively model the degradation kinetics. The interconversion reaction between the two THU epimers is acid catalyzed with a first-order rate constant for conversion of epimer 1(1) to epimer 1(2) of (7.4 +/- 0.3) x 10(-3) h(-1) and an equilibrium constant ([1(2)]/[1(1)] of 1.7 +/- 0.1 at pH 7.4 and 25 degrees C. Epimer interconversion was therefore sufficiently slow at pH 7.4 to allow the isolation of each and evaluation of their CDA inhibitory activities utilizing 1% (w/v) mouse kidney homogenates as a source for cytidine deaminase and cytidine as a substrate. Inhibition constants for the two THU epimers (1(1) and 1(2)) were determined to be 8 +/- 1 x 10(-7) M and 6.2 +/- 0.2 x 10(-8) M, respectively. Studies at elevated temperature suggested that THU degradation from epimer mixtures is biphasic with the initial rate of disappearance being acid catalyzed and first order in initial THU concentration, thus ruling out dimerization as a potential reaction mechanism. NMR/MS analyses revealed that the major degradation products included the beta-ribopyranosyl THU isomers (two epimers), the reduced pyrimidinone base (tetrahydrouracil), and various anomers of D-ribose formed through N-glycosidic bond cleavage, and the products of subsequent reactions of the base. Kinetic modeling of the data obtained from both HPLC and NMR measurements indicated that in an acidic solution THU beta-ribofuranosyl --> beta-ribopyranosyl isomerization is a rapid equilibrium reaction, which proceeds through an intermediate observable in 1H-NMR, and is followed by slower N-glycosidic bond hydrolysis. All the reactions between THU, its ribopyranosyl isomers, the intermediate, and the base are acid catalyzed and appear to proceed through the same sugar ring-opened intermediate (carbinolamine), consistent with previous literature.

Drug resistance to 5-aza-2'-deoxycytidine, 2',2'-difluorodeoxycytidine, and cytosine arabinoside conferred by retroviral-mediated transfer of human cytidine deaminase cDNA into murine cells.

PURPOSE: The hematopoietic toxicity produced by the cytosine nucleoside analogs is a critical problem that limits their effectiveness in cancer therapy. One strategy to prevent this dose-limiting toxicity would be to insert a gene for drug resistance to these analogs into normal bone marrow cells. Cytidine (CR) deaminase can deaminate and thus inactivate 5-aza-2'-deoxycytidine (5-AZA-CdR), 2',2'-difluorodeoxycytidine (dFdC) and cytosine arabinoside (ARA-C). The aim of this study was to determine if gene transfer of CR deaminase into murine fibroblast cells confers drug resistance to these cytosine nucleoside analogs and if this resistance can be prevented by the CR deaminase inhibitor, 3,4,5,6-tetrahydrouridine (THU). METHODS: NIH 3T3 murine fibroblast cells were transduced with retroviral particles containing the human CR deaminase cDNA. Assays measuring CR deaminase activity as well as the inhibitory action of 5-AZA-CdR, dFdC and ARA-C on colony formation, were performed in the presence of different concentrations of THU. RESULTS: Retroviral-mediated transfer of the CR deaminase gene into 3T3 fibroblasts produced a considerable increase in CR deaminase activity. The transduced cells also showed significant drug resistance to 5-AZA-CdR, dFdC and ARA-C, as demonstrated by a clonogenic assay. This drug resistance phenotype and elevated CR deaminase activity were reversed by THU. CONCLUSIONS: These findings indicate that the CR deaminase gene can potentially be used in cancer gene therapy for protecting normal cells against the cytotoxic actions of different cytosine nucleoside analogs. In addition, the CR deaminase-transduced cells can be used as a model for screening different CR deaminase inhibitors in an intact cellular system.

Induction of resistance to 1-beta-D-arabinofuranosylcytosine in human H9 cell line by simian immunodeficiency virus.

We examined drug sensitivity of human T cell acute lymphoblastic leukemia H9 cells chronically infected with simian immunodeficiency virus (SIVmac) and found that the retrovirus-infected H9 cells showed 8.2-fold resistance to 1-beta-D-arabinofuranosylcytosine (Ara-C). In the infected cells, Ara-CTP levels decreased to 20% of that found in uninfected H9 cells after 3 h incubation at Ara-C concentration of 1 microM, and 8.1-fold increase of cytidine deaminase activity was observed in the infected H9 cells. A competitive inhibitor of cytidine deaminase, 3, 4, 5, 6-tetrahydrouridine (THU), at 100 microM reversed Ara-C resistance in the infected cells. These results indicate that inducing increased cytidine deaminase activity by SIVmac infection conferred Ara-C resistance to H9 cells. An understanding of these cellular differences in drug sensitivity may aid in the development of therapeutic strategies against retrovirus-infected cells.

Kinetic studies on 2',2'-difluorodeoxycytidine (Gemcitabine) with purified human deoxycytidine kinase and cytidine deaminase.

Phosphorylation of cytosine analogs by deoxycytidine kinase (dCK) and deamination by cytidine deaminase (CDA) are two important processes in the activation and elimination of these drugs. We have investigated the kinetic parameters of 2',2'-difluorodeoxycytidine (dFdC) using purified enzymes from human cells. Deoxycytidine (CdR) and dFdC had Km values of 1.5 and 4.6 microM for dCK, respectively. Feedback inhibition of dCK by deoxycytidine 5'-triphosphate (dCTP) was also studied. Our results show that dCTP produced a greater inhibition of the phosphorylation of dFdC than CdR with concentrations of dCTP ranging from 1 to 25 microM. dFdC was a good substrate for CDA. Kinetic studies with this enzyme gave Km values for CdR and dFdC of 46.3 and 95.7 microM, respectively. The effect of competitive inhibitors of CDA on the deamination of dFdC was also investigated. Diazepinone riboside was a more potent inhibitor than tetrahydrouridine using either CdR or dFdC as the substrate. Inhibitors of CDA could be useful in clinical trials in patients with cancer to increase the chemotherapeutic effectiveness of dFdC.

Activity of 2',2'-difluorodeoxycytidine (Gemcitabine) against human tumor colony forming units.

2',2'-Difluorodeoxycytidine (LY 188011, Gemcitabine) is a novel pyrimidine antimetabolite with promising activity in preclinical models for leukemia and solid tumors. Phase I clinical trials with the agent are ongoing. In order to better define types of tumors with clinical sensitivity to Gemcitabine (to help target phase II trials), we have studied the antitumor effects of this agent against a variety of freshly explanted human tumor specimens using an in vitro capillary soft agar cloning system. Final concentrations of 2.0-200 micrograms/ml were used for short-term (1 h) and continuous incubations experiments. Using a short-term incubation, 94/215 (44%) tumor specimens were evaluable for the determination of antitumor activity. The most common tumor types studied included colorectal, breast, non-small cell lung, ovarian cancer, kidney and melanoma. A concentration-dependent increase in the frequency of inhibited tumor specimens was noted (2 micrograms/ml: 6/94 specimens, 20 micrograms/ml: 13/94 specimens, 200 micrograms/ml:33/94 specimens; p less than 0.0001). A similar increase in tumor growth inhibition was found using a continuous incubation (2 micrograms/ml: 0/14 specimens, 20 micrograms/ml: 1/14 specimens, 200 micrograms/ml: 7/14 specimens; p less than 0.001). We conclude that Gemcitabine is an active antitumor agent against tumor colony forming units from a variety of human malignancies if sufficiently high concentrations can be achieved. The agent should be evaluated for Phase II clinical activity against those tumor types.