James A. Kelley

Interleukin 6 Supports the Maintenance of p53 Tumor Suppressor Gene Promoter Methylation

A strong association exists between states of chronic inflammation and cancer, and it is believed that mediators of inflammation may be responsible for this phenomenon. Interleukin 6 (IL-6) is an inflammatory cytokine known to play a role in the growth and survival of many types of tumors, yet the mechanisms employed by this pleomorphic cytokine to accomplish this feat are still poorly understood. Another important factor in tumor development seems to be the hypermethylation of CpG islands located within the promoter regions of tumor suppressor genes. This common epigenetic alteration enables tumor cells to reduce or inactivate the expression of important tumor suppressor and cell cycle regulatory genes. Here we show that in the IL-6-responsive human multiple myeloma cell line KAS 6/1, the promoter region of p53 is epigenetically modified by methyltransferases, resulting in decreased levels of expression. Furthermore, cells treated with IL-6 exhibit an increase in the expression of the DNA maintenance methylation enzyme, DNMT-1. The DNA methyltransferase inhibitor zebularine reverses the methylation of the p53 promoter, allowing the resumption of its expression. However, when zebularine is withdrawn from the cells, the reestablishment of the original CpG island methylation within the p53 promoter does not occur in the absence of IL-6, and cells which do not receive IL-6 eventually die, as p53 expression continues unchecked by remethylation. Interestingly, this loss of viability seems to involve not the withdrawal of cytokine, but the inability of the cell to resilence the promoter. Consistent with this model, when cells that express IL-6 in an autocrine fashion are subjected to identical treatment, p53 expression is reduced shortly after withdrawal of zebularine. Therefore, it seems IL-6 is capable of maintaining promoter methylation thus representing one of the possible mechanisms used by inflammatory mediators in the growth and survival of tumors.

Microarray Analysis of Epigenetic Silencing of Gene Expression in the KAS-6/1 Multiple Myeloma Cell Line

The epigenetic control of gene transcription in cancer has been the theme of many recent studies and therapeutic approaches. Carcinogenesis is frequently associated with hypermethylation and consequent down-regulation of genes that prevent cancer, e.g., those that control cell proliferation and apoptosis. We used the demethylating drug zebularine to induce changes in DNA methylation, then examined patterns of gene expression using cDNA array analysis and Restriction Landmark Genomic Scanning followed by RNase protection assay and reverse transcription-PCR to confirm the results. Microarray studies revealed that many genes were epigenetically regulated by methylation. We concluded that methylation decreased the expression of, or silenced, several genes, contributing to the growth and survival of multiple myeloma cells. For example, a number of genes (BAD, BAK, BIK, and BAX) involved in apoptosis were found to be suppressed by methylation. Sequenced methylation-regulated DNA fragments identified by Restriction Landmark Genomic Scanning were found to contain CpG islands, and some corresponded to promoters of genes that were regulated by methylation. We also observed that after the removal of the demethylating drug, the addition of interleukin 6 restored CpG methylation and re-established previously silenced gene patterns, thus implicating a novel role of interleukin 6 in processes regulating epigenetic gene repression and carcinogenesis.

Zebularine: A Unique Molecule for an Epigenetically Based Strategy in Cancer Chemotherapy

1‐(β‐d‐ribofuranosyl)‐1,2‐dihydropyrimidin‐2‐one (zebularine) corresponds structurally to cytidine minus the exocyclic 4‐amino group. The increased electrophilic character of its simple aglycon endows the molecule with unique biologic properties as a potent inhibitor of both cytidine deaminase and DNA cytosine methyltransferase. The latter activity makes zebularine a promising antitumor agent that is hydrolytically stable, preferentially targets cancer cells, and shows activity both in vitro and in experimental animals, even after oral administration.

Phase I clinical trial of continuous infusion cyclopentenyl cytosine.

Cyclopentenyl cytosine (CPE-C) is an investigational drug that is active against human solid tumor xenografts. The 5′-triphosphate of CPE-C inhibits CTP synthase, and depletes CTP and dCTP pools. We conducted a phase I clinical trial of CPE-C given as a 24-h continuous i. v. infusion every 3 weeks in 26 adults with solid tumors. The starting dose rate, 1 mg/m2 per h, was selected on the basis of both preclinical studies and pharmacokinetic data from two patients obtained after a test dose of 24 mg/m2 CPE-C as an i. v. bolus. Dose escalation was guided by clinical toxicity. A total of 87 cycles were given, and ten patients received four or more cycles. The mean CPE-C steady-state plasma levels (Cpss) increased linearly from 0.4 μM to 3.1 μM at dose levels ranging from 1 to 5.9 mg/m2 per h (actual body weight); the mean total body clearance was 146±38 ml/min per m2. CPE-C was eliminated by both renal excretion of intact drug and deamination to cyclopentenyl uracil in an apparent 2∶1 ratio. CTP synthase activity in intact bone marrow mononuclear cells was inhibited by 58% to 100% at 22 h compared to matched pretreatment samples at all CPE-C dose levels. When all data were combined, flux through CTP synthase was decreased by 89.6%±3.1% at 22h (mean ± SE,n=16), and remained inhibited by 67.6%±7.7% (n=10) for at least 24 h post-CPE-C infusion. Granulocyte and platelet toxicities were dose-dependent, and dose-limiting myelosuppression occurred during the initial cycle in two of three patients treated with 5.9 mg/m2 per h. Four of 11 patients (4 of 20 cycles) who received 4.7 mg/m2 per h CPE-C experienced hypotension 24–48 h after completion of the CPE-C infusion during their first (n=2), third (n=1) and sixth cycles (n=1), respectively. Two of these patients died with refractory hypotension despite aggressive hydration and cardiopulmonary resuscitation. One of 12 patients (28 total cycles) treated with 3.5 mg/m2 per h CPE-C experienced orthostatic hypotension during cycle 1, and this patient had a second episode of orthostatic hypotension at a lower dose (3.0 mg/m2per h). Hypotension was not seen in patients receiving ≤2.5 mg/m2 per h CPE-C. The occurrence of hypotension did not directly correlate with either CPE-C Cpss, CPE-U plasma levels, pretreatment cytidine plasma levels, baseline CTP synthase activity, or with the degree of enzyme inhibition during treatment. While the hypotension appeared to be dose-related, its unpredictable occurrence and the uncertainty concerning the mechanism preclude a recommendation of a tolerable dose for future studies.

ZEBULARINE: A UNIQUE MOLECULE FOR AN EPIGENETICALLY BASED STRATEGY IN CANCER CHEMOTHERAPY. THE MAGIC OF ITS CHEMISTRY AND BIOLOGY

1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) is structurally 4-deamino cytidine. The increased electrophilic character of this simple aglycon endows the molecule with unique chemical and biological properties, making zebularine a versatile starting material for the synthesis of complex nucleosides and an effective inhibitor of cytidine deaminase and DNA cytosine methyltransferase. Zebularine is a stable, antitumor agent that preferentially targets cancer cells and shows activity both in vitro and in experimental animals, even after oral administration.

A Novel Side-Bridged Hybrid Phosphonate/Acetate Pendant Cyclam: Synthesis, Characterization, and 64Cu Small Animal PET Imaging

Copper-64 (t(1/2)=12.7h; beta(+): 0.653 MeV, 17.4%; beta(-): 0.578 MeV, 39%) is produced in a biomedical cyclotron and has applications in both imaging and therapy. Macrocyclic chelators are widely used as bifunctional chelators to bind copper radionuclides to antibodies and peptides owing to their relatively high kinetic stability. A novel side-bridged cyclam featuring both pendant acetate and phosphonate groups was synthesized using a Kabachnik-Fields approach followed by hydrobromic acid deprotection. The Cu(II) complex of the novel ligand was synthesized, radiolabeling with (64)Cu was demonstrated, and in vitro (serum) stability was performed. In addition, in vivo distribution and clearance of the (64)Cu-labeled complex was visualized by positron emission tomography (PET) imaging. This novel chelate may be useful in (64)Cu-mediated diagnostic positron emission tomography (PET) imaging as well as targeted radiotherapeutic applications.

A Rapid Microscale Method for the Determination of Partition Coefficients by HPLC

Abstract A rapid, reliable and simple microscale method for the determination of octanol-water partition coefficients has been developed and evaluated. Rapid solute partitioning and facile octanol-water phase separation is accomplished in a commercially available mixer-separator device. The relative concentration of solute in each phase is then measured directly by computer-based reverse phase HPLC. The procedure requires only 10 μg of sample, which need not be pure, and uses 1 ml or less of both n-octanol and pH 7.0 phosphate buffer. Log P values of 26 compounds, mainly nucleoside analogues with anti-HIV and antitumor activity, have been determined in the range 0.7 to -2.4 with a precision better than ±0.04 log units. For compounds with literature data available, measured log P values are in good agreement (better than ±0.2 log units) with those values obtained by traditional “shake-flask” methodology. The described method is applicable to both single compound analysis and simultaneous multiple compound ...

Diaziquone, 2,5‐diaziridinyl‐3,6‐biscarboethoxyamino‐1,4‐benzoquinone, plasma and cerebrospinal fluid kinetics

Plasma and cerebrospinal fluid (CSF) kinetics of diaziquone, 2,5‐diaziridinyl‐3,6‐biscarboethoxyamino‐1,4‐benzoquinone (AZQ), were evaluated after intravenous injection in patients with implanted Ommaya reservoirs. After a 30‐min infusion plasma disappearance was very rapid, with a disposition half‐life of 2 to 6 min and an elimination half‐life of 25 to 35 min. Area under the concentration‐time curve for CSF was 22% to 42% of the corresponding plasma area. Based upon total plasma AZQ concentration, volume of distribution for AZQ was 2.0 to 10.0 l/m2. Plasma binding of AZQ was 79% in one normal subject. When the effect of plasma binding is considered, distribution volumes are more plausible and it appears that free plasma AZQ is completely available to the CSF.

Metabolites of alanosine, an antitumor antibiotic

Abstract The metabolism of alanosine. dl -2-amino-3-( N -hydroxy, N -nitrosamino) propionic acid (NSC-143647), a new antitumor antibiotic, was studied in mice, rats, monkeys and dogs. Urine is the principal excretory vehicle for the drug in these four species. In rats, unchanged alanosine is the principal excretory product. In the other species, a second, more acidic component accounts for the major fraction of the drug-derived radioactivity in urine; this product retains the characteristic u.v. spectrum and both carbons, 1 and 3, of alanosine: it is chromatographically and spectrally indistinguishable from the compound generated by the action of NADH and malate dehydrogenase on the product resulting from the incubation of l -alanosine with l -glutamate oxaloacetate transaminase (GOT) (EC 2.6.1.1) and α-ketoglutarate. On the basis of this evidence, this metabolite is concluded to be the α-hydroxy counterpart of l -alanosine. The antibiotic was susceptible to transamination in vitro by extract of organs rich in GOT; heart was pre-eminent in this regard, and α-ketoglutaric acid was found to be the preferred α-keto partner in the reaction. Crystalline GOT catalyzed an identical reaction in vitro , and the product, like oxaloacetic acid, was susceptible to enzymatic condensation with acetyl CoA, in the presence of citrate synthase. Inability to detect the -α- keto analogue of alanosine. 2-oxo-3-( N -hydroxy, N -nitrosamino) propionic acid, in tissues and excreta is attributed to the facile decomposition of this metabolite in vivo . In vitro , alanosine was susceptible to decarboxylation by homogenates of mouse brain and by purified l -glutamate decarboxylase (EC 4.1.1.15) from Escherichia coli . No evidence could be adduced for denitrosation of the antibiotic nor for reduction of the nitroso function in a system containing hepatic microsomes and NADPH. However, l -amino acid oxidase (EC 1.4.3.2) and high concentrations of pyridoxal phosphate catalyzed the deamination of alanosine at alkaline pH. In confirmation of the observations of Hurlbert et al , ¦ Proc. Am. Ass. Cancer Res. 18 , 234,(1977)¦. alanosine was found to be used by phosphoribosylaminoimi-dazole-succinocarboxamide synthetase (EC 6.3.2.6) as a fraudulent substrate. Also observed was the condensation of alanosine with IMP. catalyzed by a partially purified preparation of adenylosuccinate synthetase (EC 6.3.4.4) from rabbit muscle; this anabolite exhibited chromatographic properties quite similar to adenylosuccinic acid. Inasmuch as a substantial percentage of the administered dose of alanosine was found to associate with carcass-macromolecules for protracted periods, attempts were made to determine the basis for this fate. Equivalent labeling was produced irrespective of whether dl -[1- 14 C] or dl -[3- 14 C] alanosine was the injectate, so that reutilization of metabolically generated [ 14 C]O 2 is not likely to explain macromolecular retention of the antibiotic. In vitro , no esterification of alanosine to tRNA was observed, but the drug did bind to tRNA in an ATP-independent reaction. In vivo , ten times more dl -[3- 14 C] alanosine was incorporated into the hemoglobin of animals recovering from phenylhydrazine anemia than was observed in their saline-treated counterparts. Isolated reticulocytes incorporated only minor amounts of purified dl -[I- 14 C] alanosine into their molecules; this process was insensitive to inhibition by cycloheximide. In vitro , alanosine (used in lieu of l -aspartic acid) neither supported nor inhibited globin synthesis by rabbit reticulocyte lysates. These results leave unsettled the question of whether macromolecular association of alanosine reflects incorporation or affiliation.

Potent Inhibition of HhaI DNA Methylase by the Aglycon of 2-(1H)-Pyrimidinone Riboside (Zebularine) at the GCGC Recognition Domain

Abstract: A short oligodeoxynucleotide (ODN) with 2‐(1H)‐pyrimidinone at the HhaI DNA methyltransferase target site (GCGC) is shown to induce a level of inhibition of methyl transfer and thermal stability of the complex with the enzyme identical to that achieved with a similar ODN substituted with 5‐azacytosine. The drugs responsible for these effects—zebularine and 5‐azacytidine/2′‐deoxy‐5‐azacytidine—are contrasted in terms of chemical stability and possible metabolic activation by a brief structure‐activity analysis.