Deoxycytosine methylation within CpG islands of tumor suppressor genes plays a prominent role in the development and progression of drug-resistant ovarian cancer. Consequently, epigenetic therapies directed toward tumor suppressor demethylation/reexpression could potentially reverse malignant phenotypes and chemosensitize recalcitrant tumors. In this report, we examined the demethylating agent zebularine [1-(B-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one], in comparison with the well-known methylation inhibitor 5-aza-2V -deoxycytidine (5-aza-dC), for its ability to inhibit ovarian cancer cell proliferation and to demethylate and induce tumor suppressor genes. Zebularine exerted significant (>5-aza-dC) antiproliferative effects against the ovarian cancer cell lines Hey, A2780, and the cisplatin-resistant A2780/CP in a dose-dependent manner (65% versus 35% inhibition at 48 hours, zebularine versus 5-aza-dC). Moreover, 48-hour treatment with 0.2 mmol/L zebularine significantly induced demethylation of the tumor suppressors ras-associated domain family 1A and human MutL homologue-1. RASSF1A gene reexpression was also observed, as was reexpression of two other tumor suppressors, ARHI and BLU, although levels differed from those induced by 5-aza-dC. Global analyses of DNA methylation revealed similar overall demethylation (2.5-to 3-fold) by 5-aza-dC and zebularine as determined by methyl acceptance assay. However, differences in demethylation of individual loci were observed as determined by differential methylation hybridization. Finally, we found that zebularine could resensitize the drugresistant cell line A2780/CP to cisplatin, with a 16-fold reduction in the IC 50 of that conventional agent. In summary, zebularine seems to be a promising clinical candidate, singly or combined with conventional regimens, for the therapy of drug-resistant ovarian cancer.

We have determined the mutational spectrum of N-ethyl-N-nitrosourea (ENU) in exon 3 of the hypoxanthine guanine) phosphoribosyltransferase gene (Hprt) in splenic T cells following in vivo exposure of male B6C3F1 mice (5-7 weeks old) to ENU. Hpir mutants were isolated by culturing splenic T cells in microtiter dishes containing medium supplemented with interleukin 2, concanavalin A, and 6-thiouanine. DNA was extracted from 6-thoa ne-sistant colonies and amplified by the polymerase chain reaction (PCR) using primers flanking Hprt exon 3. Identification of mutant sequences and purification of mutant DNA from contaminating wild-type Hprt DNA was accomplished by denaturing-adient gel electrophoresis. Purified mutant DNA was then sequenced. Treatment of mice with ENU at 40 mg/kg of body weight produced a Hprr mutant frequency of 7.3 x 10-5 in splenic T cells, =35-fold above background levels. Sixty-nine of the 521 HprF mutants analyzed contained mutations in exon 3 (13%). Tranversions and transitions at AFT base pais dominated the spectrum; 62 of the 69 exon 3 mutations were at AKT base pairs (14 different sites). Thirteen of 14 thymine bases undergoing mutation (61 of 62 mutations at AFT bases) were located on the nontranscribed stand of exon 3. The majority of the remaining mutations (6 of 69) were transitions at a single G'C base -i. These results suggest the importance of thymidine alkylation in ENU-induced mutagenesis in vivo. The mouse HpW-T-cell cloning/sequencing assay described here may represent a useful system for studying the molecular mechanism of chemically induced mutation occurring in vivo in an endogenous gene.Chemically induced mutation in vivo is the end result of a complicated cascade of events including compound uptake and distribution, metabolic activation/detoxification, compound interaction with DNA, DNA repair, and cell replication. The complexity of this pathway suggests that in vivo mutagenesis assays may be more relevant than in vitro systems for modeling possible mutagenic consequences in humans. In vivo mutation assays based on the cloning of hypoxanthine (guanine) phosphoribosyltransferase (HPRT)-negative T cells have been developed in the mouse (1-3), rat (4), monkey (5), and human (6, 7). Recently, transgenic mouse systems have also been described that contain bacterial transgenes as mutational targets (8, 9). These systems provide excellent opportunities to quantitate and compare the type and frequency of mutations occurring in vivo in different sequence contexts.The type of base-pair changes produced in DNA can offer important insight regarding the specific DNA adducts and mutagenic mechanisms involved in the mutagenic event. To this end, our group has developed a method to sequence Hprt mutations induced in vivo in splenic T cells of B6C3F1 mice, the strain presently used in the National Toxicology Program cancer bioassays. The approach utilizes denaturing-gradient gel electrophoresis (DGGE) (10-12) to purify mutant sequences for analysis. DGGE is based on the fact that the ...

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Cytokine production has been implicated in the antiviral response to interferon-alpha (IFN-alpha) in hepatitis C and in the development of IFN-alpha-related side effects. We characterized acute changes in serum cytokine levels following administration of a single dose of consensus IFN (IFN-con1) and during continuous treatment of chronic hepatitis C patients. Serum samples were collected at baseline, at multiple times early after IFN administration, and weekly thereafter. Viral RNA titers were assessed by RT-PCR, and viral kinetics were followed. ELISA assays were used to measure IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-4, IL-6, and IL-16. Serum cytokine levels were low at baseline. IL-6 was detected in patients with hepatitis C but not in healthy control subjects by either ELISA or RT-PCR, indicating that low levels of circulating IL-6 were associated with hepatitis C infection. None of the cytokines measured increased significantly after IFN administration except for IL-6. IL-6 levels rose rapidly, peaked at 6-15 h in a dose-dependent manner, and returned to baseline by 48 h in both patients receiving a single dose of IFN and those receiving continuous treatment. This was confirmed by RT-PCR. Pretreatment IL-6 levels were directly correlated with area under the curve (AUC) for IL-6 during the 24 h after IFN dosing (r = 0.611, p = 0.007). Viral titers decreased within 24-48 h after a single dose of IFN-con1. Changes in hepatitis C RNA titers were not significantly associated with pretreatment IL-6 levels or with changes in IL-6 levels. In conclusion, (1) baseline serum cytokine levels, except for IL-6, were low or within the normal range in patients with hepatitis C, (2) IL-6 levels were detected in some patients with hepatitis C before treatment but not in healthy controls, (3) IL-6 levels increased acutely after a single dose of IFN-alpha, and IL-6 induction was related to baseline IL-6 level, and (4) changes in IL-6 levels did not correlate with the early virologic response to IFN.

Precipitation of calcium phosphate in neonatal total parenteral nutrition (TPN) solutions remains a significant problem. Whereas numerous studies have attempted to establish guidelines for maximum concentrations of various combinations that can be mixed, differences in study design and reliance upon subjective visual assessment severely limit their applicability. The purpose of this study was to quantitatively determine calcium and phosphate compatibility in commonly used neonatal TPN solutions containing a final concentration of either 1 or 2% amino acids. The final dextrose concentration was 10%. Electrolytes, heparin, and pediatric vitamins and trace minerals were also added. Calcium gluconate (10%) and potassium phosphate (mono and dibasic) were added by calibrated micropipetors. Calcium concentrations ranged from 5 to 60 mEq/L and phosphate from 5 to 40 mM/L with a minimum of 84 combinations tested for each amino acid concentration. Calcium concentrations were measured in duplicate for each tested combination. Control solutions containing calcium but no phosphate were included to validate the assay methodology. All samples were stored at room temperature for 23.5 hours and then placed in a water bath at 37 degrees C for 30 minutes to simulate incubator conditions encountered during TPN infusion. Calcium determinations were then repeated and precipitation was judged to have occurred whenever calcium concentrations fell below 90% of the initial measured values. These data allowed plotting a calcium and phosphorus reference curve for TPN solutions containing 1 and 2% amino acids based on quantitative assessment. These reference curves should allow pharmacists to avoid compounding TPN solutions that will precipitate, thus saving considerable cost to the pharmacy and preventing complications.