Synthetic Studies on Formamidopyrimidines Related to Clofarabine

Degradation of clofarabine (3) in 0.9% saline solution at 100°C afforded three degradation products which were determined to be formamidopyrimidines 4–6.Compounds 4 and 5 were assigned as C_1′ anomers on the basis of one-dimensional and two-dimensional NMR experiments, whereas 6 was found to be the formamidopyrimidine lacking the sugar moiety. An improved procedure for the synthesis of formamidopyrimidines was developed, wherein benzoylated clofarabine (11) was treated with allyl chloroformate, followed by deprotection of the alloc group with catalytic Pd(PPh₃)₄ and dimedone. A synthesis of compound 6 from 4 is also described.     

Synthesis and biological activity of 4'-C-hydroxymethyl-2'-fluro-D-arabinofuranosylpurine nucleosides

A series of 4'-C-hydroxymethyl-2'-fluoro-D-arabinofuranosylpurine nucleosides was prepared and evaluated for cytotoxicity. The details of a convenient synthesis of the carbohydrate precursor 4-C-hydroxymethyl-3,5-di-O-benzoyl-2-fluoro-alpha-D-arabinofuranosyl bromide (13) are presented. Proof of the structure and configuration at all chiral centers of the sugars and the nucleosides were obtained by proton NMR. All five target nucleosides were evaluated for cytotoxicity in human tumor cell lines. The 4'-C-hydroxymethyl clofarabine analogue (16beta) showed slight cytotoxicity in CCRF-CEM leukemia cells.     

Clofarabine in adult acute leukemias: clinical success and pharmacokinetics

Clofarabine is a deoxyadenosine analog synthesized with the intention of retaining the favorable mechanistic properties of fludarabine and cladribine while eliminating their undesirable characteristics. Phase I studies among 32 patients with acute leukemia defined a maximum tolerated dose (MTD) of 40 mg/m2/d given as a one hour infusion daily for 5 days. The dose limiting toxicity (DLT) was transient hepatotoxicity. In a phase II study, 62 patients with acute leukemias received clofarabine at the MTD over 1 hour daily for 5 days. Twenty patients (32%) achieved complete response (CR), 1 had a partial response (PR), and 9 had a CR but without platelet recovery (CRp), for an overall response rate of 48%. Pharmacokinetic studies in the phase I trial revealed marked heterogeneity in peak levels of clofarabine among patients at the end of infusion, however; there was a linear, dose dependent increase in clofarabine concentration in the plasma. Pharmacodynamically, at the MTD, DNA synthesis was inhibited by more than 80% at the end of infusion. In phase II studies, the relationship between the pharmacokinetics of clofarabine triphosphate accumulation and clinical response at the MTD was explored, revealing an accumulation advantage of the cytotoxic triphosphate in leukemia cells of responders. The circulating leukemia blasts of patients who respond to clofarabine therapy exhibited a favorable pharmacokinetic profile. In conclusion, clofarabine is an active agent in the treatment of acute leukemias and MDS, and cellular pharmacokinetics has prognostic significance.     

Clofarabine in Adult Acute Leukemias: Clinical Success and Pharmacokinetics

Clofarabine is a deoxyadenosine analog synthesized with the intention of retaining the favorable mechanistic properties of fludarabine and cladribine while eliminating their undesirable characteristics. Phase I studies among 32 patients with acute leukemia defined a maximum tolerated dose (MTD) of 40 mg/m²/d given as a one hour infusion daily for 5 days. The dose limiting toxicity (DLT) was transient hepatotoxicity. In a phase II study, 62 patients with acute leukemias received clofarabine at the MTD over 1 hour daily for 5 days. Twenty patients (32%) achieved complete response (CR), 1 had a partial response (PR), and 9 had a CR but without platelet recovery (CRp), for an overall response rate of 48%. Pharmacokinetic studies in the phase I trial revealed marked heterogeneity in peak levels of clofarabine among patients at the end of infusion, however; there was a linear, dose dependent increase in clofarabine concentration in the plasma. Pharmacodynamically, at the MTD, DNA synthesis was inhibited by more than 80% at the end of infusion. In phase II studies, the relationship between the pharmacokinetics of clofarabine triphosphate accumulation and clinical response at the MTD was explored, revealing an accumulation advantage of the cytotoxic triphosphate in leukemia cells of responders. The circulating leukemia blasts of patients who respond to clofarabine therapy exhibited a favorable pharmacokinetic profile. In conclusion, clofarabine is an active agent in the treatment of acute leukemias and MDS, and cellular pharmacokinetics has prognostic significance.     

Progesterone and RU486 regulation of uterine complement C3 after prior induction with estradiol.

Previous results demonstrated that progesterone (P4) given simultaneously with estradiol (E2) prevented stimulation by E2 of complement C3 expression in the immature rat uterus. Northern blot analysis revealed that simultaneous administration of P4 was able to prevent the E2-stimulated increase in C3 mRNA concentration in the luminal epithelial cells. The purpose of the present investigation was to determine whether progesterone modulates C3 expression after the gene has been induced by prior administration of E2 and also to determine the reversibility of this effect by the concomitant administration of RU38486, 17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]estra-4,9,-dien-3-one (RU486). This regulation was studied by examination of protein synthesis as well as mRNA concentrations. Immature 21-day-old female rats were treated with E2 for 2 days (1 microgram/day), followed 24 h later by P4 (500 micrograms) or vehicle. Uteri were removed 6, 9, and 18 h after progesterone treatment and the radiolabeled secreted proteins were analyzed by SDS-PAGE and immunoprecipitation using a goat anti-rat C3 antibody. In animals treated with vehicle, E2-stimulated C3 synthesis remained elevated at 6 and 9 h and returned to control values by 18 h. In contrast, the administration of P4 resulted in a decrease in C3 synthesis at 6 and 9 h with the greatest decrease observed at 9 h. Similar results were obtained when C3 mRNA concentrations were examined. E2-stimulated C3 mRNA concentrations were decreased in rats treated with progesterone compared to those treated with vehicle alone.2     

Exclusive leukotriene C4 synthesis by purified human eosinophils induced by opsonized zymosan

Purified human eosinophils were challenged with N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, platelet-activating-factor, valyl-glycyl-seryl-glutamic acid, phorbol myristate acetate, zymosan, opsonized zymosan and the calcium ionophore A23187 to induce leukotriene synthesis. Reversed-phase high performance liquid chromatography analysis demonstrated the almost exclusive synthesis of leukotriene C4 by eosinophils of 11 healthy donors after challenge with opsonized zymosan [(22 +/- 4) X 10(6) molecules LTC4/cell, mean +/- SE] or the calcium ionophore A23187 [(54 +/- 7) X 10(6) molecules LTC4/cell, mean +/- SE]. The other agents were not capable of inducing leukotriene formation. When in addition to opsonized zymosan N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor were added a significant increase of the leukotriene C4 synthesis by eosinophils was observed. These results suggest that eosinophils might be triggered to produce considerable amounts of the spasmogenic leukotriene C4 in vivo by C3b- and/or IgG-mediated mechanisms e.g. phagocytosis.     

Effects of dietary lipids on behaviour, lipid biosynthesis and lipid composition, in rat platelets

Rats of either sex were fed for 18 and 34 weeks respectively diets containing 40% (by weight) lipids with polyunsaturated fatty acids representing 1.34% or 13.2% of total calories. Platelet reactivity to thrombin, platelet fatty acid composition and incorporation of [14C]acetate into platelet lipids were investigated. Diets rich in saturated fatty acids markedly increased platelet sensitivity to thrombin. The concentration of 20:3 and 22:3 of the (n - 9) series and of 20:3 and 22:5 of the (n - 6) series were increased at the expense of 18:2 and 22:4 of the (n - 6) family in platelet lipids. 20:4 (n - 6) was unchanged. The fatty acid changes were more pronounced in male rats and after 34 weeks. [14C]Acetate incorporation into total platelet lipids and particularly into choline phosphoglycerides and ceramides was lower in animals fed saturated fats. This diet reduced the synthesis of 16:0 and of 22:4(n - 6) in platelet total fatty acids, while that of 22:3(n - 9) was markedly enhanced. This study showed that long-term feeding of high-saturated-low-polyunsaturated fat diets in rats induced marked changes in platelet lipid synthesis and composition, in both sexes. The lipid synthesis modification appears to be more pronounced in males than in females. The changes in the fatty acids 20:3(n - 9), 22:3(n - 9) and 22:4(n - 6) appeared to be closely related to platelet behaviour. The balance between the content and synthesis of these last fatty acids might be of significance for the effect of diet on thrombogenesis.     

Preparation of partially 2H/13C-labelled RNA for NMR studies. Stereo-specific deuteration of the H5" in nucleotides

An effective in vitro enzymatic synthesis is described for the production of nucleoside triphosphates (NTPs) which are stereo-specifically deuterated on the H5" position with high selectivity (>98%), and which can have a variety of different labels (13C, 15N, 2H) in other positions. The NTPs can subsequently be employed in the enzymatic synthesis of RNAs using T7 polymerase from a DNA template. The stereo-specific deuteration of the H5" immediately provides the stereo-specific assignment of H5' resonances in NMR spectra, giving access to important structural parameters. Stereo-chemical H-exchange was used to convert commercially available 1,2,3,4,5,6,6-2H-1,2,3,4,5,6-13C-D-glucose (d7-13C6-D-glucose) into [1,2,3,4,5,6(R)-2H-1,2,3,4,5,6-13C]-D-glucose (d6-13C6-D-glucose). [1',3',4',5"-2H-1',2',3',4',5'-13C]GTP (d4-13C5-GTP) was then produced from d6-13C6-D-glucose and guanine base via in vitro enzymatic synthesis employing enzymes from the pentose-phosphate, nucleotide biosynthesis and salvage pathways. The overall yield was approximately 60 mg NTP per 1 g glucose, comparable with the yield of NTPs isolated from Escherichia coli grown on enriched media. The d4-13C5-GTP, together with in vitro synthesised d5-UTP, d5-CTP and non-labelled ATP, were used in the synthesis of a 31 nt RNA derived from the primer binding site of hepatitis B virus genomic RNA. (13C,1H) hetero-nuclear multiple-quantum spectra of the specifically deuterated sample and of a non-deuterated uniformly 13C/15N-labelled sample demonstrates the reduced spectral crowding and line width narrowing compared with 13C-labelled non-deuterated RNA.     

The biosynthesis in vitro of chondroitin sulphate in neonatal rat epiphysial cartilage

1. A system is described, which was used to incubate neonatal rat epiphysial cartilage in vitro with [U-(14)C]glucose and [(35)S]sulphate. 2. The acid glycosaminoglycans of neonatal rat epiphyses were extracted and fractionated on cetylpyridinium chloride-cellulose columns. The major components were chondroitin 4-sulphate (65%), chondroitin 6-sulphate (15%), hyaluronic acid (4%) and keratan sulphate (2%). 3. The acid-soluble nucleotides and intermediates of glycosaminoglycan synthesis were separated on a Dowex 1 (formate) system. The tissue contents and cellular concentrations of these metabolites were determined. 4. The rates of synthesis of UDP-glucuronic acid and UDP-N-acetyl-hexosamine from [U-(14)C]glucose were found to be 0.79+/-0.16 and 3.2+/-0.08nmol/min per g wet wt. respectively. 5. The incorporation of [U-(14)C]glucose into the uronic acid and hexosamine moieties of the polymers was also measured and the turnover rates of the glycosaminoglycans were calculated. It was found that chondroitin sulphate was turning over in about 70h and hyaluronic acid in about 120h. 6. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from [(35)S]sulphate incorporation and were found to be in good agreement with those obtained from [U-(14)C]glucose labelling.     

Protein synthesis and degradation in isolated muscle. Effect of omega 3 and omega 6 fatty acids.

The ability of derivatives of the essential fatty acids linoleic acid (C18:2, omega 6) and alpha-linolenic acid (C18:3, omega 3) to stimulate rates of protein synthesis and degradation was investigated in isolated intact muscles from fasted rabbits. Both omega 6 derivatives examined, arachidonic acid (C20:4, omega 6) and dihomo-gamma-linolenic acid (C20:3, omega 6), when added at concentrations up to 1 microM, stimulated the rate of protein synthesis and the release of prostaglandin F2 alpha (PGF2 alpha). Metabolites of the omega 6 series, namely eicosapentaenoic acid (C20:5, omega 3) and docosahexaenoic acid (C22:6, omega 3), were without effect on the rate of protein synthesis and resulted in a decrease in the release of PGF2 alpha. None of the fatty acids had a significant effect on the rate of protein degradation. Although insulin (100 mu units/ml) also stimulated rates of protein synthesis when added alone, none of the omega 3 or omega 6 fatty acids, when added with insulin at concentrations of 0.2 microM, potentiated the effect of the hormone.     

synthesis of progesterone and prostaglandin F by luteal tissue and prostaglandin F by endometrial tissue from the pig

The relationship between progesterone (P₄) synthesis by luteal tissue and prostaglandin F (PGF) synthesis by endometrium and luteal tissue from two stages of the cycle, Days 7 to 8 and 15 to 16, was determined. Luteal and endometrial tissues were collected from pigs in three experimental groups at two stages of the cycle: (A) 6 pigs on Days 7 to 8 with spontaneous, 5 to 6 day old corpora lutea (CL); (B) 5 pigs on Days 15 to 16 with spontaneous, 13 to 14 day old CL; and (C) 6 pigs on Days 15 to 16 with spontaneous, 13 to 14 day old CL and 5 to 6 day old CL induced by pregnant mares serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) injections. Pigs with spontaneous, 13 to 14 day old CL of the cycle and PMSG-HCG induced accessory, 5 to 6 day old CL were used so that P₄ and PGF synthesis in tissue from old and new CL could be compared in the same pig on Day 15 to 16 of the cycle. Tissues (100 mg minces) were incubated in 5 ml of Krebs Ringer solution in an atmosphere of 95% 0₂:5% CO₂ for 2 hours at 0° C, 37° C, or 37° C with 1.3 x 10⁻⁴M indomethacin (IND). An aliquot of the incubation medium and an aliquot of the supernatant after homogenization of the tissue in the remaining medium of each flask was quantified for P₄ and PGF by radioimmunoassay. P₄ and PGF release into the medium and total accumulation of P₄ and PGF in the flasks indicated that synthesis had occured at 37° C. Compared to tissue from 13 to 14 day old CL, tissue from 5 to 6 day old CL synthesized more P₄ per flask (53.9 25.0 ng/mg tissue, P<.001) and released more P₄ into the medium (20.8 8.8 ng/mg, P<.001). P₄ synthesis by luteal tissue from 5 to 6 day old and 13 to 14 day old CL from pigs in group C was similar to P₄ synthesis by luteal tissue from pigs in group A and group B, respectively. Luteul PGF synthesis was not affected significantly by either the age of the CL or by PMSG-HCG treatment. For endometrial samples, the synthesis of PGF was not significantly different among pigs in groups A, B and C. If uterine PGF is involved in luteal regression in the pig, the sensitivity of the CL to PGF may be more important than an increase in PGF secretion during the late luteal phase of the estrous cycle.     

Association of poly(ADP-rib) synthesis with cessation of DNA synthesis and DNA fragmentation

CHO cells and cs-4-D3 cells were used to investigate the association between poly(ADP-rib) synthesis and the cessation of DNA synthesis and DNA fragmentation. The cs4-D3 cells are cold-sensitive DNA synthesis arrest mutants of CHO cells. Upon incubation at 33 degrees C, DNA synthesis in the cs4-D3 cells stops and the cells enter a prolonged G1 or G0 phase. The events that occurred when cs4 cells were incubated at 33 degrees C were similar to those that occurred when wild-type CHO cells grew to high density. (1) In both cases, DNA synthesis and cell growth stopped. (2) The NAD+ concentration/cell was 20-25% lower in growth-arrested cells than in logarithmically growing cells. (3) Poly(ADP-rib) synthesis was 3-4 fold higher in growth-arrested cells than in logarithmically growing cells. (4) The growth-inhibited cells developed DNA strand breaks which resulted in large percentages of their DNA appearing in the low molecular weight range of alkaline sucrose gradients. (5) Both the increased rate of poly(ADP-rib) synthesis and the development of DNA strand breaks appears to be characteristic of the G1 phase of the cell cycle. (6) When growth-inhibited cells were restored to conditions favorable for DNA synthesis and cell growth, the DNA strand breaks were repaired. (7) Prolonged incubation under growth-restrictive conditions resulted in the accumulation of more DNA strand breaks than the cells could repair. This was followed by cell death when the cells were restored to conditions favorable for cell growth.     

SYNTHESIS AND BIOLOGICAL ACTIVITY OF 4′-C-HYDROXYMETHYL-2′-FLUORO- D-ARABINOFURANOSYLPURINE NUCLEOSIDES

A series of 4′-C-hydroxymethyl-2′-fluoro-D-arabinofuranosylpurine nucleosides was prepared and evaluated for cytotoxicity. The details of a convenient synthesis of the carbohydrate precursor 4-C-hydroxymethyl-3,5-di-O-benzoyl-2-fluoro-α-D-arabinofuranosyl bromide (13) are presented. Proof of the structure and configuration at all chiral centers of the sugars and the nucleosides were obtained by proton NMR. All five target nucleosides were evaluated for cytotoxicity in human tumor cell lines. The 4′-C-hydroxymethyl clofarabine analogue (16β) showed slight cytotoxicity in CCRF-CEM leukemia cells.     

Effect of diets enriched in Delta6 desaturated fatty acids (18:3n-6 and 18:4n-3), on growth, fatty acid composition and highly unsaturated fatty acid synthesis in two populations of Arctic charr (Salvelinus alpinus L.)

This study aimed to test the hypothesis that diets containing relatively high amounts of the Delta6 desaturated fatty acids stearidonic acid (STA, 18:4n-3) and gamma-linolenic acid (GLA, 18:3n-6), may be beneficial in salmonid culture. The rationale being that STA and GLA would be better substrates for highly unsaturated fatty acid (HUFA) synthesis as their conversion does not require the activity of the reputed rate-limiting enzyme, fatty acid Delta6 desaturase. Duplicate groups of two Arctic charr (Salvelinus alpinus L.) populations with different feeding habits, that had been reported previously to show differences in HUFA biosynthetic capacity, were fed for 16 weeks on two fish meal based diets containing 47% protein and 21% lipid differing only in the added lipid component, which was either fish oil (FO) or echium oil (EO). Dietary EO had no detrimental effect on growth performance and feed efficiency, mortalities, or liver and flesh lipid contents in either population. The proportions of 18:2n-6, 18:3n-3, 18:3n-6, 18:4n-3, 20:3n-6 and 20:4n-3 in total lipid in both liver and flesh were increased by dietary EO in both populations. However, the percentages of 20:5n-3 and 22:6n-3 were reduced by EO in both liver and flesh in both strains, whereas 20:4n-6 was only significantly reduced in flesh. In fish fed FO, HUFA synthesis from both [1-(14)C]18:3n-3 and [1-(14)C]20:5n-3 was significantly higher in the planktonivorous Coulin charr compared to the demersal, piscivorous Rannoch charr morph. However, HUFA synthesis was increased by EO in Rannoch charr, but not in Coulin charr. In conclusion, dietary EO had differential effects in the two populations of charr, with HUFA synthesis only stimulated by EO in the piscivorous Rannoch morph, which showed lower activities in fish fed FO. However, the hypothesis was not proved as, irrespective of the activity of the HUFA synthesis pathway in either population, feeding EO resulted in decreased tissue levels of n-3HUFA and 20:4n-6. This has been observed previously in salmonids fed vegetable oils, and thus the increased levels of Delta6 desaturated fatty acids in EO did not effectively compensate for the lack of dietary HUFA.     

ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3

We hypothesized that reduction/loss of very long chain PUFAs (VLC-PUFAs) due to mutations in the ELOngase of very long chain fatty acid-4 (ELOVL4) protein contributes to retinal degeneration in autosomal dominant Stargardt-like macular dystrophy (STGD3) and age-related macular degeneration; hence, increasing VLC-PUFA in the retina of these patients could provide some therapeutic benefits. Thus, we tested the efficiency of elongation of C20-C22 PUFA by the ELOVL4 protein to determine which substrates are the best precursors for biosynthesis of VLC-PUFA. The ELOVL4 protein was expressed in pheochromocytoma cells, while green fluorescent protein-expressing and nontransduced cells served as controls. The cells were treated with 20:5n3, 22:6n3, and 20:4n6, either individually or in equal combinations. Both transduced and control cells internalized and elongated the supplemented FAs to C22-C26 precursors. Only ELOVL4-expressing cells synthesized C28-C38 VLC-PUFA from these precursors. In general, 20:5n3 was more efficiently elongated to VLC-PUFA in the ELOVL4-expressing cells, regardless of whether it was in combination with 22:6n3 or with 20:4n6. In each FA treatment group, C34 and C36 VLC-PUFAs were the predominant VLC-PUFAs in the ELOVL4-expressing cells. In summary, 20:5n3, followed by 20:4n6, seems to be the best precursor for boosting the synthesis of VLC-PUFA by ELOVL4 protein.     

Semliki Forest virus capsid protein acts as a pleiotropic regulator of host cellular protein synthesis

The Semliki Forest virus capsid (C) protein was introduced into various target cells by electroporation-, liposome-, and erythrocyte-ghost-mediated delivery. Data are presented which show that the incorporated C protein is biologically active and, at low concentrations (10(3) to 10(4) molecules per cell), markedly induces host cellular protein synthesis (average value, up to 90%). On the other hand, high concentrations (10(5) to 10(6) molecules per cell) led to a significant inhibition (average value, up to 60%). The cellular response to C protein was found to be identical in P3X63Ag8 suspension cells, CV-1 cells, and GpBind4 cells. Following electroporation-mediated delivery of C-protein molecules, both induction and repression of cellular protein synthesis were immediate, whereas with liposome-mediated delivery these events were delayed by about 1 h. Maximum stimulation and repression occurred between 0 and 1 h after delivery of C protein and decreased thereafter to reach control values at about 4 h. The analysis of the proteins synthesized suggests that low amounts of microinjected C protein are responsible for the induction of classes with specific Mrs, whereas high amounts lead to an inhibition of overall protein synthesis.     

Kaurenolides and fujenoic acids are side products of the gibberellin P450-1 monooxygenase in Gibberella fujikuroi

The steps involved in kaurenolide and fujenoic acids biosynthesis, from ent-kauradienoic acid and ent-6alpha,7alpha-dihydroxykaurenoic acid, respectively, are demonstrated in the gibberellin (GA)-deficient Gibberella fujikuroi mutant SG139, which lacks the entire GA-biosynthesis gene cluster, complemented with the P450-1 gene of GA biosynthesis (SG139-P450-1). ent-[2H]Kauradienoic acid was efficiently converted into 7beta-hydroxy[2H]kaurenolide and 7beta,18-dihydroxy[2H]kaurenolide by the cultures while 7beta-hydroxy[2H]kaurenolide was transformed into 7beta,18-dihydroxy[2H]kaurenolide. The limiting step was found to be hydroxylation at C-18. In addition, SG139-P450-1 transformed ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid into [14C4]fujenoic acid and [14C4]fujenoic triacid. Fujenal was also converted into the same products but was demonstrated not to be an intermediate in this sequence. All the above reactions were absent in the mutant SG139 and were suppressed in the wild-type strain ACC917 by disruption of the P450-1 gene. Kaurenolide and fujenoic acids synthesis were associated with the microsomal fraction and showed an absolute requirement for NADPH or NADH, all properties of cytochrome P450 monooxygenases. Only 7beta-hydroxy[14C4]kaurenolide synthesis and not further 18-hydroxylation was detected in the microsomal fraction. The substrates for the P450-1 monooxygenase, ent-kaurenoic acid and [2H]GA12, efficiently inhibited kaurenolide synthesis with I50 values of 3 and 6 microM, respectively. Both substrates also inhibited ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid metabolism by SG139-P450-1. Conversely, [14C4]GA14 synthesis from [14C4]GA12-aldehyde was inhibited by ent-[2H]kauradienoic acid and fujenal with I50 values of 10 and 30 microM, respectively. These results demonstrate that kaurenolides and seco-ring B kaurenoids are formed by the P450-1 monooxygenase (GA14 synthase) of G. fujikuroi and are thus side products that probably result from stabilization of radical intermediates involved in GA14 synthesis.     

Measurement of formamidopyrimidines in DNA

Formamidopyrimidines, 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), are among major lesions in DNA generated by hydroxyl radical attack, UV radiation, or photosensitization in vitro and in vivo. FapyAde and FapyGua exist in living cells at detectable background levels and are formed by exposure of cells to DNA-damaging agents. Numerous prokaryotic and eukaryotic DNA glycosylases exist for the repair of formamidopyrimidines by base excision repair pathways in cells, indicating their biological significance. Moreover, they are premutagenic lesions, albeit to different extents, revealing a possible role in disease processes. Methodologies using gas chromatography/mass spectrometry (GC/MS) with capillary columns have been developed to accurately measure FapyAde and FapyGua in DNA in vitro and in vivo. Stable isotope-labeled analogues of these compounds have been synthesized and are commercially available to be used as internal standards for accurate quantification. GC/MS with isotope dilution provides excellent sensitivity and selectivity for positive identification and accurate quantification, and has widely been applied in the past to the measurement of formamidopyrimidines under numerous experimental conditions. This paper reports on the details of this GC/MS methodology.     

Effect of cycloheximide or puromycin on induction of thermotolerance by sodium arsenite in Chinese hamster ovary cells: involvement of heat shock proteins

After sodium arsenite (100 microM) treatment, the synthesis of three major heat shock protein families (HSPs; Mr = 110,000, 87,000, and 70,000), as studied with one-dimensional gels, was enhanced twofold relative to that of unheated cells. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed as a 100,000-fold increase in survival from 10(-6) to 10(-1) after 4 hr at 43 degrees C, and as a thermotolerance ratio (TTR) of 2-3 at 10(-3) isosurvival for heating at 45.5 degrees C. Cycloheximide (CHM: 10 micrograms/ml) or puromycin (PUR: 100 micrograms/ml), which inhibited total protein synthesis and HSP synthesis by 95%, completely suppressed the development of thermotolerance when either drug was added after sodium arsenite treatment and removed prior to the subsequent heat treatment. Therefore, thermotolerance induced by arsenite treatment correlated with an increase in newly synthesized HSPs. However, with or without arsenite treatment, CHM or PUR added 2-6 hr before heating and left on during heating caused a 10,000-100,000-fold enhancement of survival when cells were heated at 43 degrees C for 4 hr, even though very little synthesis of heat shock proteins occurred. Moreover, these cells manifesting resistance to heating at 43 degrees C after CHM treatment were much different than those manifesting resistance to 43 degrees C after arsenite treatment. Arsenite-treated cells showed a great deal of thermotolerance (TTR of about 10) when they were heated at 45 degrees C after 5 hr of heating at 43 degrees C, compared with less thermotolerance (TTR of about 2) for the CHM-treated cells heated at 45 degrees C after 5 hr of heating at 43 degrees C. Therefore, there are two different phenomena. The first is thermotolerance after arsenite treatment (observed at 43 degrees C or 45.5 degrees C) that apparently requires synthesis of HSPs. The second is resistance to heat after CHM or PUR treatment before and during heating (observed at 43 degrees C with little resistance at 45.5 degrees C) that apparently does not require synthesis of HSPs. This phenomenon not requiring the synthesis of HSPs also was observed by the large increase in thermotolerance to 45 degrees C caused by heating at 43 degrees C, with or without CHM, after cells were incubated for 6 hr following arsenite pretreatment. For both phenomena, a model based on synthesis and redistribution of HSPs is presented.     

A novel strategy for combination of clofarabine and pictilisib is synergistic in gastric cancer

Gastric cancer (GC) is frequently characterized by resistance to standard chemotherapeutic regimens and poor clinical outcomes. We aimed to identify a novel therapeutic approach using drug sensitivity testing (DST) and our computational SynerySeq pipeline. DST of GC cell lines was performed with a library of 215 Federal Drug Administration (FDA) approved compounds and identified clofarabine as a potential therapeutic agent. RNA-sequencing (RNAseq) of clofarabine treated GC cells was analyzed according to our SynergySeq pipeline and identified pictilisib as a potential synergistic agent. Clonogenic survival and Annexin V assays demonstrated increased cell death with clofarabine and pictilisib combination treatment (P<0.01). The combination induced double strand breaks (DSB) as indicated by phosphorylated H2A histone family member X (γH2AX) immunofluorescence and western blot analysis (P<0.01). Pictilisib treatment inhibited the protein kinase B (AKT) cell survival pathway and promoted a pro-apoptotic phenotype as evidenced by quantitative real time polymerase chain reaction (qRT-PCR) analysis of the B-cell lymphoma 2 (BCL2) protein family members (P<0.01). Patient derived xenograft (PDX) data confirmed that the combination is more effective in abrogating tumor growth with prolonged survival than single-agent treatment (P<0.01). The novel combination of clofarabine and pictilisib in GC promotes DNA damage and inhibits key cell survival pathways to induce cell death beyond single-agent treatment.