Christopher L. Bacon

Antiproliferative action of valproate is associated with aberrant expression and nuclear translocation of cyclin D3 during the C6 glioma G1 phase

Cell cycle progression is tightly regulated by cyclins, cyclin‐dependent kinases (cdks) and related inhibitory phophatases. Here, we employed mitotic selection to synchronize the C6 glioma cell cycle at the start of the G1 phase and mapped the temporal regulation of selected cyclins, cdks and inhibitory proteins throughout the 12 h of G1 by immunoblot analysis. The D‐type cyclins, D3 and D1, were differentially expressed during the C6 glioma G1 phase. Cyclin D1 was up‐regulated in the mid‐G1 phase (4–6 h) while cyclin D3 expression emerged only in late G1 (9–12 h). The influence of the anticonvulsant agent valproic acid (VPA) on expression of cyclins and related proteins was determined, since its teratogenic potency has been linked to cell cycle arrest in the mid‐G1 phase. Exposure of C6 glioma to VPA induced a marked up‐regulation of cyclin D3 and decreased expression of the proliferating cell nuclear antigen. In synchronized cell populations, increased expression of cyclin D3 by VPA was detected in the mid‐G1 phase (3–5 h). Immunocytochemical localization demonstrated rapid intracellular translocation of cyclin D3 to the nucleus following VPA exposure, suggesting that VPA‐induced cell cycle arrest may be mediated by precocious activation of cyclin D3 in the G1 phase.

Correlation of in vitro anti-proliferative potential with in vivo teratogenicity in a series of valproate analogues

The prediction that an anti‐proliferative effect coupled with a pro‐differentiative action will detect a neural tube teratogen has been validated by comparison of these in vitro endpoints with in vivo teratogenicity in a series of closely allied valproate structural analogues. The majority of the compounds significantly inhibited C6 glioma proliferation, the most potent compounds being ranked as octanoic acid >2‐propylhexanoic acid≥2‐ethylhexanoic acid≥valproic acid. The anti‐proliferative potency of these compounds did not correlate strictly to their relative in vivo teratogenic potential. Valproic acid exhibited an anti‐proliferative ic50 of 1.45 mM, whereas 2‐propyl‐2‐pentenoic acid and 2‐propyl‐4‐pentenoic acid were virtually indistinguishable, exhibiting significantly lower ic50 values of 2.5 and 2.55 mM, respectively. The concanavalin A lectin affinity assay was employed to establish whether an anti‐proliferative action was coupled with an increased state of cell differentiation. In this lectin affinity assay, the most potent analogues to significantly attenuate the affinity of exposed C6 glioma cells for concanavalin A lectin‐coated plastic included 2‐butylhexanoic acid, 2‐propyl‐4‐pentenoic acid, 2‐propylhexanoic acid and 2‐ethylhexanoic acid in a manner which can be related to their relative teratogenic potencies in vivo. All compounds screened positive in both the anti‐proliferative and pro‐differentiative assays exhibited in vivo exencephalic rates of 5–44%. These included valproic acid, 2‐ethylhexanoic acid, 2‐propylhexanoic acid and 2‐butylhexanoic acid. It would appear that combined anti‐proliferative and pro‐differentiative screens provide a promising detection system for teratogenic status in a series of valproate analogues.

Low risk of inhibitor formation in haemophilia A patients following en masse switch in treatment to a third generation full length plasma and albumin-free recombinant factor VIII product (ADVATE®).

Summary.  Previous studies have suggested that development of inhibitors in previously treated patients (PTPs) may be attributable to a switch in factor VIII (FVIII) therapeutic product. Consequently, it is widely recognized that inhibitor development must be assessed in PTPs following the introduction of any new FVIII product. Following a national tender process in 2006, all patients with haemophilia A in Ireland changed their FVIII treatment product en masse to a plasma and albumin‐free recombinant full‐length FVIII product (ADVATE®). In this study, we retrospectively reviewed the case records of Irish PTPs to evaluate risk of inhibitor formation following this treatment switch. One hundred and thirteen patients participated in the study. Most patients (89%) had severe haemophilia. Only one of 96 patients with no inhibitor history developed an inhibitor. Prior to the switch in his recombinant FVIII (rFVIII) treatment of choice, this child had only experienced three exposure days (EDs). Consequently, in total he had only received 6 EDs when his inhibitor was first diagnosed. In keeping with this lack of de novo inhibitor development, we observed no evidence of any recurrent inhibitor formation in any of 16 patients with previously documented inhibitors. Similarly, following a previous en masse switch, we have previously reported that changing from a Chinese hamster ovary cell‐produced to a baby hamster kidney cell‐produced rFVIII was also associated with a low risk of inhibitor formation in PTPs. Our cumulative findings from these two studies clearly emphasizes that the risk of inhibitor development for PTPs following changes in commercial rFVIII product is low, at least in the Irish population.

A doctor(s) dilemma: ETV6-ABL1 positive acute lymphoblastic leukaemia.

Sanz, M., Wieland, S., Barber, J.R. & Kantarjian, H.M. (2009) A phase II study of oral tamibarotene in acute promyelocytic leukemia (APL) patients (PTS) who have received prior therapy with all-trans retinoic acid and arsenic trioxide (STAR-1 trial). Blood (ASH Annual Meeting Abstracts), 114, 2050. Ohnishi, K. (2007) PML-RARa inhibitors (ATRA, tamibaroten, arsenic troxide) for acute promyelocytic leukemia. International Journal of Clinical Oncology, 12, 313–317. Sanz, M.A., Lo-Coco, F., Martı́n, G., Avvisati, G., Rayón, C., Barbui, T., Dı́az-Mediavilla, J., Fioritoni, G., González, J.D., Liso, V., Esteve, J., Ferrara, F., Bolufer, P., Bernasconi, C., Gonzalez, M., Rodeghiero, F., Colomer, D., Petti, M.C., Ribera, J.M. & Mandelli, F. (2000) Definition of relapse risk and role of nonanthracycline drugs for consolidation in patients with acute promyelocytic leukemia: a joint study of the PETHEMA and GIMEMA cooperative groups. Blood, 96, 1247–1253. Sanz, M.A., Grimwade, D., Tallman, M.S., Lowenberg, B., Fenaux, P., Estey, E.H., Naoe, T., Lengfelder, E., Büchner, T., Döhner, H., Burnett, A.K. & Lo-Coco, F. (2009) Management of acute promyelocytic leukemia: recommendations from an expert panel on behalf of the European LeukemiaNet. Blood, 113, 1875–1891. Tobita, T., Takeshita, A., Kitamura, K., Ohnishi, K., Yanagi, M., Hiraoka, A., Karasuno, T., Takeuchi, M., Miyawaki, S., Ueda, R., Naoe, T. & Ohno, R. (1997) Treatment with a new synthetic retinoid, Am80, of acute promyelocytic leukemia relapsed from complete remission induced by all-trans retinoic acid. Blood, 90, 967–973.

Antiproliferative actions of inhalational anesthetics: comparisons to the valproate teratogen

The antiproliferative potential of the volatile anesthetics isoflurane, enflurane and sevoflurane was determined and compared to the valproate teratogen. The in vitro system employed, a G1 phase proliferative arrest endpoint in C6 glioma, has served previously to discriminate agents with known teratogenic potential in vivo. Based on estimated IC50 values that were within twice the estimated minimum aveolar concentration value, the rank antiproliferative potency of the inhalational anesthetics employed was isoflurane=enflurane≫sevoflurane. Flow cytometric analysis of growth‐arrested cell populations failed to reveal specific accumulation in any cell cycle phase and the lack of a G1 phase‐specific effect was confirmed by the absence of a transient, time‐dependent sialylation event in synchronized cells. The antiproliferative mechanism of volatile anesthetics, and valproate, was mediated at hydrophobic binding sites, as increasing the hydration sphere of the drug‐micelle complex, using the hygroscopic qualities of the dimethylsulfoxide vehicle, completely reversed this effect. Our findings suggest inhalational anesthetics lack the specific in vitro characteristics of the valproate teratogen.

Valproic acid suppresses G1 phase-dependent sialylation of a 65 kDa glycoprotein in the C6glioma cell cycle

The influence of valproate on invitro glycosylation events in C6 glioma has been investigated, as this major human teratogen restricts proliferation in the mid‐G1 phase of the cycle and alters the prevalence and/or glycosylation state of cell surface glycoproteins with the potential to mediate cell–cell and cell–matrix interactions critical to development. C6 glioma cultured continuously in the presence of 1 mM valproate exhibited a significant depression of exponential growth but attained confluency one day later, when the majority of cells entered the G1 phase of the cycle. Glycoprotein sialyltransferase, which exhibited a four‐fold increase during exponential growth and a small decrease at confluency, was markedly attenuated in valproate‐exposed cells in a manner which was indirect. This was associated with an inhibition of transient α2,3 sialylation of a 65 kDa glycoprotein expressed maximally at 4 hr into the G1 phase of the cell cycle. This effect was cell‐cycle phase‐specific, as exposure of synchronized cells to valproate inhibited transient sialylation at 4 and 5 hr into the G1 phase. Inhibition of the 65 kDa glycoprotein sialylation by valproate is suggested to arise from impaired signal transduction preceding the eventual arrest by the drug at a 5–6 hr G1 phase restriction point.

Cell proliferation, migration and CAM-dependent neurite outgrowth as developmental in vitro endpoints for screening teratogenic potential: Application to valproic acid and related analogues of varying potency

The in vivo teratogenic potential of valproic acid (VPA) and related teratogenic and non-teratogenic analogues has been correlated with their effects on specific in vitro endpoints of cell proliferation, migration and CAM-dependent neurite outgrowth, as these events are common to crucial epochs of development. The (+/-)-2-n-propyl-4-pentynoic acid [(+/-)-4-yn-VPA] and S-2-n-propyl-4-pentynoic acid [S(-)-4-yn-VPA] analogues increased the incidence of neural tube defects in mouse embryos exposed to a single dose, whereas the E-2-n-propyl-2-pentenoic acid (E-2-en-VPA) analogue and R-2-n-propyl-4-pentynoic acid [R( + )-4-yn-VPA] enantiomer were without effect. VPA and related analogues tested exerted comparable G1 phase antiproliferative effects in C6 glioma and limb bud cells in a dose range of 0-3 mM; however, their relative potency did not correlate with in vivo teratogenicity. In contrast, VPA and all teratogenic analogues, at 3 mM, inhibited neuronal cell aggregation and limb bud chondrocyte differentiation in a manner that exhibited a reasonable correlation with their in vivo teratogenicity. The teratogenic S(-)-4-yn-VPA and non-teratogenic R( + )-4-yn-VPA enantiomers exhibited a differential inhibition of primary neurone outgrowth of neuntes stimulated by cell adhesion molecules [L1 and N-cadherin (NCAD)]. Half-maximal inhibition was observed at approximately 150 muM for the teratogenic S(-)-4-yn-VPA enantiomer, but not the non-teratogenic R( + )-4-yn-VPA form. These results suggest that in vitro perturbations of differentiation are likely to provide the greatest discriminatory power for in vivo teratogenicity.

Syntheses of 1,2-diamino and 1,2-aminoalcohol derivatives in the piperidine and pyrrolidine series as anti-amnesic agents.

Tacrine, one of the drugs available for Alzheimers disease based on the cholinergic approach, suffers from considerable toxicity. Many analogues of tacrine has been prepared which retain the pharmacologically rich aminopyridine or aminoquinoline motifs. The current research is a continuation of our efforts in the area of 11-aminobenzoquinolizidines (4) and 10-aminobenzoindolizidines (5) (cf. ref9). A serendipitous discovery led us to the biologically active open chain analogue 9, and we proceeded to elaborate on this molecule. Overall, the compounds we prepared were poor inhibitors of acetylcholinesterase as compared to tacrine. The single exception was compound 20 which exhibited an effect comparable to that of tacrine, but only at a dose in the order of 10(-3) M. However, despite the poor acetylcholinesterase inhibition by 9, this compound was found to be an effective antiamnesic agent.

Syntheses of benzoquinolizidine and benzoindolizidine derivatives as anti-amnesic agents.

Tacrine, one of the drugs available for Alzheimers disease based on the cholinergic approach, suffers from considerable toxicity. Many analogues of tacrine have been prepared which retain the pharmacologically rich aminopyridine or aminoquinoline motifs. The current research was undertaken to produce an acetylcholinesterase inhibitor by employing 11-aminobenzoquinolizidines (4) and 10-aminobenzoindolizidines (5) as templates. Thus, we aimed to achieve three goals relative to tacrine: eliminate the pyridine and quinoline moieties and render the molecule less flat. Overall, the compounds we prepared were poorer inhibitors of acetylcholinesterase compared to tacrine. The single exception was compound 6f which exhibited an effect comparable to that of tacrine, but only at a dose of the order of 10(-3) M. However, despite the poor acetylcholinesterase inhibition by 6b, this compound proved to be an effective anti-amnesic agent at 45 mg/kg dose.

Does vaccine dose predict response to the monovalent pandemic H1N1 influenza a vaccine in children with acute lymphoblastic leukemia? A single-centre study

Vaccination against influenza is an important strategy in preventing severe infection among children with acute lymphoblastic leukemia (ALL). Successful vaccination depends on both vaccine and host‐related factors. We conducted a study on factors predicting the immunogenicity of the monovalent pandemic H1N1 (pH1N1) influenza A vaccine in children with ALL.