Inhibition of histone deacetylases 1 and 6 enhances cytarabine-induced apoptosis in pediatric acute myeloid leukemia cells

Background

Pediatric acute myeloid leukemia (AML) remains a challenging disease to treat even with intensified cytarabine-based chemotherapy. Histone deacetylases (HDACs) have been reported to be promising therapeutic targets for treating AML. However, HDAC family members that are involved in chemotherapy sensitivities remain unknown. In this study, we sought to identify members of the HDAC family that are involved in cytarabine sensitivities, and to select the optimal HDACI that is most efficacious when combined with cytarabine for treating children with AML.

Methodology

Expression profiles of classes I, II, and IV HDACs in 4 pediatric AML cell lines were determined by Western blotting. Inhibition of class I HDACs by different HDACIs was measured post immnunoprecipitation. Individual down-regulation of HDACs in pediatric AML cells was performed with lentiviral shRNA. The effects of cytarabine and HDACIs on apoptosis were determined by flow cytometry analysis.

Results

Treatments with structurally diverse HDACIs and HDAC shRNA knockdown experiments revealed that down-regulation of both HDACs 1 and 6 is critical in enhancing cytarabine-induced apoptosis in pediatric AML, at least partly mediated by Bim. However, down-regulation of HDAC2 may negatively impact cytarabine sensitivities in the disease. At clinically achievable concentrations, HDACIs that simultaneously inhibited both HDACs 1 and 6 showed the best anti-leukemic activities and significantly enhanced cytarabine-induced apoptosis.

Conclusion

Our results further confirm that HDACs are bona fide therapeutic targets for treating pediatric AML and suggest that pan-HDACIs may be more beneficial than isoform-specific drugs.     

Aberrant immune responses in a mouse with behavioral disorders

BTBR T+tf/J (BTBR) mice have recently been reported to have behaviors that resemble those of autistic individuals, in that this strain has impairments in social interactions and a restricted repetitive and stereotyped pattern of behaviors. Since immune responses, including autoimmune responses, are known to affect behavior, and individuals with autism have aberrant immune activities, we evaluated the immune system of BTBR mice, and compared their immunity and degree of neuroinflammation with that of C57BL/6 (B6) mice, a highly social control strain, and with F1 offspring. Mice were assessed at postnatal day (pnd) 21 and after behavioral analysis at pnd70. BTBR mice had significantly higher amounts of serum IgG and IgE, of IgG anti-brain antibodies (Abs), and of IgG and IgE deposited in the brain, elevated expression of cytokines, especially IL-33 IL-18, and IL-1β in the brain, and an increased proportion of MHC class II-expressing microglia compared to B6 mice. The F1 mice had intermediate levels of Abs and cytokines as well as social activity. The high Ab levels of BTBR mice are in agreement with their increased numbers of CD40(hi)/I-A(hi) B cells and IgG-secreting B cells. Upon immunization with KLH, the BTBR mice produced 2-3 times more anti-KLH Abs than B6 mice. In contrast to humoral immunity, BTBR mice are significantly more susceptible to listeriosis than B6 or BALB/c mice. The Th2-like immune profile of the BTBR mice and their constitutive neuroinflammation suggests that an autoimmune profile is implicated in their aberrant behaviors, as has been suggested for some humans with autism.     

Lysosomal carboxypeptidase A

Lysosomal carboxypeptidase A (cathepsin A) is synthetized in the form of preproenzyme, which undergoes to active enzyme as a result of post-translational modification. It splits off C-terminal amino acid residues from peptides and proteins and synergizes with other proteases in degradation of cellular proteins in lysosomes. Lysosomal carboxypeptidase A has an effect on peptide hormones and peptides of biological activity of tissues and body fluids as well. It forms complexes with some glycosidases that protects them against proteolytic degradation. Deficiency of this enzyme induces storage diseases. Lysosomal carboxypeptidase A as multifunctional enzyme plays an important regulatory role in organismal metabolism.     

Poly(ethylene glycol)-conjugated human serum albumin including iron porphyrins: surface modification improves the O2-transporting ability

Artificial O2-carrying hemoprotein composed of human serum albumin including tetrakis(o-amidophenyl)porphinatoiron(II) (Fe4P or Fe3P) [HSA-FeXP] has been modified by maleimide- or succinimide-terminated poly(ethylene glycol) (PEG), and the formed PEG bioconjugates have been physicochemically characterized. 2-Iminothiolane (IMT) reacted with the amino groups of Lys to create active thiol groups, which bind to alpha-maleimide-omega-methoxy PEG [Mw: 2-kDa (PEG(M2)), 5-kDa (PEG(M5))]. On the other hand, alpha-succinimidyl-omega-methoxy PEG [Mw: 2-kDa (PEG(S2)), 5-kDa (PEG(S5))] directly binds to Lys residues. MALDI-TOF MS of the PEG-conjugated HSA-FeXP showed distinct molecular ion peaks, which provide an accurate number of the PEG chains. In the case of PEG(MY)(HSA-FeXP), the spectroscopic assay of the thiol groups also provided the mean of the binding numbers of the polymers, and the degree of the modification was controlled by the ratio of [IMT]/[HSA]. The viscosity and colloid osmotic pressures of the 2-kDa PEG conjugates (phosphate-buffered saline solution, [HSA] = 5 g dL(-1)) were almost the same as that of the nonmodified one, whereas the 5-kDa PEG binding increased the rheological parameters. The presence of flexible polymers on the HSA surface retarded the association reaction of O2 to FeXP and stabilized the oxygenated complex. Furthermore, PEG(MY)(HSA-FeXP) exhibited a long circulation lifetime of FeXP in rats (13-16 h). On the basis of these results, it can be concluded that the surface modification of HSA-FeXP by PEG has improved its comprehensive O2-transporting ability. In particular the PEG(MY)(HSA-FeXP) solution could be a promising material for entirely synthetic O2-carrying plasma expander as a red cell substitute.     

PPAR‐β facilitating maturation of hepatic‐like tissue derived from mouse embryonic stem cells accompanied by mitochondriogenesis and membrane potential retention

Relatively little is known about mitochondria metabolism in differentiating embryonic stem (ES) cells. Present research focused on several elements of cellular energy metabolism in hepatic‐like tissue derived from mouse ES cells. We demonstrated that mitochondrial location patterns and mitochondrial membrane potential (ΔΨ_m) existed in subsequent differentiation of the tissue. Mitochondriogenesis appeared at the early stage and kept a normal ΔΨ_m in differentiated mature hepatocytes. Peroxisome proliferator‐activated receptor‐α (PPAR‐α) expression was transitorily increased at the beginning, and kept a relatively low level later, which accompanied by expression of PPAR‐γ coactivator (PGC)‐1α, a master regulator of mitochondrial biogenesis. PPAR‐β expression showed robust up‐regulation in the late differentiation course. Enhanced co‐expressions of PPAR‐β and albumin with catalysis of UDP‐glucuronosyltransferases (UGTs) were observed at mature stage. While PPAR‐γ expression changed little before and after differentiation. Mitochondriogenesis could be accelerated by PPAR‐α specific agonist WY14643 and abolished by its antagonist GW6471 at the early stage. Neither of them affected mitochondrial ΔΨ_m and albumin generation in the differentiated hepatocytes. Furthermore, maturation of hepatic‐like tissue and mitochondriogenesis in hepatocyte could be efficiently stimulated by PPAR‐β specific agonist L165041 and abolished by PPAR‐β specific antagonist GSK0660, but not affected by PPAR‐γ specific agonist GW1929. In conclusion, the derived hepatic tissue morphologically possessed cellular energy metabolism features. PPAR‐α seemed only necessary for early mitochondriogenesis, while less important for ΔΨ_m retention in the mature tissue derived. The stimulation of PPAR‐β but not ‐γ enhanced hepatogenesis, hepatocytes maturation, and mitochondriogenesis. PPAR‐β took an important role in cellular energy metabolism of hepatogenesis. J. Cell. Biochem. 109: 498–508, 2010. © 2009 Wiley‐Liss, Inc.     

Wound ballistics of the pig mandibular angle: A preliminary finite element analysis and experimental study

To study wound ballistics of the mandibular angle, a combined hexahedral-tetrahedral finite element (FE) model of the pig mandible was developed to simulate ballistic impact. An experimental study was carried out by measuring impact load parameters from 14 fresh pig mandibles that were shot at the mandibular angle by a standard 7.62 mm M43 bullet. FE analysis was executed through the LS-DYNA code under impact loads similar to those obtained from the experimental study. The resulting residual velocity, the transferred energy from the bullet to the mandible, and the surface area of the entrance wound had no statistical differences between the FE simulation and the experimental study. However, the mean surface area of the exit wounds in the experimental study was significantly larger than that in the simulation. According to the FE analysis, the stress concentrated zones were mainly located at the region of impact, condylar neck, coronoid process and mandibular body. The simulation results also indicated that trabecular bone had less stress concentration and a lower speed of stress propagation compared with cortical bone. The FE model is appropriate and conforms to the basic principles of wound ballistics. This modeling system will be helpful for further investigations of the biomechanical mechanisms of wound ballistics.     

Calcium-dependent Association of Calmodulin with the Rubella Virus Nonstructural Protease Domain

The rubella virus (RUBV) nonstructural (NS) protease domain, a Ca²⁺- and Zn²⁺-binding papain-like cysteine protease domain within the nonstructural replicase polyprotein precursor, is responsible for the self-cleavage of the precursor into two mature products, P150 and P90, that compose the replication complex that mediates viral RNA replication; the NS protease resides at the C terminus of P150. Here we report the Ca²⁺-dependent, stoichiometric association of calmodulin (CaM) with the RUBV NS protease. Co-immunoprecipitation and pulldown assays coupled with site-directed mutagenesis demonstrated that both the P150 protein and a 110-residue minidomain within NS protease interacted directly with Ca²⁺/CaM. The specific interaction was mapped to a putative CaM-binding domain. A 32-mer peptide (residues 1152–1183, denoted as RUBpep) containing the putative CaM-binding domain was used to investigate the association of RUBV NS protease with CaM or its N- and C-terminal subdomains. We found that RUBpep bound to Ca²⁺/CaM with a dissociation constant of 100–300 nm. The C-terminal subdomain of CaM preferentially bound to RUBpep with an affinity 12.5-fold stronger than the N-terminal subdomain. Fluorescence, circular dichroism and NMR spectroscopic studies revealed a “wrapping around” mode of interaction between RUBpep and Ca²⁺/CaM with substantially more helical structure in RUBpep and a global structural change in CaM upon complex formation. Using a site-directed mutagenesis approach, we further demonstrated that association of CaM with the CaM-binding domain in the RUBV NS protease was necessary for NS protease activity and infectivity.