The anti-cancer drug chlorambucil as a substrate for the human polymorphic enzyme glutathione transferase P1-1: kinetic properties and crystallographic characterisation of allelic variants

The commonly used anti-cancer drug chlorambucil is the primary treatment for patients with chronic lymphocytic leukaemia. Chlorambucil has been shown to be detoxified by human glutathione transferase Pi (GST P1-1), an enzyme that is often found over-expressed in cancer tissues. The allelic variants of GST P1-1 are associated with differing susceptibilities to leukaemia and differ markedly in their efficiency in catalysing glutathione (GSH) conjugation reactions. Here, we perform detailed kinetic studies of the allelic variants with the aid of three representative co-substrates. We show that the differing catalytic properties of the variants are highly substrate-dependent. We show also that all variants exhibit the same temperature stability in the range 10 °C to 45 °C. We have determined the crystal structures of GST P1-1 in complex with chlorambucil and its GSH conjugate for two of these allelic variants that have different residues at positions 104 and 113. Chlorambucil is found to bind in a non-productive mode to the substrate-binding site (H-site) in the absence of GSH. This result suggests that under certain stress conditions where GSH levels are low, GST P1-1 can inactivate the drug by sequestering it from the surrounding medium. However, in the presence of GSH, chlorambucil binds in the H-site in a productive mode and undergoes a conjugation reaction with GSH present in the crystal. The crystal structure of the GSH-chlorambucil complex bound to the *C variant is identical with the *A variant ruling out the hypothesis that primary structure differences between the variants cause structural changes at the active site. Finally, we show that chlorambucil is a very poor inhibitor of the enzyme in contrast to ethacrynic acid, which binds to the enzyme in a similar fashion but can act as both substrate and inhibitor.     

A positive role for the Ku complex in DNA replication following strand break damage in mammals

Ku70-Ku80 complex is the regulatory subunit of DNA-dependent protein kinase (DNA-PK) and plays an essential role in double-strand break repair following ionizing radiation (IR). It preferentially interacts with chromosomal breaks and protects DNA ends from nuclease attack. Here we show evidence that cells defective in Ku80 exhibit a significantly slow S phase progression following DNA damage. IR-induced retardation in S phase progression in Ku80-/- cells was not due to the lack of DNA-PK kinase activity because both wild-type cells and DNA-PKcs-deficient cells showed no such symptom. Instead, proliferating cell nuclear antigen (PCNA) dissociated from chromosomes following IR in Ku80-deficient cells but not in wild-type or DNA-PKcs-deficient cells. Treatment of HeLa cells with IR induced colocalization of the Ku complex with PCNA on chromosomes. Together, these results suggest that binding of the Ku complex at chromosomal breaks may be necessary to maintain the sliding clamps (PCNA) on chromatin, which would allow cells to resume DNA replication without a major delay following IR.     

Evolutionary and clinical neocentromeres: two faces of the same coin?

It has been hypothesized that human clinical neocentromeres and evolutionary novel centromeres (ENC) represent two faces of the same phenomenon. However, there are only two reports of loci harboring both a novel centromere and a clinical neocentromere. We suggest that only the tip of the iceberg has been scratched because most neocentromerization events have a very low chance of being observed. In support of this view, we report here on a neocentromere at 9q33.1 that emerged in a ring chromosome of about 12 Mb. The ring was produced by a balanced rearrangement that was fortuitously discovered because of its malsegregation in the propositus. Chromatin-immunoprecipitation-on-chip experiments using anti-centromere protein (CENP)-A and anti-CENP-C antibodies strongly indicated that a novel centromeric domain was present in the ring, in a chromosomal domain where an ENC emerged in the ancestor to Old World monkeys.     

The CD85/LIR-1/ILT2 inhibitory receptor is expressed by all human T lymphocytes and down-regulates their functions

The inhibitory molecule CD85/LIR-1/ILT2 has been detected previously on the surface of a small proportion of T lymphocytes. In this study, evidence is provided that, although only a fraction of CD3+ cells are stained by mAb specific for CD85/LIR-1/ILT2 on their surface, this inhibitory receptor is present in the cytoplasm of all T lymphocytes, and that it is detectable on the surface of all T cell clones by the M402 mAb. Biochemical analyses further demonstrate that CD85/LIR-1/ILT2 is present in all T clones analyzed, and that the protein is tyrosine-phosphorylated. Expression of mRNA coding for CD85/LIR-1/ILT2 has been assessed by RT-PCR. Notably, in the NKL cell line and in one T cell clone, amplification of the messenger required 30 cycles only, whereas, in other T cell clones, an amplification product was detected by increasing the number of cycles. CD85/LIR-1/ILT2 inhibits CD3/TCR-mediated activation in both CD4+ and CD8+ clones, and it down-regulates Ag recognition by CD8+ cells in a clonally distributed fashion. Addition of anti-ILT2 HP-F1 mAb in the cytolytic assay enhances target cell lysis mediated by Ag-specific CTL. This could be due to interference of the mAb with receptor/ligand interactions. In contrast, HP-F1 mAb cross-linking triggers inhibitory signals that reduce cytotoxicity. CD85/LIR-1/ILT2 also controls responses to recall Ags and, in low responders, its engagement sharply increases T cell proliferation. The inhibitory function of the molecule is also confirmed by its ability to reduce CD3/TCR-induced intracellular Ca2+ mobilization.     

Mitochondrial respirasome works as a single unit and the cross-talk between complexes I,III2 and IV stimulates NADH dehydrogenase activity

Ustilago maydis is an aerobic basidiomycete that depends on oxidative phosphorylation for its ATP supply, pointing to the mitochondrion as a key player in its energy metabolism. Mitochondrial respiratory complexes I, III₂, and IV occur in supramolecular structures named respirasome. In this work, we characterized the subunit composition and the kinetics of NADH:Q oxidoreductase activity of the digitonine-solubilized respirasome (1600 kDa) and the free-complex I (990 kDa). In the presence of 2,6-dimethoxy-1,4-benzoquinone (DBQ) and cytochrome c, both the respirasome NADH:O₂ and the NADH:DBQ oxidoreductase activities were inhibited by rotenone, antimycin A or cyanide. A value of 2.4 for the NADH oxidized/oxygen reduced ratio was determined for the respirasome activity, while ROS production was less than 0.001% of the oxygen consumption rate. Analysis of the NADH:DBQ oxidoreductase activity showed that respirasome was 3-times more active and showed higher affinity than free-complex I. The results suggest that the contacts between complexes I, III₂ and IV in the respirasome increase the catalytic efficiency of complex I and regulate its activity to prevent ROS production.     

Effects of resveratrol on nucleotide degrading enzymes in streptozotocin-induced diabetic rats

AimsDiabetes mellitus is associated with platelet alterations that may contribute to the development of cardiovascular complications. The present study investigates the effects of resveratrol (RSV), an important compound with cardioprotective activities, on NTPDase, ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP), 5′-nucleotidase and adenosine deaminase (ADA) activities in platelets from streptozotocin (STZ)-induced diabetic rats.Main methodsThe animals were divided into six groups (n = 8): control/saline; control/RSV 10 mg/kg; control/RSV 20 mg/kg; diabetic/saline; diabetic/RSV 10 mg/kg; diabetic/RSV 20 mg/kg. RSV was administered during 30 days and after this period the blood was collected for enzymatic assay.Key findingsThe results demonstrated that NTPDase, E-NPP and 5′-nucleotidase activities were significantly higher in the diabetic/saline group (P < 0.05) compared to control/saline group. Treatment with RSV significantly increased NTPDase, 5′-nucleotidase and E-NPP activities in the diabetic/RSV10 and diabetic/RSV20 groups (P < 0.05) compared to diabetic/saline group. When RSV was administered per se there was also an increase in the activities of these enzymes in the control/RSV10 and control/RSV20 groups (P < 0.05) compared to control/saline group. ADA activity was significantly increased in the diabetic/saline group (P < 0.05) compared to control/saline group. The treatment with RSV prevented this increase in the diabetic/RSV10 and diabetic/RSV20 groups. No significant differences in ADA activity were observed in the control/RSV10 and control/RSV20 compared to control/saline group.SignificanceThe present findings demonstrate alterations in nucleotide hydrolysis in platelets of STZ-induced diabetic rats and treatment with RSV was able to modulate adenine nucleotide hydrolysis, which may be important in the control of the platelet coagulant status in diabetes.     

The potential role of thioredoxin 1 and CD30 systems as multiple pathway targets and biomarkers in tumor therapy

Our progress in understanding pathological disease mechanisms has led to the identification of biomarkers that have had a considerable impact on clinical practice. It is hoped that the move from generalized to stratified approaches, with the grouping of patients into clinical/therapeutic subgroups according to specific biomarkers, will lead to increasingly more effective clinical treatments in the near future. This success depends on the identification of biomarkers that reflect disease evolution and can be used to predict disease state and therapy response, or represent themselves a target for treatment. Biomarkers can be identified by studying relationships between serum, tissue, or tumor microenvironment parameters and clinical or therapeutic parameters at onset and during the progression of the disease, using systems biology. Given that multiple pathways, such as those responsible for redox and immune regulation, are deregulated or altered in tumors, the future of tumor therapy could lie in the simultaneous targeting of these pathways using extracellular and intracellular targets and biomarkers. With this aim in mind, we evaluated the role of thioredoxin 1, a key redox regulator, and CD30, a cell membrane receptor, in immune regulation. Our results lead us to suggest that the combined use of these biomarkers provides more detailed information concerning the multiple pathways affected in disease and hence the possibility of more effective treatment.     

Methionine and methionine sulfoxide alter parameters of oxidative stress in the liver of young rats: in vitro and in vivo studies

Hyperglycemia- and oxidative stress-induced modification of circulating lipoproteins is being increasingly recognized as an important pathogenetic factor for diabetic cardiovascular damages. This study was designed to investigate the impact of modified very low-density lipoprotein and high-density lipoprotein on phagocyte adhesion to endothelial cells and the involvement of scavenger receptor class B type 1 (SR-BI) in this process. Native lipoproteins were isolated by density gradient ultracentrifugation and in vitro glycoxidative or oxidative modification was performed in the presence of glucose or sodium hypochlorite, respectively. One hour co-incubation experiments with lipoproteins, freshly prepared polymorphonuclear leukocytes (PMN), and venous endothelial cells (HUVEC) were performed in the presence or absence of different scavenger receptors and signal transduction inhibitors. PMN adhesion to HUVEC was quantified fluorimetrically. We demonstrated that oxidized and glycoxidized lipoproteins promote adhesion of PMN to HUVEC from 1.5- to 2.5-fold with oxidized lipoproteins having the greatest effect. Treatment with the highly specific SR-BI inhibitor, BLT-1 produced substantial reduction of lipoprotein-induced adhesion to endothelial cells. Native and modified lipoproteins recruited extracellular signal-regulated kinase (ERK 1/2), p38 mitogen-activated protein kinase, and Janus kinase 2 as downstream signaling pathways for adhesion. From this study, it could be concluded that modification of lipoproteins plays a crucial role in atherosclerotic progression and SR-BI may be considered as a potential therapeutic target for the prevention of diabetic cardiovascular complications.     

MicroRNAs in Myeloid Hematological Malignancies

MicroRNAs are 19-24 nucleotides noncoding RNAs which silence modulate the expression of target genes by binding to the messenger RNAs. Myeloid malignancies include a broad spectrum of acute and chronic disorders originating from from the clonal transformation of a hematopoietic stem cell. Specific genetic abnormalities may define myeloid malignancies, such as translocation t(9;22) that represent the hallmark of chronic myeloid leukemia. Although next-generation sequencing pro-vided new insights in the genetic characterization and pathogenesis of myeloid neoplasms, the molecular mechanisms underlying myeloid neoplasms are lacking in most cases. Recently, several studies have demonstrated that the expression levels of specific miRNAs may vary among patients with myeloid malignancies compared with healthy individuals and partially unveiled how miRNAs participate in the leukemic transformation process. Finally, in vitro experiments and pre-clinical model provided preliminary data of the safety and efficacy of miRNA inhibitory molecules, opening new avenue in the treatment of myeloid hematological malignancies.