Integrative Analysis of Epigenetic Modulation in Melanoma Cell Response to Decitabine: Clinical Implications

Decitabine, an epigenetic modifier that reactivates genes otherwise suppressed by DNA promoter methylation, is effective for some, but not all cancer patients, especially those with solid tumors. It is commonly recognized that to overcome resistance and improve outcome, treatment should be guided by tumor biology, which includes genotype, epigenotype, and gene expression profile. We therefore took an integrative approach to better understand melanoma cell response to clinically relevant dose of decitabine and identify complementary targets for combined therapy. We employed eight different melanoma cell strains, determined their growth, apoptotic and DNA damage responses to increasing doses of decitabine, and chose a low, clinically relevant drug dose to perform whole-genome differential gene expression, bioinformatic analysis, and protein validation studies. The data ruled out the DNA damage response, demonstrated the involvement of p21^Cip1 in a p53-independent manner, identified the TGFβ pathway genes CLU and TGFBI as markers of sensitivity to decitabine and revealed an effect on histone modification as part of decitabine-induced gene expression. Mutation analysis and knockdown by siRNA implicated activated β-catenin/MITF, but not BRAF, NRAS or PTEN mutations as a source for resistance. The importance of protein stability predicted from the results was validated by the synergistic effect of Bortezomib, a proteasome inhibitor, in enhancing the growth arrest of decitabine in otherwise resistant melanoma cells. Our integrative analysis show that improved therapy can be achieved by comprehensive analysis of cancer cells, identified biomarkers for patient''s selection and monitoring response, as well as targets for improved combination therapy.     

Influence of Hsp70s and their regulators on yeast prion propagation

Propagation of yeast prions requires normal abundance and activity of many protein chaperones. Central among them is Hsp70, a ubiquitous and essential chaperone involved in many diverse cellular processes that helps promote proper protein folding and acts as a critical component of several chaperone machines. Hsp70 is regulated by a large cohort of co-chaperones, whose effects on prions are likely mediated through Hsp70. Hsp104 is another chaperone, absent from mammalian cells, that resolubilizes proteins from aggregates. This activity, which minimally requires Hsp70 and its co-chaperone Hsp40, is essential for yeast prion replication. Although much is known about how yeast prions can be affected by altering protein chaperones, mechanistic explanations for these effects are uncertain. We discuss the variety of effects Hsp70 and its regulators have on different prions and how the effects might be due to the many ways chaperones interact with each other and with amyloid.Key words: Hsp70, Hsp40, chaperone, prion, yeast     

Dynamic nature of a wheat centromere with a functional gene

Centromeric regions of higher eukaryotes are comprised mainly of tandem and non-tandem repeat sequences with variable copy number, spacing, order and orientation; are heterochromatic in nature, and are believed to be devoid of actively transcribing genes. Here, we report an actively transcribing wheat homolog of HSP70 gene that maps in the functional wheat centromere, and copy number of which seems to change in response to centromeric breaks. The HSP70 gene physically maps on the short arm of chromosomes 1A and 1D of Chinese Spring (CS) and 1R of rye. Whereas, on chromosome 1B in both ‘CS’ and Pavon background, the gene maps in the functional centromere as evident from its presence in both cytologically confirmed true ditelosomic lines Dt1BS and Dt1BL. Sequence comparison of 11 ESTs showed three sequence patterns suggesting that all three homoeologous copies of the gene are expressing. The cDNA-single stranded conformation polymorphism analysis confirmed expression of the ‘CS’ 1B copy of the gene. Observed in two independently developed Dt1BL lines, the 1B copy number of the gene showed three to fivefold increase in response to chromosomal breaks around the centromere. Putative gene duplications seem to involve large chromosomal segments as only one of the ten restriction enzymes used for DNA gel-blot analysis showed unique extra fragment band in the Dt1BL line. Further investigations are warranted to uncover the nature and mechanism of these duplications.     

LXR-α genomics programmes neuronal death observed in Alzheimer’s disease

Keeping in view the fact that the most pathognomonic feature of Alzheimer’s disease is the abnormal processing of neuronal cell membrane amyloid precursor protein accompanied by significantly elevated human serum and CSF levels of 24-hydroxycholesterol recognised widely as the specific endogenous ligand of Liver X receptor (LXR-α), the present study was addressed to explore the epigenomic-pathway (if any) that connects LXR-α activation with the genes recognised to be involved in the regulation of aberrant Abeta production leading to the generation of toxic and inflammatory mediators responsible for neuronal death. The results of such a study revealed that LXR-α activation by its specific endogenous or exogenous ligands within neuroblastoma cells resulted in the over-expression of PAR-4 gene accompanied by suppression of AATF gene through its inherent capacity to regulate genes coding for SREBP and NF-κB. Over-expression of PAR-4 gene was accompanied by aberrant Abeta production followed by ROS generation and subsequent death of neuroblastoma cells used in the present study as a cellular model for neurons. Further based upon these results, it was proposed that Abeta-induced heme oxygenase-1 can ensure cholesterol-oxidation to provide endogenous ligands for the sustained activation of neuronal LXR-α dependent epigenomic-pathway leading to neuronal death observed in Alzheimer’s disease.     

An incoming nucleotide imposes an anti to syn conformational change on the templating purine in the human DNA polymerase-iota active site

Substrate-induced conformational change of the protein is the linchpin of enzymatic reactions. Replicative DNA polymerases, for example, convert from an open to a closed conformation in response to dNTP binding. Human DNA polymerase-iota (hPoliota), a member of the Y family of DNA polymerases, differs strikingly from other polymerases in its much higher proficiency and fidelity for nucleotide incorporation opposite template purines than opposite template pyrimidines. We present here a crystallographic analysis of hPoliota binary complexes, which together with the ternary complexes show that, contrary to replicative DNA polymerases, the DNA, and not the polymerase, undergoes the primary substrate-induced conformational change. The incoming dNTP "pushes" templates A and G from the anti to the syn conformation dictated by a rigid hPoliota active site. Together, the structures posit a mechanism for template selection wherein dNTP binding induces a conformational switch in template purines for productive Hoogsteen base pairing.     

A Mycobacterium tuberculosis Sigma Factor Network Responds to Cell-Envelope Damage by the Promising Anti-Mycobacterial Thioridazine

Background

Novel therapeutics are urgently needed to control tuberculosis (TB). Thioridazine (THZ) is a candidate for the therapy of multidrug and extensively drug-resistant TB.

Methodology/Principal Findings

We studied the impact of THZ on Mycobacterium tuberculosis (Mtb) by analyzing gene expression profiles after treatment at the minimal inhibitory (1x MIC) or highly inhibitory (4x MIC) concentrations between 1–6 hours. THZ modulated the expression of genes encoding membrane proteins, efflux pumps, oxido-reductases and enzymes involved in fatty acid metabolism and aerobic respiration. The Rv3160c-Rv3161c operon, a multi-drug transporter and the Rv3614c/3615c/3616c regulon, were highly induced in response to THZ. A significantly high number of Mtb genes co-expressed with σ^B (the σ^B regulon) was turned on by THZ treatment. σ^B has recently been shown to protect Mtb from envelope-damage. We hypothesized that THZ damages the Mtb cell-envelope, turning on the expression of the σ^B regulon. Consistent with this hypothesis, we present electron-microscopy data which shows that THZ modulates cell-envelope integrity. Moreover, the Mtb mutants in σ^H and σ^E, two alternate stress response sigma factors that induce the expression of σ^B, exhibited higher sensitivity to THZ, indicating that the presence and expression of σ^B allows Mtb to resist the impact of THZ. Conditional induction of σ^B levels increased the survival of Mtb in the presence of THZ.

Conclusions/Significance

THZ targets different pathways and can thus be used as a multi-target inhibitor itself as well as provide strategies for multi-target drug development for combination chemotherapy. Our results show that the Mtb sigma factor network comprising of σ^H, σ^E and σ^B plays a crucial role in protecting the pathogen against cell-envelope damage.     

Downregulation of ADIPOQ and PPARγ2 Gene Expression in Subcutaneous Adipose Tissue of Obese Adolescents With Hepatic Steatosis

Hepatic steatosis is associated with hypoadiponectinemia. The mechanism(s) resulting in lower serum adiponectin levels in obese adolescents with fatty liver is unknown. In two groups of equally obese adolescents, but discordant for hepatic fat content, we measured adiponectin, leptin, peroxisome proliferator–activated receptor γ 2 (PPARγ2) and tumor necrosis factor‐α (TNFα) gene expression in the abdominal subcutaneous adipose tissue (SAT). Twenty six adolescents with similar degrees of obesity underwent a subcutaneous periumbilical adipose tissue biopsy, in addition to metabolic (oral glucose tolerance test, and hyperinsulinemic—euglycemic clamp), and imaging studies (magnetic resonance imaging (MRI), DEXA). Using quantitative real‐time‐PCR; adiponectin, PPARγ2, TNFα, and leptin mRNA were measured. Based on a hepatic fat content (hepatic fat fraction, HFF) >5.5%, measured by fast MRI, the subjects were divided into low and high HFF group. In addition to the hypoadiponectinemia in the high HFF group, we found that the expression of adiponectin as well as PPARγ2 in the SAT was significantly decreased in this group. No differences were noted for TNFα and leptin plasma or mRNA levels between the groups. An inverse relationship was observed between adiponectin or PPARγ2 expression and hepatic fat content, whereas, adiponectin expression was positively related to PPARγ2 expression. Independent of overall obesity, a reduced expression of adiponectin and PPARγ2 in the abdominal SAT is associated with high liver fat content, as well as with insulin resistance in obese adolescents.