Identification of genomic regions contributing to etoposide-induced cytotoxicity

Etoposide is routinely used in combination-based chemotherapy for testicular cancer and small-cell lung cancer; however, myelosuppression, therapy-related leukemia and neurotoxicity limit its utility. To determine the genetic contribution to cellular sensitivity to etoposide, we evaluated cell growth inhibition in Centre d’ Etude du Polymorphisme Humain lymphoblastoid cell lines from 24 multi-generational pedigrees (321 samples) following treatment with 0.02–2.5 μM etoposide for 72 h. Heritability analysis showed that genetic variation contributes significantly to the cytotoxic phenotypes (h ² = 0.17–0.25, P = 4.9 × 10⁻⁵–7.3 × 10⁻³). Whole genome linkage scans uncovered 8 regions with peak LOD scores ranging from 1.57 to 2.55, with the most significant signals being found on chromosome 5 (LOD = 2.55) and chromosome 6 (LOD = 2.52). Linkage-directed association was performed on a subset of HapMap samples within the pedigrees to find 22 SNPs significantly associated with etoposide cytotoxicity at one or more treatment concentrations. UVRAG, a DNA repair gene, SEMA5A, SLC7A6 and PRMT7 are implicated from these unbiased studies. Our findings suggest that susceptibility to etoposide-induced cytotoxicity is heritable and using an integrated genomics approach we identified both genomic regions and SNPs associated with the cytotoxic phenotypes. W. K. Bleibel and S. Duan equally contributed to this work.     

GSK3beta modulates PACAP-induced neuritogenesis in PC12 cells by acting downstream of Rap1 in a caveolae-dependent manner

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neurotrophic peptide. Here, we show that PACAP recruits Rap1 into caveolin-enriched membrane subdomains in PC12 cells and activates Rap1, nuclear ERK1/2, Elk-1 and CREB in a caveolae-dependent manner. We reveal that GSK3beta is a novel modulator in PACAP signalling. PACAP induces phosphorylation of serine 9 in GSK3beta, which is inhibited by silencing Rap1. Lithium and valproate promote but wortmannin and LY294002 attenuate PACAP-induced phosphorylation of both GSK3beta and ERK1/2, whereas MEK inhibitor PD98059 inhibits nerve growth factor- but not PACAP-induced phosphorylation of GSK3beta, suggesting that GSK3beta operates downstream of Rapt 1 but upstream of ERK1/2 in PACAP signalling. Inhibition or stimulation of GSK3beta results in a 2-fold increase and 6-fold decrease in PACAP-induced neurite outgrowth, respectively. These results reveal an important role of caveolae in the signal transduction of PACAP and that GSK3beta is a critical regulator in PACAP-induced neuronal differentiation.     

Purification of Schwann cells from adult rats by differential detachment

Differential detachment by collagenase treatment is a new and efficient method for Schwann cell (SC) purification. As its effect on adult animals remains unclear, we have investigated the possibility of SC purification from adult rats. To avoid any systematic bias, Schwann cell purity before and after purification were compared by morphology, immunostaining of P75^NTR and S100 and flow cytometric analysis. The final SC purities reached 99% as confirmed by three independent analyses SC purity and the cell yields were above 10⁶ cells after two rounds of purification. The method of differential detachment is also suitable for SC purification in adult rats and could be useful for research and clinical applications.     

Determination of the residues of 50 anabolic hormones in muscle,milk and liver by very-high-pressure liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive and specific method for determination of the residues of 50 anabolic hormones in muscle (pork, beef, shrimp), milk and pig liver was developed. Analytes were separated and acquired by liquid chromatography coupled with an electrospray ionization tandem mass spectrometer (LC–ESI–MS/MS). Target compounds were simultaneously extracted with methanol after enzyme hydrolysis, and purified using a graphitized carbon-black solid-phase extraction (SPE) and followed by NH₂ SPE cartridge. Limits of quantification were 0.04–2.0 μg kg^?1; average recoveries were 76.9–121.3%; and the relative standard deviation was 2.4–21.2%. This method has been successfully applied in real samples.     

Dishevelled-DEP domain interacting protein (DDIP) inhibits Wnt signaling by promoting TCF4 degradation and disrupting the TCF4/β-catenin complex

The TCF4/β-catenin complex, the executor of canonical Wnt/β-catenin signaling, is regulated by a variety of factors. Among these, Dishevelled (Dvl) is a critical regulator that releases β-catenin from degradation and stabilizes TCF4/β-catenin complex. Here, we report that DDIP (Dishevelled-DEP domain Interacting Protein, also named as Spats1, spermatogenesis associated, serine-rich 1), a novel protein that interacts with Dvl, regulates Wnt signaling. We provide evidence that DDIP suppresses Lef-1 luciferase reporter activity stimulated by Wnt1, Dvl2 or β-catenin, interacts with the TCF4/β-catenin complex, and disrupts the interaction of TCF4 and β-catenin by promoting TCF4 degradation through the proteasome pathway. Our results indicate that DDIP is a negative regulator of the canonical Wnt signaling.     

TaqIB polymorphism in the CETP gene modulates the impact of HC/LF diet on the HDL profile in healthy Chinese young adults

The aim of this study was to investigate the interactions of genetic variants in the genes of cholesterol ester transfer protein (CETP) and low-density lipoprotein receptor (LDLR) with high carbohydrate and low fat (HC/LF) diet on lipid profiles in a young and healthy Chinese Han population. Fifty-six healthy subjects (22.89±1.80 years) were given washout diets of 31% fat and 54% carbohydrate for 7 days, followed by HC/LF diets of 15% fat and 70% carbohydrate for 6 days, with no total energy restriction. Serum lipid profiles at baseline, after washout and following HC/LF diets, as well as CETP and LDLR polymorphisms were analyzed. Carriers of B2 allele of CETP TaqIB polymorphism had significantly higher levels of high density lipoprotein cholesterol (HDL-C) and apo A-I in the whole study population after the diet intervention. Notably, males with CETP TaqIB B1B1 experienced significantly increased HDL-C and apo A-I after HC/LF diet. Regarding the LDLR Pvu II polymorphism, both P1P1 subjects and P2 carriers experienced decreased total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels after HC/LF diet with no statistically significant differences between the genotypes. Our results demonstrate that the elevated HDL-C levels after HC/LF diet in healthy Chinese Han youth are associated with CETP TaqI B2 allele while males with B1B1 genotype are more susceptible to the influence of HC/LF diet on their HDL-C levels. The decreased TC and LDL-C levels after HC/LF diet are not associated with LDLR Pvu II polymorphism.     

Effects of phenylalanine on the survival and neurite outgrowth of rat cortical neurons in primary cultures: possible involvement of brain-derived neurotrophic factor

Bax inhibitor-1 (BI-1), a newly identified apoptosis inhibitor, has recently been found to be overexpressed in several human carcinomas and its specific down-regulation by RNA interference (RNAi) could lead to cell death. The purpose of this study is to investigate the role of BI-1 in apoptosis-resistance and the underlying mechanisms in human nasopharyngeal carcinoma (NPC) cells. Our results showed that BI-1 was expressed in two different human NPC cell lines, CNE-2Z and CNE-1, and specific inhibition of BI-1 expression by siRNA caused a significant increase in spontaneous apoptosis in both cell lines. Mechanistically, we demonstrated that down-regulation of BI-1 protein expression decreased the ratio of Bcl-X_L/Bcl-2 with Bax protein as determined by Western blot and increased the activity of caspase-3 by colorimetric analysis, thus leading to the activation of the associated cell death pathways. Taken together, these results have provided evidence that BI-1 could serve as an important molecular target gene for the development of new therapeutic strategy against human NPCs.     

Differential Proteomics Analysis of Specific Carbonylated Proteins in the Temporal Cortex of Aged Rats: The Deterioration of Antioxidant System

Oxidative stress plays a pivotal role in normal brain aging and various neurodegenerative diseases, including Alzheimer’s disease (AD). Irreversible protein carbonylation, a widely used marker for oxidative stress, rises during aging. The temporal cortex is essential for learning and memory and particularly susceptible to oxidative stress during aging and in AD patients. In this study, we used 2-DE, MALDI-TOF/TOF MS, and Western blotting to analyze the differentially carbonylated proteins in the rat temporal cortex between 1-month-old and 24-month-old. We showed that the carbonyl levels of ten protein spots corresponding to six gene products: SOD1, SOD2, peroxiredoxin 1, peptidylprolyl isomerase A, cofilin 1, and adenylate kinase 1, significantly increased in the temporal cortex of aged rats. These proteins are associated with antioxidant defense, the cytoskeleton, and energy metabolism. Several oxidized proteins identified in aged rat brain are known to be involved in neurodegenerative disorders as well. Our findings indicate that these carbonylated proteins may be implicated in the decline of normal brain aging process and provide insights into the mechanisms underlying age-associated dysfunction of temporal cortex.