Genetically regulated expression underlies cellular sensitivity to chemotherapy in diverse populations.

Most cancer chemotherapeutic agents are ineffective in a subset of patients; thus, it is important to consider the role of genetic variation in drug response. Lymphoblastoid cell lines (LCLs) in 1000 Genomes Project populations of diverse ancestries are a useful model for determining how genetic factors impact the variation in cytotoxicity. In our study, LCLs from three 1000 Genomes Project populations of diverse ancestries were previously treated with increasing concentrations of eight chemotherapeutic drugs, and cell growth inhibition was measured at each dose with half-maximal inhibitory concentration (IC50) or area under the dose-response curve (AUC) as our phenotype for each drug. We conducted both genome-wide association studies (GWAS) and transcriptome-wide association studies (TWAS) within and across ancestral populations. We identified four unique loci in GWAS and three genes in TWAS to be significantly associated with the chemotherapy-induced cytotoxicity within and across ancestral populations. In the etoposide TWAS, increased STARD5 predicted expression associated with decreased etoposide IC50 (P = 8.5 × 10-8). Functional studies in A549, a lung cancer cell line, revealed that knockdown of STARD5 expression resulted in the decreased sensitivity to etoposide following exposure for 72 (P = 0.033) and 96 h (P = 0.0001). By identifying loci and genes associated with cytotoxicity across ancestral populations, we strive to understand the genetic factors impacting the effectiveness of chemotherapy drugs and to contribute to the development of future cancer treatment.

Identification of Novel Protein Expression Changes Following Cisplatin Treatment and Application to Combination Therapy.

Determining the effect of chemotherapeutic treatment on changes in protein expression can provide important targets for overcoming resistance. Due to challenges in simultaneously measuring large numbers of proteins, a paucity of data exists on global changes. To overcome these challenges, we utilized microwestern arrays that allowed us to measure the abundance and modification state of hundreds of cell signaling and transcription factor proteins in cells following drug exposure. HapMap lymphoblastoid cell lines (LCLs) were exposed to cisplatin, a chemotherapeutic agent commonly used to treat testicular, head and neck, non-small cell lung, and gynecological cancers. We evaluated the expression of 259 proteins following 2, 6, and 12 h of cisplatin treatment in two LCLs with discordant sensitivity to cisplatin. Of these 259 proteins, 66 displayed significantly different protein expression changes (p < 0.05). Fifteen of these proteins were evaluated in a second pair of LCLs with discordant sensitivities to cisplatin; six demonstrated significant differences in expression. We then evaluated a subset of 63 proteins in a second set of LCLs with discordant sensitivity, and 40% of those that were significant in the first pair were also significant in the second part with concordant directionality (p < 0.05). We functionally validated one of the top proteins identified, PDK1, and demonstrated a synergistic relationship between cisplatin and a PDK1 inhibitor in multiple lung cancer lines. This study highlights the potential for identifying novel targets through an understanding of cellular changes in protein expression and modification following drug treatments.

Linking the genetic architecture of cytosine modifications with human complex traits.

Interindividual variation in cytosine modifications could contribute to heterogeneity in disease risks and other complex traits. We assessed the genetic architecture of cytosine modifications at 283,540 CpG sites in lymphoblastoid cell lines (LCLs) derived from independent samples of European and African descent. Our study suggests that cytosine modification variation was primarily controlled in local by single major modification quantitative trait locus (mQTL) and additional minor loci. Local genetic epistasis was detectable for a small proportion of CpG sites, which were enriched by more than 9-fold for CpG sites mapped to population-specific mQTL. Genetically dependent CpG sites whose modification levels negatively (repressive sites) or positively (facilitative sites) correlated with gene expression levels significantly co-localized with transcription factor binding, with the repressive sites predominantly associated with active promoters whereas the facilitative sites rarely at active promoters. Genetically independent repressive or facilitative sites preferentially modulated gene expression variation by influencing local chromatin accessibility, with the facilitative sites primarily antagonizing H3K27me3 and H3K9me3 deposition. In comparison with expression quantitative trait loci (eQTL), mQTL detected from LCLs were enriched in associations for a broader range of disease categories including chronic inflammatory, autoimmune and psychiatric disorders, suggesting that cytosine modification variation, while possesses a degree of cell linage specificity, is more stably inherited over development than gene expression variation. About 11% of unique single-nucleotide polymorphisms reported in the Genome-Wide Association Study Catalog were annotated, 78% as mQTL and 31% as eQTL in LCLs, which covered 37% of the investigated diseases/traits and provided insights to the biological mechanisms.

Association of an inherited genetic variant with vincristine-related peripheral neuropathy in children with acute lymphoblastic leukemia.

IMPORTANCE: With cure rates of childhood acute lymphoblastic leukemia (ALL) exceeding 85%, there is a need to mitigate treatment toxicities that can compromise quality of life, including peripheral neuropathy from vincristine treatment. OBJECTIVE: To identify genetic germline variants associated with the occurrence or severity of vincristine-induced peripheral neuropathy in children with ALL. DESIGN, SETTING, AND PARTICIPANTS: Genome-wide association study of patients in 1 of 2 prospective clinical trials for childhood ALL that included treatment with 36 to 39 doses of vincristine. Genome-wide single-nucleotide polymorphism (SNP) analysis and vincristine-induced peripheral neuropathy were assessed in 321 patients from whom DNA was available: 222 patients (median age, 6.0 years; range, 0.1-18.8 years) enrolled in 1994-1998 in the St Jude Children's Research Hospital protocol Total XIIIB with toxic effects follow-up through January 2001, and 99 patients (median age, 11.4 years; range, 3.0-23.8 years) enrolled in 2007-2010 in the Children's Oncology Group (COG) protocol AALL0433 with toxic effects follow-up through May 2011. Human leukemia cells and induced pluripotent stem cell neurons were used to assess the effects of lower CEP72 expression on vincristine sensitivity. EXPOSURE: Treatment with vincristine at a dose of 1.5 or 2.0 mg/m2. MAIN OUTCOMES AND MEASURES: Vincristine-induced peripheral neuropathy was assessed at clinic visits using National Cancer Institute criteria and prospectively graded as mild (grade 1), moderate (grade 2), serious/disabling (grade 3), or life threatening (grade 4). RESULTS: Grade 2 to 4 vincristine-induced neuropathy during continuation therapy occurred in 28.8% of patients (64/222) in the St Jude cohort and in 22.2% (22/99) in the COG cohort. A SNP in the promoter region of the CEP72 gene, which encodes a centrosomal protein involved in microtubule formation, had a significant association with vincristine neuropathy (meta-analysis P = 6.3×10(-9)). This SNP had a minor allele frequency of 37% (235/642), with 50 of 321 patients (16%; 95% CI, 11.6%-19.5%) homozygous for the risk allele (TT at rs924607). Among patients with the high-risk CEP72 genotype (TT at rs924607), 28 of 50 (56%; 95% CI, 41.2%-70.0%) developed at least 1 episode of grade 2 to 4 neuropathy, a higher rate than in patients with the CEP72 CC or CT genotypes (58/271 patients [21.4%; 95% CI, 16.9%-26.7%]; P = 2.4×10(-6)). The severity of neuropathy was greater in patients homozygous for the TT genotype compared with patients with the CC or CT genotype (2.4-fold by Poisson regression [P<.0001] and 2.7-fold based on mean grade of neuropathy: 1.23 [95% CI, 0.74-1.72] vs 0.45 [95% CI, 0.3-0.6]; P = .004 by t test). Reducing CEP72 expression in human neurons and leukemia cells increased their sensitivity to vincristine. CONCLUSIONS AND RELEVANCE: In this preliminary study of children with ALL, an inherited polymorphism in the promoter region of CEP72 was associated with increased risk and severity of vincristine-related peripheral neuropathy. If replicated in additional populations, this finding may provide a basis for safer dosing of this widely prescribed anticancer agent.

An eQTL-based method identifies CTTN and ZMAT3 as pemetrexed susceptibility markers.

Pemetrexed, approved for the treatment of non-small cell lung cancer and malignant mesothelioma, has adverse effects including neutropenia, leucopenia, thrombocytopenia, anemia, fatigue and nausea. The results we report here represent the first genome-wide study aimed at identifying genetic predictors of pemetrexed response. We utilized expression quantitative trait loci (eQTLs) mapping combined with drug-induced cytotoxicity data to gain mechanistic insights into the observed genetic associations with pemetrexed susceptibility. We found that CTTN and ZMAT3 expression signature explained >30% of the pemetrexed susceptibility phenotype variation for pemetrexed in the discovery population. Replication using PCR and a semi-high-throughput, scalable assay system confirmed the initial discovery results in an independent set of samples derived from the same ancestry. Furthermore, functional validation in both germline and tumor cells demonstrates a decrease in cell survival following knockdown of CTTN or ZMAT3. In addition to our particular findings on genetic and gene expression predictors of susceptibility phenotype for pemetrexed, the work presented here will be valuable to the robust discovery and validation of genetic determinants and gene expression signatures of various chemotherapeutic susceptibilities.

Acquired resistance to immunotherapy and chemoradiation in MYC amplified head and neck cancer.

The proto-oncogene MYC encodes a nuclear transcription factor that has an important role in a variety of cellular processes, such as cell cycle progression, proliferation, metabolism, adhesion, apoptosis, and therapeutic resistance. MYC amplification is consistently observed in aggressive forms of several solid malignancies and correlates with poor prognosis and distant metastases. While the tumorigenic effects of MYC in patients with head and neck squamous cell carcinoma (HNSCC) are well known, the molecular mechanisms by which the amplification of this gene may confer treatment resistance, especially to immune checkpoint inhibitors, remains under-investigated. Here we present a unique case of a patient with recurrent/metastatic (R/M) HNSCC who, despite initial response to nivolumab-based treatment, developed rapidly progressive metastatic disease after the acquisition of MYC amplification. We conducted comparative transcriptomic analysis of this patient's tumor at baseline and upon progression to interrogate potential molecular processes through which MYC may confer resistance to immunotherapy and/or chemoradiation and used TCGA-HNSC dataset and an institutional cohort to further explore clinicopathologic features and key molecular networks associated with MYC amplification in HNSCC. This study highlights MYC amplification as a potential mechanism of immune checkpoint inhibitor resistance and suggest its use as a predictive biomarker and potential therapeutic target in R/M HNSCC.

Population differences in microRNA expression and biological implications.

Population differences observed for complex traits may be attributed to the combined effect of socioeconomic, environmental, genetic and epigenetic factors. To better understand population differences in complex traits, genome-wide genetic and gene expression differences among ethnic populations have been studied. Here we set out to evaluate population differences in small non-coding RNAs through an evaluation of microRNA (miRNA) baseline expression in HapMap lymphoblastoid cell lines (LCLs) derived from 53 CEU (Utah residents with northern and western European ancestry) and 54 YRI (African from Ibadan, Nigeria). Using the Exiqon miRCURYTM LNA arrays, we found that 16% of all miRNAs evaluated in our study differ significantly between these 2 ethnic groups (pBonferroni corrected< 0.05). Furthermore, we explored the potential biological function of these observed differentially expressed miRNAs by comprehensively examining their effect on the transcriptome and their relationship with cellular sensitivity drug phenotypes. After multiple testing adjustment (false discovery rate (FDR)< 0.1), we found that 55% and 88% of the differentially expressed miRNAs were significantly and inversely correlated with an mRNA expression phenotype in the CEU and YRI samples, respectively. Interestingly, a substantial proportion (64%) of these miRNAs correlated with cellular sensitivity to chemotherapeutic agents (FDR< 0.05). Lastly, upon performing a genome-wide association study between SNPs and miRNA expression, we identified a large number of SNPs exhibiting different allele frequencies that affect the expression of these differentially expressed miRNAs, suggesting the role of genetic variants in mediating the observed population differences.

Identification of small molecules that mitigate vincristine-induced neurotoxicity while sensitizing leukemia cells to vincristine.

Vincristine (VCR) is one of the most widely prescribed medications for treating solid tumors and acute lymphoblastic leukemia (ALL) in children and adults. However, its major dose-limiting toxicity is peripheral neuropathy that can disrupt curative therapy. Peripheral neuropathy can also persist into adulthood, compromising quality of life of childhood cancer survivors. Reducing VCR-induced neurotoxicity without compromising its anticancer effects would be ideal. Here, we show that low expression of NHP2L1 is associated with increased sensitivity of primary leukemia cells to VCR, and that concomitant administration of VCR with inhibitors of NHP2L1 increases VCR cytotoxicity in leukemia cells, prolongs survival of ALL xenograft mice, but decreases VCR effects on human-induced pluripotent stem cell-derived neurons and mitigates neurotoxicity in mice. These findings offer a strategy for increasing VCR's antileukemic effects while reducing peripheral neuropathy in patients treated with this widely prescribed medication.

Procedures for the Synthesis and Capping of Metal Nanoparticles.

The increasing impact of metallic nanoparticles on life sciences has stimulated the development of new techniques and multiple improvements to the existing methods of manufacturing nanoparticles with tailored properties. Nanoparticles can be synthesized through a variety of physical and chemical methods. The choice of preparation procedure will depend on the physical and chemical characteristics required in the final product, such as size, dispersion, chemical miscibility, and optical properties, among others. Here we review basic practical procedures used for the preparation of protected and unprotected metallic nanoparticles and describe a number of experimental procedures based on colloidal chemistry methods. These include gold nanoparticle synthesis by reduction with trisodium citrate, ascorbic acid, or sugars in aqueous phase and nanoparticle passivation with alkanethiols, CTAB, or BSA. We also describe microwave-assisted synthesis, nanoparticle synthesis in ethylene glycol, and template-assisted synthesis with dendrimers, and, briefly, how to control nanoparticle shape (star-shaped and branched nanoparticles).

Pt-Ni/ZnO-rod catalysts for hydrogen production by steam reforming of methanol with oxygen.

Ni, Pt and a mixture of Ni and Pt supported on ZnO-rods were evaluated in autothermal steam reforming of methanol (ASRM) for hydrogen production as a function of the reaction temperature. The catalytic materials were characterized by SEM-EDS, XRD, TEM, HRTEM, TPR and BET. Analysis by SEM and TEM showed structural modifications on the surface of the ZnO rods after Ni impregnation. The reactivity of the catalytic materials in the range of 200-500 °C showed that the bimetallic sample had better catalytic activity among all the catalysts studied. This finding could be associated to PtZn and NiZn alloys present in this catalyst, which were identified by XRD and HRTEM analyses. Catalyst characterization by XRD after the catalytic testing showed that the intermetallic PtZn phase was stable during the reaction in the Pt/ZnO-rod sample. The cubic Ni0.75-Zn0.25 structure identified in the Ni/ZnO-rod sample was transformed to Zn0.1-Ni0.9-O and metallic Ni phases, respectively. On the bimetallic PtNi/ZnO-rod sample, the cubic Ni0.75-Zn0.25 structure remained, although the tetragonal NiZn structure is unstable and was destroyed during the ASRM reaction and then a new phase of Ni0.7Pt0.3 emerged. The promotion effect of Pt and/or Ni on the ZnO-rod was clearly shown.

Clinical and Genome-wide Analysis of Cisplatin-induced Tinnitus Implicates Novel Ototoxic Mechanisms.

PURPOSE: Cisplatin, a commonly used chemotherapeutic, results in tinnitus, the phantom perception of sound. Our purpose was to identify the clinical and genetic determinants of tinnitus among testicular cancer survivors (TCS) following cisplatin-based chemotherapy. EXPERIMENTAL DESIGN: TCS (n = 762) were dichotomized to cases (moderate/severe tinnitus; n = 154) and controls (none; n = 608). Logistic regression was used to evaluate associations with comorbidities and SNP dosages in genome-wide association study (GWAS) following quality control and imputation (covariates: age, noise exposure, cisplatin dose, genetic principal components). Pathway over-representation tests and functional studies in mouse auditory cells were performed. RESULTS: Cisplatin-induced tinnitus (CisIT) significantly associated with age at diagnosis (P = 0.007) and cumulative cisplatin dose (P = 0.007). CisIT prevalence was not significantly greater in 400 mg/m2-treated TCS compared with 300 (P = 0.41), but doses >400 mg/m2 (median 580, range 402-828) increased risk by 2.61-fold (P < 0.0001). CisIT cases had worse hearing at each frequency (0.25-12 kHz, P < 0.0001), and reported more vertigo (OR = 6.47; P < 0.0001) and problems hearing in a crowd (OR = 8.22; P < 0.0001) than controls. Cases reported poorer health (P < 0.0001) and greater psychotropic medication use (OR = 2.4; P = 0.003). GWAS suggested a variant near OTOS (rs7606353, P = 2 × 10-6) and OTOS eQTLs were significantly enriched independently of that SNP (P = 0.018). OTOS overexpression in HEI-OC1, a mouse auditory cell line, resulted in resistance to cisplatin-induced cytotoxicity. Pathway analysis implicated potassium ion transport (q = 0.007). CONCLUSIONS: CisIT associated with several neuro-otological symptoms, increased use of psychotropic medication, and poorer health. OTOS, expressed in the cochlear lateral wall, was implicated as protective. Future studies should investigate otoprotective targets in supporting cochlear cells.

Predicting Acute Renal Injury in Cancer Patients Receiving Cisplatin Using Urinary Neutrophil Gelatinase-Associated Lipocalin and Cystatin C.

Acute kidney injury (AKI) limits cisplatin use. We tested whether urine cystatin C (uCyC) and neutrophil gelatinase-associated lipocalin (uNGAL) can preidentify patients at risk for AKI. Patients initiating cisplatin-based chemotherapy were prospectively enrolled. uNGAL/uCyC were measured pre/post-cisplatin administration and compared with serum creatinine (sCr). AKI was defined as sCr increase ≥50% or ≥0.3 mg/dL above baseline. In all, 102 patients were enrolled; 95 provided evaluable data. Twenty-five patients developed AKI. Median baseline and pre-cisplatin uNGAL levels were significantly higher in AKI patients. Although immediate changes in uNGAL/uCyC 2 h after cisplatin were not detectable, post-cisplatin peak values over the course of therapy were markedly and significantly elevated in AKI patients. In multivariate modeling with age, baseline glomerular filtration rate, and histology, maximum uCyC was a significant independent AKI predictor. These findings suggest pre-cisplatin uNGAL and peak uCyC levels can identify patients with increased AKI risk, potentially allowing for tailored modification of cisplatin-based treatment regimens.

Gene and MicroRNA Perturbations of Cellular Response to Pemetrexed Implicate Biological Networks and Enable Imputation of Response in Lung Adenocarcinoma.

Pemetrexed is indicated for non-small cell lung carcinoma and mesothelioma, but often has limited efficacy due to drug resistance. To probe the molecular mechanisms underlying chemotherapeutic response, we performed mRNA and microRNA (miRNA) expression profiling of pemetrexed treated and untreated lymphoblastoid cell lines (LCLs) and applied a hierarchical Bayesian method. We identified genetic variation associated with gene expression in human lung tissue for the most significant differentially expressed genes (Benjamini-Hochberg [BH] adjusted p < 0.05) using the Genotype-Tissue Expression data and found evidence for their clinical relevance using integrated molecular profiling and lung adenocarcinoma survival data from The Cancer Genome Atlas project. We identified 39 miRNAs with significant differential expression (BH adjusted p < 0.05) in LCLs. We developed a gene expression based imputation model of drug sensitivity, quantified its prediction performance, and found a significant correlation of the imputed phenotype generated from expression data with survival time in lung adenocarcinoma patients. Differentially expressed genes (MTHFD2 and SUFU) that are putative targets of differentially expressed miRNAs also showed differential perturbation in A549 fusion lung tumor cells with further replication in A549 cells. Our study suggests pemetrexed may be used in combination with agents that target miRNAs to increase its cytotoxicity.

Application of stem cell derived neuronal cells to evaluate neurotoxic chemotherapy.

The generation of induced pluripotent stem cells (iPSCs) and differentiation to cells composing major organs has opened up the possibility for a new model system to study adverse toxicities associated with chemotherapy. Therefore, we used human iPSC-derived neurons to study peripheral neuropathy, one of the most common adverse effects of chemotherapy and cause for dose reduction. To determine the utility of these neurons in investigating the effects of neurotoxic chemotherapy, we measured morphological differences in neurite outgrowth, cell viability as determined by ATP levels and apoptosis through measures of caspase 3/7 activation following treatment with clinically relevant concentrations of platinating agents (cisplatin, oxaliplatin and carboplatin), taxanes (paclitaxel, docetaxel and nab-paclitaxel), a targeted proteasome inhibitor (bortezomib), an antiangiogenic compound (thalidomide), and 5-fluorouracil, a chemotherapeutic that does not cause neuropathy. We demonstrate differential sensitivity of neurons to mechanistically distinct classes of chemotherapeutics. We also show a dose-dependent reduction of electrical activity as measured by mean firing rate of the neurons following treatment with paclitaxel. We compared neurite outgrowth and cell viability of iPSC-derived cortical (iCell® Neurons) and peripheral (Peri.4U) neurons to cisplatin, paclitaxel and vincristine. Goshajinkigan, a Japanese herbal neuroprotectant medicine, was protective against paclitaxel-induced neurotoxicity but not oxaliplatin as measured by morphological phenotypes. Thus, we have demonstrated the utility of human iPSC-derived neurons as a useful model to distinguish drug class differences and for studies of a potential neuroprotectant for the prevention of chemotherapy-induced peripheral neuropathy.

Evaluation of inter-batch differences in stem-cell derived neurons.

Differentiated cells retain the genetic information of the donor but the extent to which phenotypic differences between donors or batches of differentiated cells are explained by variation introduced during the differentiation process is not fully understood. In this study, we evaluated four separate batches of commercially available neurons originating from the same iPSCs to investigate whether the differentiation process used in manufacturing iPSCs to neurons affected genome-wide gene expression and modified cytosines, or neuronal sensitivity to drugs. No significant changes in gene expression, as measured by RNA-Seq, or cytosine modification levels, as measured by the Illumina 450K arrays, were observed between batches relative to changes over time. As expected, neurotoxic chemotherapeutics affected neuronal outgrowth, but no inter-batch differences were observed in sensitivity to paclitaxel, vincristine and cisplatin. As a testament to the utility of the model for studies of neuropathy, we observed that genes involved in neuropathy had relatively higher expression levels in these samples across different time points. Our results suggest that the process used to differentiate iPSCs into neurons is consistent, resulting in minimal intra-individual variability across batches. Therefore, this model is reasonable for studies of human neuropathy, druggable targets to prevent neuropathy, and other neurological diseases.

Pharmacogenetic Discovery in CALGB (Alliance) 90401 and Mechanistic Validation of a VAC14 Polymorphism that Increases Risk of Docetaxel-Induced Neuropathy.

PURPOSE: Discovery of SNPs that predict a patient's risk of docetaxel-induced neuropathy would enable treatment individualization to maximize efficacy and avoid unnecessary toxicity. The objectives of this analysis were to discover SNPs associated with docetaxel-induced neuropathy and mechanistically validate these associations in preclinical models of drug-induced neuropathy. EXPERIMENTAL DESIGN: A genome-wide association study was conducted in metastatic castrate-resistant prostate cancer patients treated with docetaxel, prednisone and randomized to bevacizumab or placebo on CALGB 90401. SNPs were genotyped on the Illumina HumanHap610-Quad platform followed by rigorous quality control. The inference was conducted on the cumulative dose at occurrence of grade 3+ sensory neuropathy using a cause-specific hazard model that accounted for early treatment discontinuation. Genes with SNPs significantly associated with neuropathy were knocked down in cellular and mouse models of drug-induced neuropathy. RESULTS: A total of 498,081 SNPs were analyzed in 623 Caucasian patients, 50 (8%) of whom experienced grade 3+ neuropathy. The 1,000 SNPs most associated with neuropathy clustered in relevant pathways including neuropathic pain and axonal guidance. An SNP in VAC14 (rs875858) surpassed genome-wide significance (P = 2.12 × 10-8, adjusted P = 5.88 × 10-7). siRNA knockdown of VAC14 in stem cell-derived peripheral neuronal cells increased docetaxel sensitivity as measured by decreased neurite processes (P = 0.0015) and branches (P < 0.0001). Prior to docetaxel treatment, VAC14 heterozygous mice had greater nociceptive sensitivity than wild-type litter mate controls (P = 0.001). CONCLUSIONS: VAC14 should be prioritized for further validation of its potential role as a predictor of docetaxel-induced neuropathy and biomarker for treatment individualization. Clin Cancer Res; 22(19); 4890-900. ©2016 AACR.

2.45 GHz radiofrequency fields alter gene expression in cultured human cells.

The biological effect of radiofrequency (RF) fields remains controversial. We address this issue by examining whether RF fields can cause changes in gene expression. We used the pulsed RF fields at a frequency of 2.45 GHz that is commonly used in telecommunication to expose cultured human HL-60 cells. We used the serial analysis of gene expression (SAGE) method to measure the RF effect on gene expression at the genome level. We observed that 221 genes altered their expression after a 2-h exposure. The number of affected genes increased to 759 after a 6-h exposure. Functional classification of the affected genes reveals that apoptosis-related genes were among the upregulated ones and the cell cycle genes among the downregulated ones. We observed no significant increase in the expression of heat shock genes. These results indicate that the RF fields at 2.45 GHz can alter gene expression in cultured human cells through non-thermal mechanism.

Temperature effect on the synthesis of Au-Pt bimetallic nanoparticles.

Pt-Au bimetallic nanoparticles have been synthesized by the polyol method and stabilized with poly(vinylpyrrolidone) (PVP), modifying the temperature of synthesis. Interesting structure changes were observed in the nanoparticles as the temperature was varied. At lower temperatures no bimetallic nanoparticles were detected, but as the temperature increased bimetallic nanoparticles started to appear, commonly obtaining core-shell nanoparticles, always covered by the polymer. This originates the modification of the optical response of the system in the UV-visible region. An absorption peak centered at 520 nm at low temperatures was observed (100-110 degrees C); at higher temperatures (130-170 degrees C) there were non detectable absorption peaks, and finally at the two highest temperatures (180-190 degrees C) the reappearance of an absorption feature centered at 510 nm was noticed. These UV-visible results indirectly imply the composition of the surface of the particle. The structure of the particles has been determined using transmission electron microscopy and high-angle annular dark field (HAADF), the latter being a powerful technique to determine the structural composition of the particles and allowing a direct correlation of the optical response with their structural composition. X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) studies were also performed on the samples and their results support the idea of a Pt(core)-Au(shell) structure with the elements segregated from each other. The combination of these experimental techniques with calculated UV-vis absorption spectra allowed, in a reliable way, the elucidation of the nanoparticles structure and elemental distribution.

Modeling chemotherapeutic neurotoxicity with human induced pluripotent stem cell-derived neuronal cells.

There are no effective agents to prevent or treat chemotherapy-induced peripheral neuropathy (CIPN), the most common non-hematologic toxicity of chemotherapy. Therefore, we sought to evaluate the utility of human neuron-like cells derived from induced pluripotent stem cells (iPSCs) as a means to study CIPN. We used high content imaging measurements of neurite outgrowth phenotypes to compare the changes that occur to iPSC-derived neuronal cells among drugs and among individuals in response to several classes of chemotherapeutics. Upon treatment of these neuronal cells with the neurotoxic drug paclitaxel, vincristine or cisplatin, we identified significant differences in five morphological phenotypes among drugs, including total outgrowth, mean/median/maximum process length, and mean outgrowth intensity (P < 0.05). The differences in damage among drugs reflect differences in their mechanisms of action and clinical CIPN manifestations. We show the potential of the model for gene perturbation studies by demonstrating decreased expression of TUBB2A results in significantly increased sensitivity of neurons to paclitaxel (0.23 ± 0.06 decrease in total neurite outgrowth, P = 0.011). The variance in several neurite outgrowth and apoptotic phenotypes upon treatment with one of the neurotoxic drugs is significantly greater between than within neurons derived from four different individuals (P < 0.05), demonstrating the potential of iPSC-derived neurons as a genetically diverse model for CIPN. The human neuron model will allow both for mechanistic studies of specific genes and genetic variants discovered in clinical studies and for screening of new drugs to prevent or treat CIPN.

Pharmacoethnicity in Paclitaxel-Induced Sensory Peripheral Neuropathy.

PURPOSE: Paclitaxel is used worldwide in the treatment of breast, lung, ovarian, and other cancers. Sensory peripheral neuropathy is an associated adverse effect that cannot be predicted, prevented, or mitigated. To better understand the contribution of germline genetic variation to paclitaxel-induced peripheral neuropathy, we undertook an integrative approach that combines genome-wide association study (GWAS) data generated from HapMap lymphoblastoid cell lines (LCL) and Asian patients. METHODS: GWAS was performed with paclitaxel-induced cytotoxicity generated in 363 LCLs and with paclitaxel-induced neuropathy from 145 Asian patients. A gene-based approach was used to identify overlapping genes and compare with a European clinical cohort of paclitaxel-induced neuropathy. Neurons derived from human-induced pluripotent stem cells were used for functional validation of candidate genes. RESULTS: SNPs near AIPL1 were significantly associated with paclitaxel-induced cytotoxicity in Asian LCLs (P < 10(-6)). Decreased expression of AIPL1 resulted in decreased sensitivity of neurons to paclitaxel by inducing neurite morphologic changes as measured by increased relative total outgrowth, number of processes and mean process length. Using a gene-based analysis, there were 32 genes that overlapped between Asian LCL cytotoxicity and Asian patient neuropathy (P < 0.05), including BCR. Upon BCR knockdown, there was an increase in neuronal sensitivity to paclitaxel as measured by neurite morphologic characteristics. CONCLUSIONS: We identified genetic variants associated with Asian paclitaxel-induced cytotoxicity and functionally validated the AIPL1 and BCR in a neuronal cell model. Furthermore, the integrative pharmacogenomics approach of LCL/patient GWAS may help prioritize target genes associated with chemotherapeutic-induced peripheral neuropathy.