Geographic variation of mutagenic exposures in kidney cancer genomes.

International differences in the incidence of many cancer types indicate the existence of carcinogen exposures that have not yet been identified by conventional epidemiology make a substantial contribution to cancer burden1. In clear cell renal cell carcinoma, obesity, hypertension and tobacco smoking are risk factors, but they do not explain the geographical variation in its incidence2. Underlying causes can be inferred by sequencing the genomes of cancers from populations with different incidence rates and detecting differences in patterns of somatic mutations. Here we sequenced 962 clear cell renal cell carcinomas from 11 countries with varying incidence. The somatic mutation profiles differed between countries. In Romania, Serbia and Thailand, mutational signatures characteristic of aristolochic acid compounds were present in most cases, but these were rare elsewhere. In Japan, a mutational signature of unknown cause was found in more than 70% of cases but in less than 2% elsewhere. A further mutational signature of unknown cause was ubiquitous but exhibited higher mutation loads in countries with higher incidence rates of kidney cancer. Known signatures of tobacco smoking correlated with tobacco consumption, but no signature was associated with obesity or hypertension, suggesting that non-mutagenic mechanisms of action underlie these risk factors. The results of this study indicate the existence of multiple, geographically variable, mutagenic exposures that potentially affect tens of millions of people and illustrate the opportunities for new insights into cancer causation through large-scale global cancer genomics.

Mode of action, in vitro activity, and in vivo efficacy of AFN-1252, a selective antistaphylococcal FabI inhibitor.

The mechanism of action of AFN-1252, a selective inhibitor of Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI), which is involved in fatty acid biosynthesis, was confirmed by using biochemistry, macromolecular synthesis, genetics, and cocrystallization of an AFN-1252-FabI complex. AFN-1252 demonstrated a low propensity for spontaneous resistance development and a time-dependent reduction of the viability of both methicillin-susceptible and methicillin-resistant S. aureus, achieving a ≥2-log(10) reduction in S. aureus counts over 24 h, and was extremely potent against clinical isolates of S. aureus (MIC(90), 0.015 µg/ml) and coagulase-negative staphylococci (MIC(90), 0.12 µg/ml), regardless of their drug resistance, hospital- or community-associated origin, or other clinical subgroup. AFN-1252 was orally available in mouse pharmacokinetic studies, and a single oral dose of 1 mg/kg AFN-1252 was efficacious in a mouse model of septicemia, providing 100% protection from an otherwise lethal peritoneal infection of S. aureus Smith. A median effective dose of 0.15 mg/kg indicated that AFN-1252 was 12 to 24 times more potent than linezolid in the model. These studies, demonstrating a selective mode of action, potent in vitro activity, and in vivo efficacy, support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.

Reaction mechanism of melatonin oxidation by reactive oxygen species in vitro.

Melatonin (N-acetyl-5-hydroxytryptamine) is a pineal hormone widely known for its antioxidant properties, both in vivo and by direct capture of free radicals in vitro. Although some metabolites and oxidation products of melatonin have been identified, the molecular mechanism by which melatonin exerts its antioxidant properties has not been totally unravelled. This study investigated the reaction mechanism of oxidation of melatonin by radio-induced reactive oxygen species, generated by gamma radiolysis of water for aqueous solutions of melatonin (from 20 to 200 µm), in the presence or absence of molecular oxygen. The hydroxyl radical was found to be the unique species able to initiate the oxidation process, leading to three main products, e.g. N(1)-acetyl-N(2)-formyl-5-methoxykynurenin (AFMK), N(1)-acetyl-5-methoxykynurenin (AMK) and hydroxymelatonin (HO-MLT). The generation of AFMK and HO-MLT strongly depended on the presence of molecular oxygen in solution: AFMK was the major product in aerated solutions (84%), whereas HO-MLT was favoured in the absence of oxygen (86%). Concentrations of AMK remained quite low, and AMK was proposed to result from a chemical hydrolysis of AFMK in solution. A K-value of 1.1 × 10(-4) was calculated for this equilibrium. Both hydrogen peroxide and superoxide dismutase had no effect on the radio-induced oxidation of melatonin, in good accordance for the second case with the poor reactivity of the superoxide anion towards melatonin. Finally, a reaction mechanism was proposed for the oxidation of melatonin in vitro.

Melatonin protects PLPC liposomes and LDL towards radical-induced oxidation.

This study investigated the in vitro protective effects of melatonin against oxidation of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) liposomes [(PLPC) = 250 µm] and low-density lipoproteins (LDL, 3 g/L total concentration) by hydroxyl radicals produced by water gamma radiolysis. Conjugated dienes (CD) and hydroperoxides from cholesteryl esters (CEOOH) and phospholipids (PCOOH) were measured as indices of lipid peroxidation. Protein (apoB) oxidation in LDL was assessed by carbonyl groups. Two LDL antioxidants (vitamin E and ß-carotene) were monitored as a function of the radiation dose. Three concentrations of melatonin were studied in PLPC liposomes, i.e., 20, 50 and 100 µm, and one in LDL, i.e., 100 µm. Melatonin consumption was also followed up in both lipid models upon irradiation, together with the residual PLPC concentration in liposomes. In PLPC liposomes, scavenging of lipid-derived peroxyl radicals was not the only phenomenon to explain the protective properties of melatonin towards lipid peroxidation. Indeed, melatonin also reacted with hydroxyl radicals generated in aqueous phase, which led us to suggest that hydroxyl radicals reacted relatively slowly with PLPC. Melatonin was efficient in lowering lipid peroxidation in LDL, as shown by the decrease in the formation of CDs and in hydroperoxides. Moreover, melatonin clearly slowed radio-induced apolipoprotein B carbonylation and protected α-tocopherol and ß-carotene in LDL.

Liquid chromatographic/electrospray ionization mass spectrometric identification of the oxidation end-products of trans-resveratrol in aqueous solutions.

trans-Resveratrol (3,5,4'-trihydroxystilbene) is a natural polyphenolic compound that exhibits antioxidant properties. Our study aimed at studying the HO*-induced oxidation of resveratrol (100 micromol.L(-1)) in aerated aqueous solutions. Gamma radiolysis of water was used to generate HO*/O(2)(*-) free radicals (I = 10 Gy.min(-1), dose = 400 Gy). Oxidation products were identified by direct infusion mass spectrometry and high-performance liquid chromatography/mass spectrometry. For each product, structural elucidation was based on simple mass spectra, fragmentation spectra and deuterium/hydrogen exchange spectra; the comparison with mass spectra of synthetic products provided valuable information allowing the complete identification of the oxidation products. Four products resulting from the direct attack of HO* radicals towards resveratrol were identified respectively as piceatannol (trans-3,5,3',4'-tetrahydroxystilbene), 3,5-dihydroxybenzoic acid, 3,5-dihydroxybenzaldehyde and 4-hydroxybenzaldehyde.

Biobanking in the COVID-19 Era and Beyond: Part 2. A Set of Tool Implementation Case Studies.

The COVID-19 era has brought about a number of novel challenges for the global biobanking community. An array of diverse tools (e.g., standards, best practices, and plans) exists to support quality and fitness-for-purpose in biobank operations. The International Society for Biological and Environmental Repositories (ISBER) COVID-19 Response Task Force has set out to identify needs and gaps in these tools and make recommendations for the next generation of available tools, having closely examined the COVID-19-related challenges. While conducting this work to examine the relationships between tools and biobank adaptability, a subgroup of the task force conducted a parallel effort to develop and describe individual COVID-19 era case studies based on a number of operating biobanks. Each case study presents a different combination of implemented tools. Observations and lessons learned from these case studies are provided, and experiences with tool implementation are discussed. This information is supplemented by data relating to tool usefulness that was obtained through an ISBER survey discussed in a companion article. The knowledge gained from this study will be combined with other task force efforts to make recommendations to better position the biobanking community in their response to future emergencies.

Biobanking in the COVID-19 Era and Beyond: Part 1. How Early Experiences Can Translate into Actionable Wisdom.

The era of COVID-19 has brought about a number of novel challenges for the global biobanking community. To better position the biobanking community to cope with current and future challenges, the International Society for Biological and Environmental Repositories (ISBER) COVID-19 Response Task Force was convened to identify needs and gaps in biobanking tools (existing resources that support good practice), for example, standards, best practices, business, etc. and to make recommendations to benefit the community. Toward these goals, the Task Force assembled a set of questions to explore individual biobanks' experiences, with emphasis on identification of key challenges and approaches, including tools employed. A survey was designed with the use of these questions and administered by ISBER. This article presents a summary of the aggregated data obtained from the survey responses, illustrating some of the major issues encountered and identifying which tools the survey respondents found most useful. In particular, this article focuses on the challenges identified during the early months of the COVID-19 era. Recommendations are provided to support biobank emergency preparedness for the future, address lessons learned, and propose solutions to bridge identified gaps. The analysis and the complete survey dataset will also inform the larger Task Force goal to develop specific tool recommendations.

High-resolution array CGH identifies novel regions of genomic alteration in intermediate-risk prostate cancer.

Approximately one-third of prostate cancer patients present with intermediate risk disease. Interestingly, while this risk group is clinically well defined, it demonstrates the most significant heterogeneity in PSA-based biochemical outcome. Further, the majority of candidate genes associated with prostate cancer progression have been identified using cell lines, xenograft models, and high-risk androgen-independent or metastatic patient samples. We used a global high-resolution array comparative genomic hybridization (CGH) assay to characterize copy number alterations (CNAs) in intermediate risk prostate cancer. Herein, we show this risk group contains a number of alterations previously associated with high-risk disease: (1) deletions at 21q22.2 (TMPRSS2:ERG), 16q22-24 (containing CDH1), 13q14.2 (RB1), 10q23.31 (PTEN), 8p21 (NKX3.1); and, (2) amplification at 8q21.3-24.3 (containing c-MYC). In addition, we identified six novel microdeletions at high frequency: 1q42.12-q42.3 (33.3%), 5q12.3-13.3 (21%), 20q13.32-13.33 (29.2%), 22q11.21 (25%), 22q12.1 (29.2%), and 22q13.31 (33.3%). Further, we show there is little concordance between CNAs from these clinical samples and those found in commonly used prostate cancer cell models. These unexpected findings suggest that the intermediate-risk category is a crucial cohort warranting further study to determine if a unique molecular fingerprint can predict aggressive versus indolent phenotypes.

Radiolytic yield of cardiolipin peroxidation by gamma rays in large unilamellar vesicles of phosphatidylcholine.

Large unilamellar vesicles of 1-hexanoyl-2-(9Z-12Z-octadecadienoyl)-sn-glycero-3-phosphocholine (PLPC) have been used as model membrane to investigate the effect of increasing amount of cardiolipin (1',3'-bis-[1,2-Di-(9Z-12Z-octadecadienoyl)-sn-glycero-3-phospho]-sn-glycerol, CL) on the peroxidizability of the lipid phase. Hydroxyl radicals generated by gamma radiolysis of water initiated the lipid peroxidation. Both peroxidation products (conjugated dienes and hydroperoxides of PLPC, mono- and dihydroperoxides of CL) and disappearance of CL and PLPC were assessed as a function of the radiation dose (25 to 400 Gy, I = 10 Gy min(-1)). Our results show that the addition of 5% to 15% CL to large unilamellar vesicles (concentration ratio) produces almost complete inhibition of PLPC peroxidation. Thus, for 15% CL (known to be the proportion of CL in the inner mitochondrial membrane), the radiolytic yield of formation of PLPC hydroperoxides is reduced to zero, whereas it is equal to (3.1 +/- 0.2) x 10(-7) mol J(-1) for CL hydroperoxides, showing the importance of the targeted CL. For this concentration ratio (CL/ PLPC 15%), we have established the balance equation between the consumption of CL [G(-CL) = (2.8 +/- 0.1) x 10(-7) mol J(-1)] and the formation of CL hydroperoxides [G(CLOOH(T)) = (3.1 +/- 0.2) x 10(-7) mol J(-1)]. In addition, the radiolytic yields of disappearance of PLPC and CL have been determined [(1.5 +/- 0.1) x 10(-7) mol J(-1) and (2.8 +/- 0.1) x 10(-7) mol J(-1), respectively], their sum [(4.3 +/- 0.2) x 10(-7) mol J(-1)] being higher than G(HO.) (2.8 x 10(-7) mol J(-1)). However, there is no balance between the radiolytic yield of formation of PLPC hydroperoxides [G (PCOOH(T)) approximately 0] and the yield of disappearance of PLPC [(1.5 +/- 0.1) x 10(-7) mol J(-1)], likely because lipid fragments (not measured in this work) could be generated from HO(.) reaction on the polar head of PLPC. These results have been interpreted by assuming that the hydroxyl radicals attack in competition both lipid targets, i.e. PLPC and CL, with a higher sensitivity to CL oxidation. It can be concluded that a little amount of CL (10-15% CL/ PLPC concentration ratio) may exert a strong protective effect against the HO(.)-induced peroxidation of PLPC.

RNA and DNA Integrity Remain Stable in Frozen Tissue After Long-Term Storage at Cryogenic Temperatures: A Report from the Ontario Tumour Bank.

Background: It is widely assumed that the integrity of tissue specimens remains stable indefinitely if preserved at cryogenic temperatures. With biobanking reaching a level of maturity where samples are increasingly stored for 10 years and beyond, this assumption of prolonged stability should be tested. Data from such an assessment are critical to verify if samples stored for extended durations remain "fit for purpose" or if there is need to reconsider the utility of samples stored beyond a certain timeframe. The Ontario Tumour Bank has been collecting samples since 2004, and assesses a random selection of frozen samples each year for RNA and DNA integrity as a part of ongoing quality control (QC) practices. This historical quality assessment data provide a unique opportunity to assess the impact of extended storage on nucleic acid integrity using replicate samples that remain in the bank in the present day as comparators. Methods: To examine the stability of fresh-frozen tumor tissue stored at cryogenic temperatures, RNA was extracted and analyzed from 87 cases over 14 disease sites stored long term in vapor-phase liquid nitrogen (LN2) (approximately -180°C). Historical QC data were compared against new data after re-extraction of replicate samples to determine the effect of extended storage on RNA quality. In addition, DNA was extracted from a subselection of samples (n = 20) to determine the effect of prolonged storage on DNA integrity. Results: No time-dependent decrease in tissue RNA or DNA quality, as measured by RNA integrity number (RIN) and DNA integrity number, was observed over an 11-year period. As a secondary observation, RNA integrity was not predictive of DNA integrity: DNA quality may still be very good, and as such RIN scores should not be used as a substitute indicator for evaluating DNA. Conclusions: Extended cryogenic storage beyond 2-11 years remains a viable option for maintaining the high quality of specimens in biobanks.

Marked difference in cytochrome c oxidation mediated by HO(*) and/or O(2)(*-) free radicals in vitro.

Cytochrome c (cyt c) is an electron carrier involved in the mitochondrial respiratory chain and a critical protein in apoptosis. The oxidation of cytochrome c can therefore be relevant biologically. We studied whether cytochrome c underwent the attack of reactive oxygen species (ROS) during ionizing irradiation-induced oxidative stress. ROS were generated via water radiolysis under ionizing radiation (IR) in vitro. Characterization of oxidation was performed by mass spectrometry, after tryptic digestion, and UV-visible spectrophotometry. When both hydroxyl and superoxide free radicals were generated during water radiolysis, only five tryptic peptides of cyt c were reproducibly identified as oxidized according to a relation that was dependent of the dose of ionizing radiation. The same behavior was observed when hydroxyl free radicals were specifically generated (N(2)O-saturated solutions). Specific oxidation of cyt c by superoxide free radicals was performed and has shown that only one oxidized peptide (MIFAGIK+16), corresponding to the oxidation of Met80 into methionine sulfoxide, exhibited a radiation dose-dependent formation. In addition, the enzymatic site of cytochrome c was sensitive to the attack of both superoxide and hydroxyl radicals as observed through the reduction of Fe(3+), the degradation of the protoporphyrin IX and the oxidative disruption of the Met80-Fe(3+) bond. Noteworthy, the latter has been involved in the conversion of cyt c to a peroxidase. Finally, Met80 appears as the most sensitive residue towards hydroxyl but also superoxide free radicals mediated oxidation.

Development of an acrylate monolith in a cyclo-olefin copolymer microfluidic device for chip electrochromatography separation.

An acrylate monolith has been synthesized into a cyclic olefin copolymer microdevice for reversed-phase electrochromatography purposes. Microchannels, designed by hot embossing, were filled up with an acrylate monolith to serve as a hydrophobic stationary phase. A lauryl acrylate monolith was formulated to suit the hydrophobic material, by implementing 100% organic porogenic solvent. This new composition was tested in capillary prior to its transfer into the microfluidic device. Surface functionalization of the cyclic olefin copolymer surface was applied using UV-grafting technique to improve the covalent attachment of this monolith to the plastic walls of the microfluidic chip. The on-chip performances of this monolith were evaluated in detail for the reversed-phase electrochromatographic separation of polycyclic aromatic hydrocarbons, with plate heights reaching down to 10 microm when working at optimal velocity.

Chain-breaking activity of resveratrol and piceatannol in a linoleate micellar model.

This study investigated the in vitro protective effects of three derivatives of resveratrol, i.e., piceatannol (trans-3,5,3',4'-tetrahydroxystilbene), PDM2 (1,3-dichloro-5-[(1E)-2-(4-chlorophenyl)ethenyl]-benzene) and PDM11 ((E)-5-[2-(4-chlorophenyl)ethenyl]-1,3-dimethoxyphenyl-ethene), compared with resveratrol as reference compound, against oxidation of linoleate micelles (10(-2)M) initiated by radiolysis-generated hydroxyl radicals. Lipid peroxidation was monitored by conjugated dienes (differential absorbance at 234nm), and by hydroperoxides (reverse phase HPLC with chemiluminescence detection). The higher the concentration of resveratrol or piceatannol (from 10(-5)M to 10(-4)M), the stronger the antioxidant ability. Piceatannol, with the presence of an additional hydroxyl group, showed a better antioxidant effect than resveratrol for a given concentration (competition with the fatty acid to scavenge lipid peroxyl radicals LOO), whereas PDM2 and PDM11, without any hydroxyl group, did not exhibit any significant protective effect. A lower limit for the LOO rate constant has been estimated for piceatannol (>/=1.4x10(5)M(-1)s(-1)) and for resveratrol (>/=0.3x10(5)M(-1)s(-1)).

Visual detection of affordances for aperture negotiation in people with Parkinson disease.

BACKGROUND: An essential requirement for the guidance of action in cluttered environments is that people can accurately perceive what actions are afforded by particular surroundings given the person's action capabilities. Research has shown that healthy young individuals turn their shoulders when walking through a doorway when the aperture is less than a certain percentage of their shoulder width and that they are able to detect this critical width with visual inspection. These findings imply that movements are constrained by perception of the environment in body-scaled unit. OBJECTIVES: The present work examined whether the visual affordance of doorway passability is altered in people with Parkinson disease (PD). METHODS: People with PD, healthy age-matched controls, and young adults (16 participants per group) walked through a series of apertures scaled to shoulder width. Participants also had to visually judge a series of apertures to determine if they could walk through the gap with their normal gait pattern. Finally, participants had to estimate their eye height. RESULTS: Statistical analysis revealed that people with PD initiated shoulder turning to go through the doorway at larger apertures (A) relative to their shoulder (S) width (A/S = 1.61) in comparison to healthy age-matched participants (A/S = 1.41) and young adults (A/S = 1.26). In comparison to healthy participants, People with PD also judged wider apertures as impassable. Individuals with PD were less accurate in their estimation of eye height (Error = 10.1%) than the healthy older (Error = 6.29%) and younger adults (Error = 4.79%). CONCLUSIONS: PD significantly impacted the affordances for aperture negotiation. Such altered perceptual affordances may contribute to gait pattern changes in people with PD when walking through doorways. These findings suggest that some of the motor symptoms in PD might have a perceptual underpinning.

Microarray expression identification of differentially expressed genes in serous epithelial ovarian cancer compared with bulk normal ovarian tissue and ovarian surface scrapings.

The lack of reliable early detection of ovarian cancer and the absence of specific symptoms result in diagnosis of ovarian cancer at advanced stage in the majority of the patients. Through gene expression profiling we can identify important genes that may help understand the evolution from normal ovarian tissue to ovarian cancer. The gene expression profiles of 7 normal ovaries and 26 ovaries with serous epithelial ovarian cancer (SEOC) were examined by cDNA microarrays using supervised and unsupervised analysis, with sequential significance filtering. Real-time RT-PCR was used to measure and compare the expression levels of 5 selected genes: WAP four-disulfide core domain protein HE4 (WAP, up-regulated), secreted phosphoprotein 1 (SPP1, osteopontin; up-regulated), activin A receptor, type I (ACVR1, up-regulated), tumor necrosis factor (TNF superfamily, member 2; TNF, up-regulated) and decorin (DCN, down-regulated) in 4 epithelial scrapings and in 6 bulk-extracted normal ovaries. The gene expression profile of SEOC was not dependent on the stage of the disease at diagnosis. A supervised microarray data analysis identified a subset of 329 genes showing significant differential expression between SEOC samples and bulk normal ovarian tissue and ovarian surface scrapings, including several new genes such as TNFalpha and activin A receptor type I. The real-time RT-PCR for the up-regulated genes did not differ significantly between normal ovarian epithelial scrapings and bulk-extracted ovaries. However, decorin showed a statistically significant difference (P=0.0073) in expression between epithelial scrapings and bulk-extracted ovaries. Previously uncharacterized genes are associated with the malignant phenotype of SEOC. Bulk normal ovarian tissue may serve as control for SEOC tissue in gene expression profiling. Gene expression profiling and sequential statistical analyses of gene subsets can identify new genes and molecular pathways affecting development of SEOC. The genes of interest can be potential targets for future research of SEOC.

Transcriptional profiling identifies gene expression changes associated with IFN-alpha tolerance in hepatitis C-related hepatocellular carcinoma cells.

PURPOSE: Treatment with IFN-alpha therapy has been shown to exhibit antitumor effects on patients with hepatocellular carcinoma (HCC). However, individual responses remained unpredictable because of the frequent presence of intrinsic or acquired IFN-alpha resistance. Hence, delineation of molecular targets implicated in the resistant pathway holds value in refining the therapeutic benefits of IFN-alpha. EXPERIMENTAL DESIGN: The current study analyzed the effect of IFN-alpha in human HCC cells. Three hepatitis C virus (HCV)-related, five hepatitis B virus (HBV)-related and two non-B non-C-related cell lines were subjected to IFN-alpha treatment and the cytotoxic effect on cell viability was measured. Further analysis by cDNA microarray and quantitative reverse transcription-PCR were conducted to examine the gene expression changes that mediated the IFN-alpha resistance observed. RESULTS: According to the IC(50) values determined, HCV-related cell lines indicated distinct resistance (IC(50), 389-1468 units/mL) compared with the HBV-related (IC(50), 11-77 units/mL) and non-B non-C-related cell lines (IC(50), 24-108 units/mL). Unsupervised hierarchical clustering on array data indicated three HCV-related cell lines to cluster independently from the sensitive cell lines, suggesting discrete features in association with IFN-alpha tolerance. Moreover, Significance Analysis of Microarrays analysis indicated the differential expression of 149 expressed sequence tags that represented 51 up-regulated and 98 down-regulated genes in the resistant cell lines. Comparing the temporal pattern of gene expression between 6- and 24-hour treatments, candidate genes that were considerably induced with time were further highlighted in the tolerant HCV-related cell lines. These candidates were verified by quantitative reverse transcription-PCR, which confirmed the down-regulation of UBA2, ZNF185, and FOXF1 and up-regulation of UBE4B in the drug-tolerant cells. CONCLUSIONS: Our present study showed that the insensitivity to IFN-alpha therapy in HCC cells is associated with drug-inducible transcriptional alterations. Furthermore, our investigation highlighted potential candidate genes in conferring an anti-apoptotic effect toward IFN-alpha treatment.

Parallel analysis of sporadic primary ovarian carcinomas by spectral karyotyping, comparative genomic hybridization, and expression microarrays.

Analysis of ovarian carcinomas has shown that karyotypes are often highly abnormal and cannot be identified with certainty by conventional cytogenetic methods. In this study, 17 tumors derived from 13 patients were analyzed by a combination of spectral karyotyping (SKY), comparative genomic hybridization (CGH), and expression microarrays. Within the study group, a total of 396 chromosomal rearrangements could be identified by SKY and CGH analysis. When the distribution of aberrations was normalized with respect to relative genomic length, chromosomes 3, 8, 11, 17, and 21 had the highest frequencies. Parallel microarray expression studies of 1718 human cDNAs were used to analyze expression profiles and to determine whether correlating gene expression with chromosomal rearrangement would identify smaller subsets of differentially expressed genes. Within the entire set of samples, microarray expression analysis grouped together poorly differentiated tumors irrespective of histological subtype. For three patients, a comparison between genomic alterations and gene expression pattern was performed on samples of primary and metastatic tumors. Their common origin was demonstrated by the close relationship of both the SKY and CGH karyotypes and the observed profiles of gene expression. In agreement with the pattern of genomic imbalance observed for chromosome 3 in ovarian cancer, the relative expression profile with respect to a normal ovary exhibited a contiguous pattern of reduced expression of genes mapping to the 3p25.5-3p21.31 and increased expression of genes from 3q13.33-3q28. This study demonstrates that SKY, CGH, and microarray analysis can in combination identify significantly smaller subsets of differentially expressed genes for future studies.

High-resolution mapping of genomic imbalance and identification of gene expression profiles associated with differential chemotherapy response in serous epithelial ovarian cancer.

Array comparative genomic hybridization (aCGH) and microarray expression profiling were used to subclassify DNA and RNA alterations associated with differential response to chemotherapy in ovarian cancer. Two to 4 Mb interval arrays were used to map genomic imbalances in 26 sporadic serous ovarian tumors. Cytobands 1p36, 1q42-44, 6p22.1-p21.2, 7q32.1-q34 9q33.3-q34.3, 11p15.2, 13q12.2-q13.1, 13q21.31, 17q11.2, 17q24.2-q25.3, 18q12.2, and 21q21.2-q21.3 were found to be statistically associated with chemotherapy response, and novel regions of loss at 15q11.2-q15.1 and 17q21.32-q21.33 were identified. Gene expression profiles were obtained from a subset of these tumors and identified a group of genes whose differential expression was significantly associated with drug resistance. Within this group, five genes (GAPD, HMGB2, HSC70, GRP58, and HMGB1), previously shown to form a nuclear complex associated with resistance to DNA conformation-altering chemotherapeutic drugs in in vitro systems, may represent a novel class of genes associated with in vivo drug response in ovarian cancer patients. Although RNA expression change indicated only weak DNA copy number dependence, these data illustrate the value of molecular profiling at both the RNA and DNA levels to identify small genomic regions and gene subsets that could be associated with differential chemotherapy response in ovarian cancer.

Hydroperoxide characterisation as a signature of the micelle/monomer balance in radiation-induced peroxidation of arachidonate.

Archidonate peroxidation has been studied using HO* radicals radiolytically generated as initiators of this process. Irradiated aqueous solutions of arachidonate (between 0.01 and 25 mM at pH 10.5) have been characterised by means of conjugated dienes measurement (234 nm-absorption spectroscopy) and hydroperoxide detection (high-performance liquid chromatography coupled with a chemiluminescence detection). Radiation-induced peroxidation of arachidonate gives a different trend of peroxide products, depending on the degree of substrate interaction; endoperoxide and hydro-endoperoxide being favored at low concentrations (monomer/oligomer) and monohydroperoxide at high concentrations (micellar form). The experimental ratios G(Hydro2)/G(Hydro1) increase significantly only for arachidonate concentrations higher than 1 mM, i.e. in micellar medium. However, between 0.1 and 1?mM in arachidonate, G-values (for conjugated dienes, Hydro2 and Hydro1) remain nearly constant, meaning that the physical arrangement of the solution changes: Aggregation occurs. The experimental yields of conjugated dienes formation indicated that GDienes > GHO for [arachidonate]>2.5 mM, indicating that a chain propagation process had occurred. Radiolytic yields and structural identification (HPLC-MS analysis) of peroxidation products allowed us to propose a mechanism for the formation of both hydroperoxides.

Genetic alterations in doxorubicin-resistant hepatocellular carcinoma cells: a combined study of spectral karyotyping, positional expression profiling and candidate genes.

Despite a prolongation of patient survival, the overall response of doxorubicin (DX) treatment on patients with hepatocellular carcinoma (HCC) remains modest. This is largely attributed to the development of tumor drug resistance either at the onset or during the course of treatment. To investigate the genetic changes associated with DX chemo-resistance, we examined the cytotoxic effect of DX on a panel of 9 HCC cell lines (HepG2, Hep3B, PLC/PRF/5, and six in-house established, HKCI-1, 2, 3 and 4, C1 and C2). The karyotypic abnormalities were examined by spectral karyotyping (SKY) and the chromosome loci defined were investigated for underlying deregulated genes by positional expression profiling. Quantitative RT-PCR was employed to verify the profiling findings, and also used to examine a number of drug resistance-related candidate genes (MDR1, MRP1, MGMT, PTEN, BCL2, BAX, TP53 and P21). Our results indicated that the cytotoxic effect of DX in cell lines exhibited IC50 values that ranged from sensitive to resistant (0.07 to 3.55 microM). While the overall chromosome aneuploidy did not correlate with DX resistance, aberrations on chromosome 10 demonstrated significant correlation with increasing IC50 (p=0.007). Positional profiling further suggested the consistent down-regulation of CGI-18 and ECHS1 on chromosome 10q. The array findings were substantiated by quantitative RT-PCR, which further pointed to a repressed ECHS1 expression in correlation with DX resistance (p=0.021). Among the candidate genes studied, an inverse relationship of P21 (p=0.034) and BAX (p=0.002) expression with DX resistance was also indicated. Our present study highlights the usefulness of multimodality approaches in identifying genetic markers, and further describes the novel finding of ECHS1 down-regulation in the DX chemo-resistance of HCC.