Blocking the Increase of Intracellular Deuterium Concentration Prevents the Expression of Cancer-Related Genes, Tumor Development, and Tumor Recurrence in Cancer Patients.

The possible role of the naturally occurring deuterium in the regulation of cell division was first described in the 1990s. To investigate the mechanism of influence of deuterium (D) on cell growth, expression of 236 cancer-related and 536 kinase genes were tested in deuterium-depleted (40 and 80 ppm) and deuterium-enriched (300 ppm) media compared to natural D level (150 ppm). Among genes with expression changes exceeding 30% and copy numbers over 30 (124 and 135 genes, respectively) 97.3% of them was upregulated at 300 ppm D-concentration. In mice exposed to chemical carcinogen, one-year survival data showed that deuterium-depleted water (DDW) with 30 ppm D as drinking water prevented tumor development. One quarter of the treated male mice survived 344 days, the females 334 days, while one quarter of the control mice survived only 188 and 156 days, respectively. In our human retrospective study 204 previously treated cancer patients with disease in remission, who consumed DDW, were followed. Cumulative follow-up time was 1024 years, and average follow-up time per patient, 5 years (median: 3.6 years). One hundred and fifty-six patients out of 204 (77.9%) did not relapse during their 803 years cumulative follow-up time. Median survival time (MST) was not calculable due to the extremely low death rate (11 cancer-related deaths, 5.4% of the study population). Importantly, 8 out of 11 deaths occurred several years after stopping DDW consumption, confirming that regular consumption of DDW can prevent recurrence of cancer. These findings point to the likely mechanism in which consumption of DDW keeps D-concentration below natural levels, preventing the D/H ratio from increasing to the threshold required for cell division. This in turn can serve as a key to reduce the relapse rate of cancer patients and/or to reduce cancer incidence in healthy populations.

Copy number alterations analysis of primary tumor tissue and circulating tumor cells from patients with early-stage triple negative breast cancer.

Triple negative breast cancer (TNBC) is characterized by clinical aggressiveness, lack of recognized target therapy, and a dismal patient prognosis. Several studies addressed genomic changes occurring during neoadjuvant chemotherapy (NAC) focusing on somatic variants, but without including copy number alterations (CNAs). We analyzed CNA profiles of 31 TNBC primary tumor samples before and after NAC and of 35 single circulating tumor cells (CTCs) collected prior, during and after treatment by using next-generation sequencing targeted profile and low-pass whole genome sequencing, respectively. In pre-treatment tissue samples, the most common gains occurred on chromosomes 1, 2 and 8, and SOX11 and MYC resulted the most altered genes. Notably, amplification of MSH2 (4/4 versus 0/12, p < 0.01) and PRDM1 and deletion of PAX3 (4/4 versus 1/12, p < 0.01) significantly characterized primary tumors of patients with pathological complete response. All patients with paired pre- and post-NAC samples reported a change in post-treatment CNAs compared to baseline, despite they showed at least one common alteration. CNAs detected after treatment involved genes within druggable pathways such as EGFR, cell cycle process and Ras signaling. In two patients, CTCs shared more alterations with residual rather than primary tumor involving genes such as MYC, BCL6, SOX2, FGFR4. The phylogenetic analysis of CTCs within a single patient revealed NAC impact on tumor evolution, suggesting a selection of driver events under treatment pressure. In conclusion, our data showed how chemoresistance might arise early from treatment-induced selection of clones already present in the primary tumor, and that the characterization of CNAs on single CTCs informs on cancer evolution and potential druggable targets.

Distinct Epigenetic Reprogramming, Mitochondrial Patterns, Cellular Morphology, and Cytotoxicity after Bee Venom Treatment.

BACKGROUND: Bee venom is a promising agent for cancer treatment due to its selective cytotoxic potential for cancer cells through apoptotic pathways. However, there is no evidence for changes in the epigenome and mitochondrial DNA copy numbers after bee venom application. The purpose of this study was to determine the impact of bee venom on cytosine modifications and mitochondrial DNA copy number variation. METHODS: A broad range of methods was applied to elucidate the impact of bee venom on neoplastic cells. These included MTT assay for detection of cytotoxicity, immunostaining of cytosine modifications and mitochondria, assessment of cellular morphology by flow cytometry, and quantification of mitochondrial DNA copy numbers using QPCR. RESULTS: Bee venom-induced cell death was selective for cancer cells, where it triggered a response characterized by alteration of cytosine modification. In contrast, normal cells were more resistant to DNA modifications. Furthermore, application of the venom resulted in variation of mitochondrial membrane permeability and mitochondrial DNA copy numbers, together with alterations in cell morphology, manifesting as reduced affected cell size. CONCLUSION: The study findings suggest that bee venom can be used as a selective DNA (de)methylating agent in cancer. Various agents (such as decitabine and 5-azacytidine) have been synthesized and developed for cancer treatment, and a range of syntheses and preparation and application methods have been described for these patented drugs. However, to the best of our knowledge, no previous research has investigated the use of bee venom or any component thereof for epigenetic therapy in cancer cells.

Genetic polymorphisms of metabolic enzyme genes associated with leukocyte mitochondrial DNA copy number in PAHs exposure workers.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) exposure had been reported to be a risk factor of mtDNAcn in our early study. However, the effect of metabolic enzymes' genetic polymorphisms on mtDNAcn in PAHs-Exposure workers has not been fully evaluated. AIM: The aim of the study was to explore the effect of metabolic enzymes' genetic polymorphisms on mtDNAcn in PAHs-Exposure. METHODS AND RESULTS: We investigated the effects of metabolic enzymes' genetic polymorphisms on mtDNAcn among 544 coke oven workers and 238 office staffs. The mtDNAcn of peripheral blood leukocytes was measured using the Real-time quantitative polymerase chain reaction (PCR) method. PCR and restriction fragment length was used to detect five polymorphisms in GSTT1, GSTM1, GSTP1 rs1695, CYP2E1 rs6413432, and CYP2E1 rs3813867. The mtDNAcn in peripheral blood leukocytes was significantly lower in the exposure group than that in the control group (p < .001). The 1-OHPYR had an increasing trend with the genotypes AA→AG → GG of GSTP1 rs1695 in the control group. Generalized linear model indicated that the influencing factors of mtDNAcn were PAHs-exposure [ß (95% CI) = -0.420 (-0.469, -0.372), p < .001], male [ß (95% CI) = -0.058 (-0.103, -0.012), p = .013], and AA genotype for GSTP1 rs1695 [ß (95% CI) = -0.051 (-0.095, -0.008), p = .020]. CONCLUSION: The individuals carrying the AA genotype of GSTP1 rs1695 may have a lower mtDNAcn due to their weaker detoxification of PAHs.

Effect of exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs) on mitochondrial DNA (mtDNA) copy number in rats.

Mitochondria are intracellular organelles responsible for biological oxidation and energy production. These organelles are susceptible to damage from oxidative stress and compensate for damage by increasing the number of copies of their own genome, mitochondrial DNA (mtDNA). Cancer and environmental exposure to some pollutants have also been associated with altered mtDNA copy number. Since exposures to polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been shown to increase oxidative stress, we hypothesize that mtDNA copy number will be altered with exposure to these compounds. mtDNA copy number was measured in DNA from archived frozen liver and lung specimens from the National Toxicology Program (NTP) study of female Harlan Sprague Dawley rats exposed to TCDD (3, 10, or 100 ng/kg/day), dioxin-like (DL) PCB 126 (10, 100, or 1000 ng/kg/day), non-DL PCB 153 (10, 100, or 1000 µg/kg/day), and PCB 126 + PCB 153 (10 ng/kg/day + 10 µg/kg/day, 100 ng/kg/day + 100 µg/kg/day, or 1000 ng/kg/day + 1000 µg/kg/day, respectively) for 13 and 52 weeks. An increase in mtDNA copy number was observed in the liver and lung of rats exposed to TCDD and the lung of rats exposed to the mixture of PCB 126 and PCB 153. A statistically significant positive dose-dependent trend was also observed in the lung of rats exposed to PCB 126 and a mixture of PCB 153 and PCB 126, although in neither case was the control copy number significantly exceeded at any dose level. These exposures produced a range of pathological responses in these organs in the two-year NTP studies. Conversely, there was a significant decrease or no change in mtDNA copy number in the liver and lung of rats exposed to non-DL PCB 153. This is consistent with a general lack of PCB 153 mediated liver or lung injury in the NTP study, with the exception of liver hypertrophy. Together, the results suggest that an increase in mtDNA copy number may serve as a sensitive, early biomarker of mitochondrial injury and oxidative stress that contributes to the development of the toxicity of dioxin-like compounds.

PCAT: an integrated portal for genomic and preclinical testing data of pediatric cancer patient-derived xenograft models.

Although cancer is the leading cause of disease-related mortality in children, the relative rarity of pediatric cancers poses a significant challenge for developing novel therapeutics to further improve prognosis. Patient-derived xenograft (PDX) models, which are usually developed from high-risk tumors, are a useful platform to study molecular driver events, identify biomarkers and prioritize therapeutic agents. Here, we develop PDX for Childhood Cancer Therapeutics (PCAT), a new integrated portal for pediatric cancer PDX models. Distinct from previously reported PDX portals, PCAT is focused on pediatric cancer models and provides intuitive interfaces for querying and data mining. The current release comprises 324 models and their associated clinical and genomic data, including gene expression, mutation and copy number alteration. Importantly, PCAT curates preclinical testing results for 68 models and 79 therapeutic agents manually collected from individual agent testing studies published since 2008. To facilitate comparisons of patterns between patient tumors and PDX models, PCAT curates clinical and molecular data of patient tumors from the TARGET project. In addition, PCAT provides access to gene fusions identified in nearly 1000 TARGET samples. PCAT was built using R-shiny and MySQL. The portal can be accessed at http://pcat.zhenglab.info or http://www.pedtranscriptome.org.

Copy number evolution and its relationship with patient outcome-an analysis of 178 matched presentation-relapse tumor pairs from the Myeloma XI trial.

Structural chromosomal changes including copy number aberrations (CNAs) are a major feature of multiple myeloma (MM), however their evolution in context of modern biological therapy is not well characterized. To investigate acquisition of CNAs and their prognostic relevance in context of first-line therapy, we profiled tumor diagnosis-relapse pairs from 178 NCRI Myeloma XI (ISRCTN49407852) trial patients using digital multiplex ligation-dependent probe amplification. CNA profiles acquired at relapse differed substantially between MM subtypes: hyperdiploid (HRD) tumors evolved predominantly in branching pattern vs. linear pattern in t(4;14) vs. stable pattern in t(11;14). CNA acquisition also differed between subtypes based on CCND expression, with a marked enrichment of acquired del(17p) in CCND2 over CCND1 tumors. Acquired CNAs were not influenced by high-dose melphalan or lenalidomide maintenance randomization. A branching evolution pattern was significantly associated with inferior overall survival (OS; hazard ratio (HR) 2.61, P = 0.0048). As an individual lesion, acquisition of gain(1q) at relapse was associated with shorter OS, independent of other risk markers or time of relapse (HR = 2.00; P = 0.021). There is an increasing need for rational therapy sequencing in MM. Our data supports the value of repeat molecular profiling to characterize disease evolution and inform management of MM relapse.

Dihydromyricetin Improves Endothelial Dysfunction in Diabetic Mice via Oxidative Stress Inhibition in a SIRT3-Dependent Manner.

Dihydromyricetin (DHY), a flavonoid component isolated from Ampelopsis grossedentata, exerts versatile pharmacological activities. However, the possible effects of DHY on diabetic vascular endothelial dysfunction have not yet been fully elucidated. In the present study, male C57BL/6 mice, wild type (WT) 129S1/SvImJ mice and sirtuin 3 (SIRT3) knockout (SIRT3-/-) mice were injected with streptozotocin (STZ, 60 mg/kg/day) for 5 consecutive days. Two weeks later, DHY were given at the doses of 250 mg/kg by gavage once daily for 12 weeks. Fasting blood glucose (FBG) and glycosylated hemoglobin (HbA1c) level, endothelium-dependent relaxation of thoracic aorta, reactive oxygen species (ROS) production, SIRT3, and superoxide dismutase 2 (SOD2) protein expressions, as well as mitochondrial Deoxyribonucleic Acid (mtDNA) copy number, in thoracic aorta were detected. Our study found that DHY treatment decreased FBG and HbA1c level, improved endothelium-dependent relaxation of thoracic aorta, inhibited oxidative stress and ROS production, and enhanced SIRT3 and SOD2 protein expression, as well as mtDNA copy number, in thoracic aorta of diabetic mice. However, above protective effects of DHY were unavailable in SIRT3-/- mice. The study suggested DHY improved endothelial dysfunction in diabetic mice via oxidative stress inhibition in a SIRT3-dependent manner.

Molecular signatures of BRCAness analysis identifies PARP inhibitor Niraparib as a novel targeted therapeutic strategy for soft tissue Sarcomas.

Background: Patients with advanced soft tissue sarcomas (STS) have a dismal prognosis with few effective therapeutic options. A defect in the homologous recombination repair (HRR) pathway can accumulate DNA repair errors and gene mutations, which can lead to tumorigenesis. BRCAness describes tumors with an HRR deficiency (HRD) in the absence of a germline BRCA1/2 mutation. However, the characteristics of BRCAness in STS remain largely unknown. Thus, this study aimed to explore the genomic and molecular landscape of BRCAness using whole exome sequencing (WES) in STS, aiming to find a potential target for STS treatment. Methods: WES was performed in 22 STS samples from the First Affiliated Hospital of Sun Yat-sen University to reveal the possible genomic and molecular characteristics. The characteristics were then validated using data of 224 STS samples from The Cancer Genome Atlas (TCGA) database and in vitro data. The analysis of the potential biomarker for BRCAness was performed. Targeted drug susceptibility and combination therapy screening of chemotherapeutics for STS were evaluated in STS cell lines, cell-line-derived xenografts (CDX), and patient-derived xenografts (PDX). Results: Compared with 30 somatic mutation signatures of cancers, high cosine-similarity (0.75) was identified for HRD signatures in the 22 STS samples using nonnegative matrix factorization. Single nucleotide polymorphism indicated a low mutation rate of BRCA1/2 in the 22 STS samples (11.76% and 5.88%, respectively). However, copy number variation analyses demonstrated widespread chromosomal instability; furthermore, 54.55% of STS samples (12/22) carried BRCAness traits. Subsequently, similar genomic and molecular characteristics were also detected in the 224 STS samples from TCGA and in vitro. Poly (ADP-ribose) polymerases (PARP)-1 could be a promising reflection of HRD and therapeutic response. Furthermore, the level of PAR formation was found to be correlated with PARP-1. Subsequently, STS cell lines were determined to be sensitive to PARP inhibitor (PARPi), niraparib. Moreover, based on the screening test of the five common PARPis and combination test among doxorubicin, ifosfamide, dacarbazine, and temozolomide (TMZ), niraparib and TMZ were the most synergistic in STS cell lines. The synergistic effect and safety of niraparib and TMZ combination were also shown in CDX and PDX. Conclusions: BRCAness might be the common genomic and molecular characteristics of majority of STS cases. PARP-1 and PAR could be potential proper and feasible theranostic biomarkers for assessing HRD in patients. STSs were sensitive to PARPi. Moreover, the combination of niraparib and TMZ showed synergistic effect. Niraparib and TMZ could be a promising targeted therapeutic strategy for patients with STS.

Changes in peripheral mitochondrial DNA copy number in metformin-treated women with polycystic ovary syndrome: a longitudinal study.

BACKGROUND: Patients with polycystic ovarian syndrome (PCOS) are associated with known alterations in mitochondria DNA copy number (mtDNA-CN). The aim of this study is to study the change in mtDNA-CN in patients with PCOS who were treated with metformin. METHODS: This is a prospective cohort of patients with PCOS, who received metformin for one year. From 2009 to 2015, 88 women diagnosed with PCOS, based on the Rotterdam criteria, were enrolled. Serial measurements of mtDNA-CN, 8-hydroxydeoxyguanosine (8-OHdG), anthropometric, metabolic, endocrine, and inflammatory markers were obtained before and after 3, 6, and 12 months of treatment. RESULTS: A significant decrease in mtDNA-CN was seen over the course of one year. Other markers, including 8-OHdG, testosterone, free androgen index, blood pressure and liver enzymes, also decreased in the same interval. On regression analysis, there was a significant association between the change in mtDNA-CN and serum total testosterone, and no association between mtDNA-CN and metabolic factors. CONCLUSIONS: Treatment with metformin is associated with a time-dependent decrease in mtDNA-CN in patients with PCOS who are treated over the course of one year. This may signify a reduction in mitochondria dysfunction. The change in mtDNA-CN corresponds to a similar change in serum total testosterone, and suggests a possible relationship between mtDNA-CN and testosterone. TRIAL REGISTRATION: ClinicalTrials.gov , NCT00172523 . Registered September 15, 2005.

Glucose-Induced Expression of DAPIT in Pancreatic ß-Cells.

Transcript levels for selected ATP synthase membrane FO-subunits-including DAPIT-in INS-1E cells were found to be sensitive to lowering glucose down from 11 mM, in which these cells are routinely cultured. Depending on conditions, the diminished mRNA levels recovered when glucose was restored to 11 mM; or were elevated during further 120 min incubations with 20-mM glucose. Asking whether DAPIT expression may be elevated by hyperglycemia in vivo, we studied mice with hyaluronic acid implants delivering glucose for up to 14 days. Such continuous two-week glucose stimulations in mice increased DAPIT mRNA by >5-fold in isolated pancreatic islets (ATP synthase F1α mRNA by 1.5-fold). In INS-1E cells, the glucose-induced ATP increment vanished with DAPIT silencing (6% of ATP rise), likewise a portion of the mtDNA-copy number increment. With 20 and 11-mM glucose the phosphorylating/non-phosphorylating respiration rate ratio diminished to ~70% and 96%, respectively, upon DAPIT silencing, whereas net GSIS rates accounted for 80% and 90% in USMG5/DAPIT-deficient cells. Consequently, the sufficient DAPIT expression and complete ATP synthase assembly is required for maximum ATP synthesis and mitochondrial biogenesis, but not for insulin secretion as such. Elevated DAPIT expression at high glucose further increases the ATP synthesis efficiency.

Metal exposure causes rDNA copy number to fluctuate in mutation accumulation lines of Daphnia pulex.

Ribosomal (r)DNA is a highly dynamic, conserved, multigene family whose sequence homogeneity is thought to be maintained by intra- and interchromosomal recombination, which are capable of changing rDNA copy number. It is generally not known how environmental stress such as sublethal exposure to environmentally relevant concentrations of metals impacts rDNA copy number. To determine how chronic metal exposure affects rDNA, we measured copy number of the 18S rRNA gene in 355 copper and nickel-exposed samples and 132 metal-free samples derived from 325 mutation accumulation (MA) lines of two genetically distinct Daphnia pulex lineages. The MA lines were sampled at four time points over 100+ generations of clonal propagation. The copy number of rDNA was also measured in 15 individuals sampled from a metal-free non-MA control population established from the same progenitor as one of the MA lineages. We found that mean rDNA copy number fluctuated across lines exposed to metals with a tendency to decrease over time. In contrast, mean rDNA copy number in the metal-free control lines and the non-MA population remained stable over time. It is generally accepted that extreme rDNA loss results in the loss of organism fitness. Thus, fluctuations in rDNA copy number, including losses, could affect the long-term viability of natural populations of Daphnia in metal-contaminated habitats.

Exposure to the Heavy-Metal Lead Induces DNA Copy Number Alterations in Zebrafish Cells.

DNA copy number variants are associated with the development of complex neurological diseases and disorders including autism spectrum disorder, schizophrenia, Alzheimer's disease, and Parkinson's disease. Exposure to multiple environmental chemicals including various heavy metals is suggested as a risk factor in these neurological diseases and disorders, but few studies have addressed if heavy-metal exposure can result in de novo DNA copy number changes as a genetic mechanism contributing to these disease outcomes. In this study to further investigate the relationship between heavy-metal exposure and de novo copy number alterations (CNAs), zebrafish fibroblast cells were exposed to the neurotoxicant lead (Pb). A crystal violet assay was first used to determine exposure concentrations with >80% cell confluency. Then a zebrafish-specific array comparative genomic hybridization platform was used to detect CNAs following a 72 h Pb exposure (0.24, 2.4, or 24 µM). The Pb exposure resulted in 72 CNA amplifications ranging in size from 5 to 329 kb. No deletions were detected. CNAs resulted in 15 CNA regions (CNARs), leaving 7 singlet CNAs. Two of the singlets were within high repeat genomic locations. The number of CNAs tended to increase in a concentration-dependent manner. Several CNARs encompassed genes previously reported to have altered expression with Pb exposure, suggesting a mechanistic link. In addition, almost all genes are associated within a molecular network with amyloid precursor protein, a key molecular target associated with the pathophysiology of Alzheimer's disease. Overall, these findings show that Pb exposure results in de novo CNAs that could serve as a mechanism driving adverse health outcomes associated with Pb toxicity including neurological disease pathogenesis for further study.

Dietary Hizikia fusiforme enhance survival of white spot syndrome virus infected crayfish Procambarus clarkii.

The sea vegetable Hizikia fusiforme is not only a good source of dietary fiber but also enhances immunity. In this study, we investigated the effects of H. fusiforme on innate immunity in invertebrates, using white spot syndrome virus (WSSV) challenge in the crayfish, Procambarus clarkii. Supplementation with H. fusiforme significantly reduced mortality caused by WSSV infection and also reduced copy numbers of the WSSV protein VP28. Quantitative reverse transcription-polymerase chain reaction showed that supplementation of feed with H. fusiforme increased the expression of immune-related genes, including NF-κB and crustin 1. Further analysis showed that supplementation with H. fusiforme also affected three immune parameters, total hemocyte count, and phenoloxidase and superoxide dismutase activity. H. fusiforme treatment significantly increased hemocyte apoptosis rates in both WSSV-infected and uninfected crayfish. H. fusiforme thus regulates the innate immunity of crayfish, and both delays and reduces mortality after WSSV challenge. Our study demonstrates the potential for the commercial use of H. fusiforme, either therapeutically or prophylactically, to regulate the innate immunity and protect crayfish against WSSV infection.

Effects of Micronucleus Frequencies and Mitochondrial DNA Copy Numbers among Benzene-Exposed Workers in China.

To provide a more comprehensive understanding of genotoxic effects from benzene exposure, its effects on induction of mitochondrial DNA copy number (MtDNAcn) and of micronucleus (MN) were investigated using peripheral blood from workers in China. Changes in mtDNAcn and MN were determined using quantitative real-time polymerase chain reaction (PCR) and cytokinesis-block micronucleus assays (CBMN), respectively, in 58 control and 174 benzene-exposed workers in Shanghai, China. Among the exposed workers, relative mtDNAcn increased and then decreased with increasing doses of benzene exposure. Significant and dose-dependent increase in MN frequencies were observed among the different exposure groups. In addition, the relative mtDNAcn were significantly associated with the MN frequencies in the low-level exposure group (P = 0.046), but not in the high dose groups. Therefore, the mechanisms for induction of MtDNAcn and MN by benzene may be similar from exposure to low doses but different from high doses. Similar increase of MN frequencies and MtDNAcn may be due to oxidative stress induced by benzene at low concentrations, while higher concentrations may start to initiate the cell death pathway. The pathway may be associated with excessive MtDNAcn which can initiate apoptosis while MN can continue to be induced. However, the differential mechanisms need to be investigated because they may represent different levels of risk for different health consequences. On the other hand, our data indicate that induction of MtDNAcn may be a sensitive genotoxic biomarker for workers with exposure to low dose of benzene. Environ. Mol. Mutagen. 61:355-360, 2020. © 2020 Wiley Periodicals, Inc.

Neoadjuvant Chemotherapy Induces the Appearance of New Copy Number Aberrations in Breast Tumor and is Associated with Metastasis.

BACKGROUND: In this study, we examined the CNA-genetic landscape (CNA - copy number aberration) of breast cancer prior to and following neoadjuvant chemotherapy (NAC) and correlated changes in the tumor landscape with chemotherapy efficiency as well as metastasis-free survival. OBJECTIVE: Breast cancer patients (n = 30) with luminal B molecular subtypes were treated with anthracycline- based therapy. METHODS: To study CNAs in breast tumors, microarray analysis was performed. RESULTS: Three effects of NAC on tumor CNA landscape were identified: 1 - the number of CNAbearing tumor clones decreased following NAC; 2 - there were no alterations in the number of CNAcontaining clones after NAC; 3 - the treatment with NAC increased the number of CNA-bearing clones (new clones appeared). All NAC-treated patients who had new tumor clones with amplification (20%) had a 100% likelihood of metastasis formation. In these cases, NAC contributed to the emergence of potential metastatic clones. Our study identified the following loci - 5p, 6p, 7q, 8q, 9p, 10p, 10q22.1, 13q, 16p, 18Chr and 19p - that were amplified during the treatment with NAC and maybe the markers of potential metastatic clones. In other patients who showed total or partial elimination of CNA-bearing cell clones, no new amplification clones were observed after NAC, and no evidence of metastases was found with follow-up for 5 years (р = 0.00000). CONCLUSION: Our data suggest that the main therapeutic result from NAC is the elimination of potential metastatic clones present in the tumor before treatment. The results showed the necessity of an intelligent approach to NAC to avoid metastasis stimulation.

Mitochondrial and Oxidative Impacts of Short and Long-term Administration of HAART on HIV Patients.

BACKGROUND: There may be a possible link between the use of HAART and oxidative stress-related mitochondrial dysfunction in HIV patients. We evaluated the mitochondrial and oxidative impacts of short and long-term administration of HAART on HIV patients attending the Enugu State University Teaching (ESUT) Hospital, Enugu, Nigeria following short and long-term therapy. METHODS: 96 patients categorized into four groups of 24 individuals were recruited for the study. Group 1 comprised of age-matched, apparently healthy, sero-negative individuals (the No HIV group); group 2 consisted of HIV sero-positive individuals who had not started any form of treatment (the Treatment naïve group). Individuals in group 3 were known HIV patients on HAART for less than one year (Short-term treatment group), while group 4 comprised of HIV patients on HAART for more than one year (Long-term treatment group). All patients were aged between 18 to 60 years and attended the HIV clinic at the time of the study. Determination of total antioxidant status (TAS in nmol/l), malondialdehyde (MDA in mmol/l), CD4+ count in cells/µl, and genomic studies were all done using standard operative procedures. RESULTS: We found that the long-term treatment group had significantly raised the levels of MDA, as well as significantly diminished TAS compared to the Short-term treatment and No HIV groups (P<0.05). In addition, there was significantly elevated variation in the copy number of mitochondrial genes (mtDNA: D-loop, ATPase 8, TRNALEU uur) in the long-term treatment group. CONCLUSION: Long-term treatment with HAART increases oxidative stress and causes mitochondrial alterations in HIV patients.

Histone Lysine Methylation Dynamics Control EGFR DNA Copy-Number Amplification.

Acquired chromosomal DNA copy gains are a feature of many tumors; however, the mechanisms that underpin oncogene amplification are poorly understood. Recent studies have begun to uncover the importance of epigenetic states and histone lysine methyltransferases (KMT) and demethylases (KDM) in regulating transient site-specific DNA copy-number gains (TSSG). In this study, we reveal a critical interplay between a myriad of lysine methyltransferases and demethylases in modulating H3K4/9/27 methylation balance to control extrachromosomal amplification of the EGFR oncogene. This study further establishes that cellular signals (hypoxia and EGF) are able to directly promote EGFR amplification through modulation of the enzymes controlling EGFR copy gains. Moreover, we demonstrate that chemical inhibitors targeting specific KMTs and KDMs are able to promote or block extrachromosomal EGFR amplification, which identifies potential therapeutic strategies for controlling EGFR copy-number heterogeneity in cancer, and, in turn, drug response. SIGNIFICANCE: This study identifies a network of epigenetic factors and cellular signals that directly control EGFR DNA amplification. We demonstrate that chemical inhibitors targeting enzymes controlling this amplification can be used to rheostat EGFR copy number, which uncovers therapeutic opportunities for controlling EGFR DNA amplification heterogeneity and the associated drug response.This article is highlighted in the In This Issue feature, p. 161.

DNA Copy Number Variations as Markers of Mutagenic Impact.

DNA copy number variation (CNV) occurs due to deletion or duplication of DNA segments resulting in a different number of copies of a specific DNA-stretch on homologous chromosomes. Implications of CNVs in evolution and development of different diseases have been demonstrated although contribution of environmental factors, such as mutagens, in the origin of CNVs, is poorly understood. In this review, we summarize current knowledge about mutagen-induced CNVs in human, animal and plant cells. Differences in CNV frequencies induced by radiation and chemical mutagens, distribution of CNVs in the genome, as well as adaptive effects in plants, are discussed. Currently available information concerning impact of mutagens in induction of CNVs in germ cells is presented. Moreover, the potential of CNVs as a new endpoint in mutagenicity test-systems is discussed.

Comparative Genomic Hybridization (CGH) in Genotoxicology.

In the past two decades, comparative genomic hybridization (CGH) and array CGH have become indispensable tools in clinical diagnostics and toxicological risk assessment. Initially developed for the genome-wide screening of chromosomal imbalances, that is, copy-number variations in tumor cells, both CGH and array CGH have been employed in genotoxicology and most recently in toxicogenomics. The latter allows a multi-end point analysis of how particular genes react to toxic agents, revealing changes in signaling pathways and other underlying molecular mechanisms. This chapter provides background on the use of CGH and array CGH in the context of genotoxicology, and also a protocol for conventional CGH, so that the basic principles of this methodology can be better understood. Conventional and array CGH investigate DNA expression patterns, copy-number variations across the whole genome, and loss of heterozygosity after genotoxic damage. Array CGH is still cost-intensive but produces exponentially more data, requiring suitable analytical algorithms and sophisticated bioinformatic analysis. As toxicogenomics is an emerging sub-discipline of toxicology research, effectively evaluating toxicogenomic microarray data can be hugely advantageous for human risk assessment, even though international regulatory guidelines on toxicogenomics have yet to be fully agreed and implemented.