Gene Variants in Components of the microRNA Processing Pathway in Chronic Myeloid Leukemia.

Current therapy in chronic myeloid leukemia (CML) has improved patient life expectancy close to that of healthy individuals. However, molecular alterations other than BCR::ABL1 fusion gene in CML are barely known. MicroRNAs are important regulators of gene expression, and variants in some of the components of microRNA biosynthesis pathways have been associated with genetic susceptibility to different types of cancer. Thus, the aim of this study was to evaluate the association of variants located in genes involved in the biogenesis of microRNAs with susceptibility to CML. Fifteen variants in eight genes involved in the biogenesis of miRNAs were genotyped in 296 individuals with CML and 485 healthy participants using TaqMan probes. The association of gene variants with CML and clinical variables was evaluated by a Chi-square test, and odds ratios and 95% confidence intervals were estimated by logistic regression. The variant rs13078 in DICER1 was significantly higher among CML individuals than in healthy participants. In addition, the variants rs7813 and rs2740349 were significantly associated with worse prognosis, according to their Hasford scores, whereas the rs2740349 variant was also associated with a later age at diagnosis. These findings suggest that variants in components of the microRNA biogenesis pathway could be involved in CML genetic risk.

Genetic alterations in the BCR-ABL1 fusion gene related to imatinib resistance in chronic myeloid leukemia.

Use of the potent tyrosine kinase inhibitor imatinib as the first-line treatment in chronic myeloid leukemia (CML) has decreased mortality from 20% to 2%. Approximately 30% of CML patients experience imatinib resistance, however, largely because of point mutations in the kinase domain of the BCR-ABL1 fusion gene. The aim of this study was to use next-generation sequencing (NGS) to identify mutations related to imatinib resistance. The study included 22 patients diagnosed with CML and experiencing no clinical response to imatinib. Total RNA was used for cDNA synthesis, with amplification of a fragment encompassing the BCR-ABL1 kinase domain using a nested-PCR approach. Sanger and NGS were applied to detect genetic alterations. HaplotypeCaller was used for variant calling, and STAR-Fusion software was applied for fusion breakpoint identification. After sequencing analysis, F311I, F317L, and E450K mutations were detected respectively in three different participants, and in another two patients, single nucleotide variants in BCR (rs9608100, rs140506, rs16802) and ABL1 (rs35011138) were detected. Eleven patients carried e14a2 transcripts, nine had e13a2 transcripts, and both transcripts were identified in one patient. One patient had co-expression of e14a2 and e14a8 transcripts. The results identify candidate single nucleotide variants and co-expressed BCR-ABL1 transcripts in cellular resistance to imatinib.

NOTCH expression variability and relapse of breast cancer in high-risk groups.

BACKGROUND: In regards to breast cancer (BC), survival or disease-free periods are still compromised mainly in Triple Negative (TN) and HER2 tumors. The participation of estrogen receptor (ER) has been reported as crucial in the signaling pathways, including the NOTCH pathway. The study was aimed to evaluate the expression of NOTCH1 and NOTCH3 in BC and its relationship with the presence of ER, as well as with relapses. METHODS: NOTCH1 and NOTCH3 expression was evaluated in BC using Oncomine database, Breast Cancer Gene Expression Miner database and Kaplan Meier Plotter. Subsequently, detection of NOTCH1 and NOTCH3 in 100 paraffin-embedded BC samples from Mexican patients was achieved by immunohistochemistry (IHC) and RT-qPCR, a group of benign breast tumors were included as controls. Relapses were evaluated by BC subtypes and their relationship with NOTCH1 and NOTCH3 expression, as well as with ER expression. RESULTS: The analyses from public databases of TN and HER2 groups, which are estrogen receptor-negative (ERN), revealed NOTCH1 and NOTCH3 expression variability. The overexpression was associated with lower relapse-free survival (P = 0.00019). These data were concordant with results from tumor samples of patients included in this study, which showed overexpression of NOTCH1 and NOTCH3 in ERN tumors, as well as lower relapse-free survival (P < 0.0001). CONCLUSIONS: NOTCH1 and NOTCH3 were found to be overexpressed mainly in ERN tumors. HER2 and TN groups, are related to higher relapse rates. Therefore, anti-NOTCH therapy could be justified and implemented in conventional treatments of high-risk BC groups.

Arsenic exposure and non-carcinogenic health effects.

Inorganic arsenic (iAs) exposure is a serious health problem that affects more than 140 million individuals worldwide, mainly, through contaminated drinking water. Acute iAs poisoning produces several symptoms such as nausea, vomiting, abdominal pain, and severe diarrhea, whereas prolonged iAs exposure increased the risk of several malignant disorders such as lung, urinary tract, and skin tumors. Another sensitive endpoint less described of chronic iAs exposure are the non-malignant health effects in hepatic, endocrine, renal, neurological, hematological, immune, and cardiovascular systems. The present review outlines epidemiology evidence and possible molecular mechanisms associated with iAs-toxicity in several non-carcinogenic disorders.

Hexavalent chromium: Regulation and health effects.

Despite the knowledge about heavy metals toxicity on humans, its use is widely spread mainly for industrial processes. Chromium is an element that belongs to this group and although it is present in our daily diet, it can also be harmful for humans, causing skin allergies and increasing the risk of lung cancer, among other health effects reported. In this review, we highlight its nutritional role, its toxicokinetic and toxicodynamic in humans, its regulation in the industry and the biomonitoring proposal of this element in blood and urine samples with the aim to control the level of exposure of the workers in military industry and also of the general population.

Two Novel FAM20C Variants in A Family with Raine Syndrome.

Two siblings from a Mexican family who carried lethal Raine syndrome are presented. A newborn term male (case 1) and his 21 gestational week brother (case 2), with a similar osteosclerotic pattern: generalized osteosclerosis, which is more evident in facial bones and cranial base. Prenatal findings at 21 weeks and histopathological features for case 2 are described. A novel combination of biallelic FAM20C pathogenic variants were detected, a maternal cytosine duplication at position 456 and a paternal deletion of a cytosine in position 474 in exon 1, which change the reading frame with a premature termination at codon 207 and 185 respectively. These changes are in concordance with a negative detection of the protein in liver and kidney as shown in case 2. Necropsy showed absence of pancreatic Langerhans Islets, which are reported here for the first time. Corpus callosum absence is added to the few reported cases of brain defects in Raine syndrome. This report shows two new FAM20C variants not described previously, and negative protein detection in the liver and the kidney. We highlight that lethal Raine syndrome is well defined as early as 21 weeks, including mineralization defects and craniofacial features. Pancreas and brain defects found here in FAM20C deficiency extend the functional spectrum of this protein to previously unknown organs.

Curcumin differentially affects cell cycle and cell death in acute and chronic myeloid leukemia cells.

Curcumin is a phytochemical with potent anti-neoplastic properties. The antitumoral effects of curcumin in cells derived from chronic or acute myeloid leukemia have been already described. However, a comparative study of the cytostatic and cytotoxic effects of curcumin on chronic and acute myeloid leukemia cells has not yet been performed. In the present study, the cellular effects of curcumin on cell lines derived from chronic or acute myeloid leukemia were examined. Dose and time-response assays were performed with curcumin on HL-60 and K562 cells. Cell viability was evaluated with trypan blue exclusion test and cell death by flow cytometry using a fluorescent molecular probe. A cell cycle profile was analyzed, and protein markers of cell cycle progression and cell death were investigated. In the present study, the K562 cells showed a higher sensitivity to the cytostatic and cytotoxic effects of curcumin compared with HL-60. In addition, curcumin induced G1 phase arrest in HL-60 cells and G2/M phase arrest in K562 cells. Furthermore, curcumin-related cell death in HL-60 was associated with the processed forms of caspases-9 and -3 proteins, whereas in K562 cells, both the processed and the unprocessed forms were present. Accordingly, activity of these caspases was significantly higher in HL-60 cells compared with that in K562. In conclusion, curcumin elicits different cellular mechanisms in chronic or acute myeloid leukemia cells and the powerful antitumoral effect was more potent in K562 compared with HL-60 cells.

Arsenic-induced S phase cell cycle lengthening is associated with ROS generation, p53 signaling and CDC25A expression.

Cellular response to arsenic is strongly dependent on p53 functional status. Primarily arresting the cell cycle in G1 or G2/M phases, arsenic treatment also induces an increase in the S-phase time in wild-type p53 cells. In contrast, cells with a non-functional p53 display only a subtle increase in the S phase, indicating arsenic differentially affects the cell cycle depending on p53 status. Importantly, it has been reported that arsenic induces reactive oxygen species (ROS), a process counteracted by p53. To evaluate the participation of p53 in the lengthening of the S phase and the connection between the transient cell cycle arrest and oxidative stress, we evaluated the cell response to arsenic in MCF-7 and H1299 cells, and analyzed p53's role as a transcription factor in regulating genes involved in ROS reduction and S phase transition. Herein, we discovered that arsenic induced an increase in the population of S phase cells that was dependent on the presence and transcriptional activity of p53. Furthermore, for the first time, we demonstrate that arsenic activates p53-dependent transcription of ROS detoxification genes, such as SESN1, and by an indirect mechanism involving ATF3, genes that could be responsible for the S phase cell cycle arrest, such as CDC25A.

Impact of life stage and duration of exposure on arsenic-induced proliferative lesions and neoplasia in C3H mice.

Epidemiological studies suggest that chronic exposure to inorganic arsenic is associated with cancer of the skin, urinary bladder and lung as well as the kidney and liver. Previous experimental studies have demonstrated increased incidence of liver, lung, ovary, and uterine tumors in mice exposed to 85 ppm (approximately 8 mg/kg) inorganic arsenic during gestation. To further characterize age susceptibility to arsenic carcinogenesis we administered 85 ppm inorganic arsenic in drinking water to C3H mice during gestation, prior to pubescence and post-pubescence to compare proliferative lesion and tumor outcomes over a one-year exposure period. Inorganic arsenic significantly increased the incidence of hyperplasia in urinary bladder (48%) and oviduct (36%) in female mice exposed prior to pubescence (beginning on postnatal day 21 and extending through one year) compared to control mice (19 and 5%, respectively). Arsenic also increased the incidence of hyperplasia in urinary bladder (28%) of female mice continuously exposed to arsenic (beginning on gestation day 8 and extending though one year) compared to gestation only exposed mice (0%). In contrast, inorganic arsenic significantly decreased the incidence of tumors in liver (0%) and adrenal glands (0%) of male mice continuously exposed from gestation through one year, as compared to levels in control (30 and 65%, respectively) and gestation only (33 and 55%, respectively) exposed mice. Together, these results suggest that continuous inorganic arsenic exposure at 85 ppm from gestation through one year increases the incidence and severity of urogenital proliferative lesions in female mice and decreases the incidence of liver and adrenal tumors in male mice. The paradoxical nature of these effects may be related to altered lipid metabolism, the effective dose in each target organ, and/or the shorter one-year observational period.

Speciation of arsenic in exfoliated urinary bladder epithelial cells from individuals exposed to arsenic in drinking water.

BACKGROUND: The concentration of arsenic in urine has been used as a marker of exposure to inorganic As (iAs). Relative proportions of urinary metabolites of iAs have been identified as potential biomarkers of susceptibility to iAs toxicity. However, the adverse effects of iAs exposure are ultimately determined by the concentrations of iAs metabolites in target tissues. OBJECTIVE: In this study we examined the feasibility of analyzing As species in cells that originate in the urinary bladder, a target organ for As-induced cancer in humans. METHODS: Exfoliated bladder epithelial cells (BECs) were collected from urine of 21 residents of Zimapan, Mexico, who were exposed to iAs in drinking water. We determined concentrations of iAs, methyl-As (MAs), and dimethyl-As (DMAs) in urine using conventional hydride generation-cryotrapping-atomic absorption spectrometry (HG-CT-AAS). We used an optimized HG-CT-AAS technique with detection limits of 12-17 pg As for analysis of As species in BECs. RESULTS: All urine samples and 20 of 21 BEC samples contained detectable concentrations of iAs, MAs, and DMAs. Sums of concentrations of these As species in BECs ranged from 0.18 to 11.4 ng As/mg protein and in urine from 4.8 to 1,947 ng As/mL. We found no correlations between the concentrations or ratios of As species in BECs and in urine. CONCLUSION: These results suggest that urinary levels of iAs metabolites do not necessarily reflect levels of these metabolites in the bladder epithelium. Thus, analysis of As species in BECs may provide a more effective tool for risk assessment of bladder cancer and other urothelial diseases associated with exposures to iAs.

Speciation analysis of arsenic in biological matrices by automated hydride generation-cryotrapping-atomic absorption spectrometry with multiple microflame quartz tube atomizer (multiatomizer).

Analyses of arsenic (As) species in tissues and body fluids of individuals chronically exposed to inorganic arsenic (iAs) provide essential information about the exposure level and pattern of iAs metabolism. We have previously described an oxidation state-specific analysis of As species in biological matrices by hydride-generation atomic absorption spectrometry (HG-AAS), using cryotrapping (CT) for preconcentration and separation of arsines. To improve performance and detection limits of the method, HG and CT steps are automated and a conventional flame-in-tube atomizer replaced with a recently developed multiple microflame quartz tube atomizer (multiatomizer). In this system, arsines from As(III)-species are generated in a mixture of Tris-HCl (pH 6) and sodium borohydride. For generation of arsines from both As(III)- and As(V)-species, samples are pretreated with L-cysteine. Under these conditions, dimethylthioarsinic acid, a newly described metabolite of iAs, does not interfere significantly with detection and quantification of methylated trivalent arsenicals. Analytical performance of the automated HG-CT-AAS was characterized by analyses of cultured cells and mouse tissues that contained mono- and dimethylated metabolites of iAs. The capacity to detect methylated As(III)- and As(V)-species was verified, using an in vitro methylation system containing recombinant rat arsenic (+3 oxidation state) methyltransferase and cultured rat hepatocytes treated with iAs. Compared with the previous HG-CT-AAS design, detection limits for iAs and its metabolites have improved significantly with the current system, ranging from 8 to 20 pg. Recoveries of As were between 78 and 117%. The precision of the method was better than 5% for all biological matrices examined. Thus, the automated HG-CT-AAS system provides an effective and sensitive tool for analysis of all major human metabolites of iAs in complex biological matrices.

Oxidation State Specific Generation of Arsines from Methylated Arsenicals Based on L- Cysteine Treatment in Buffered Media for Speciation Analysis by Hydride Generation - Automated Cryotrapping - Gas Chromatography-Atomic Absorption Spectrometry with the Multiatomizer.

An automated system for hydride generation - cryotrapping- gas chromatography - atomic absorption spectrometry with the multiatomizer is described. Arsines are preconcentrated and separated in a Chromosorb filled U-tube. An automated cryotrapping unit, employing nitrogen gas formed upon heating in the detection phase for the displacement of the cooling liquid nitrogen, has been developed. The conditions for separation of arsines in a Chromosorb filled U-tube have been optimized. A complete separation of signals from arsine, methylarsine, dimethylarsine, and trimethylarsine has been achieved within a 60 s reading window. The limits of detection for methylated arsenicals tested were 4 ng l(-1). Selective hydride generation is applied for the oxidation state specific speciation analysis of inorganic and methylated arsenicals. The arsines are generated either exclusively from trivalent or from both tri- and pentavalent inorganic and methylated arsenicals depending on the presence of L-cysteine as a prereductant and/or reaction modifier. A TRIS buffer reaction medium is proposed to overcome narrow optimum concentration range observed for the L-cysteine modified reaction in HCl medium. The system provides uniform peak area sensitivity for all As species. Consequently, the calibration with a single form of As is possible. This method permits a high-throughput speciation analysis of metabolites of inorganic arsenic in relatively complex biological matrices such as cell culture systems without sample pretreatment, thus preserving the distribution of tri- and pentavalent species.

Arsenic stimulates sinusoidal endothelial cell capillarization and vessel remodeling in mouse liver.

UNLABELLED: Trivalent arsenic [As(III)] is a well-known environmental toxicant that causes a wide range of organ-specific diseases and cancers. In the human liver, As(III) promotes vascular remodeling, portal fibrosis, and hypertension, but the pathogenesis of these As(III)-induced vascular changes is unknown. To investigate the hypothesis that As(III) targets the hepatic endothelium to initiate pathogenic change, mice were exposed to 0 or 250 parts per billion (ppb) of As(III) in their drinking water for 5 weeks. Arsenic(III) exposure did not affect the overall health of the animals, the general structure of the liver, or hepatocyte morphology. There was no change in the total tissue arsenic levels, indicating that arsenic does not accumulate in the liver at this level of exposure. However, there was significant vascular remodeling with increased sinusoidal endothelial cell (SEC) capillarization, vascularization of the peribiliary vascular plexus (PBVP), and constriction of hepatic arterioles in As(III)-exposed mice. In addition to ultrastructural demonstration of SEC defenestration and capillarization, quantitative immunofluorescence analysis revealed increased sinusoidal PECAM-1 and laminin-1 protein expression, suggesting gain of adherens junctions and a basement membrane. Conversion of SECs to a capillarized, dedifferentiated endothelium was confirmed at the cellular level with demonstration of increased caveolin-1 expression and SEC caveolae, as well as increased membrane-bound Rac1-GTPase. CONCLUSION: These data demonstrate that exposure to As(III) causes functional changes in SEC signaling for sinusoidal capillarization that may be initial events in pathogenic changes in the liver.