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1.
Toxicol Appl Pharmacol ; 449: 116137, 2022 08 15.
Article in English | MEDLINE | ID: mdl-35750205

ABSTRACT

Workers in the oil and gas industry are at risk for exposure to a number of physical and chemical hazards at the workplace. Chemical hazard risks include inhalation of crude oil or its volatile components. While several studies have investigated the neurotoxic effects of volatile hydrocarbons, in general, there is a paucity of studies assessing the neurotoxicity of crude oil vapor (COV). Consequent to the 2010 Deepwater Horizon (DWH) oil spill, there is growing concern about the short- and long-term health effects of exposure to COV. NIOSH surveys suggested that the DWH oil spill cleanup workers experienced neurological symptoms, including depression and mood disorders, but the health effects apart from oil dispersants were difficult to discern. To investigate the potential neurological risks of COV, male Sprague-Dawley rats were exposed by whole-body inhalation to COV (300 ppm; Macondo surrogate crude oil) following an acute (6 h/d × 1 d) or sub-chronic (6 h/d × 4 d/wk. × 4 wks) exposure regimen. At 1, 28 or 90 d post-exposure, norepinephrine (NE), epinephrine (EPI), dopamine (DA) and serotonin (5-HT) were evaluated as neurotransmitter imbalances are associated with psychosocial-, motor- and cognitive- disorders. Sub-chronic COV exposure caused significant reductions in NE, EPI and DA in the dopaminergic brain regions, striatum (STR) and midbrain (MB), and a large increase in 5-HT in the STR. Further, sub-chronic exposure to COV caused upregulation of synaptic and Parkinson's disease-related proteins in the STR and MB. Whether such effects will lead to neurodegenerative outcomes remain to be investigated.


Subject(s)
Neurotoxicity Syndromes , Petroleum Pollution , Petroleum , Water Pollutants, Chemical , Animals , Gases , Male , Neurotoxicity Syndromes/etiology , Neurotransmitter Agents , Rats , Rats, Sprague-Dawley , Serotonin , Water Pollutants, Chemical/toxicity
2.
Toxicol Appl Pharmacol ; 409: 115300, 2020 12 15.
Article in English | MEDLINE | ID: mdl-33141058

ABSTRACT

Hydraulic fracturing (fracking) is a process used to recover oil and gas from shale rock formation during unconventional drilling. Pressurized liquids containing water and sand (proppant) are used to fracture the oil- and natural gas-laden rock. The transportation and handling of proppant at well sites generate dust aerosols; thus, there is concern of worker exposure to such fracking sand dusts (FSD) by inhalation. FSD are generally composed of respirable crystalline silica and other minerals native to the geological source of the proppant material. Field investigations by NIOSH suggest that the levels of respirable crystalline silica at well sites can exceed the permissible exposure limits. Thus, from an occupational safety perspective, it is important to evaluate the potential toxicological effects of FSD, including any neurological risks. Here, we report that acute inhalation exposure of rats to one FSD, i.e., FSD 8, elicited neuroinflammation, altered the expression of blood brain barrier-related markers, and caused glial changes in the olfactory bulb, hippocampus and cerebellum. An intriguing observation was the persistent reduction of synaptophysin 1 and synaptotagmin 1 proteins in the cerebellum, indicative of synaptic disruption and/or injury. While our initial hazard identification studies suggest a likely neural risk, more research is necessary to determine if such molecular aberrations will progressively culminate in neuropathology/neurodegeneration leading to behavioral and/or functional deficits.


Subject(s)
Inflammation/chemically induced , Inflammation/metabolism , Inhalation Exposure/adverse effects , Sand/chemistry , Synapses/drug effects , Synapses/metabolism , Aerosols/adverse effects , Animals , Biomarkers/metabolism , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Dust , Environmental Monitoring/methods , Hydraulic Fracking/methods , Male , Occupational Exposure/adverse effects , Rats , Rats, Sprague-Dawley
3.
Toxicology ; 328: 168-78, 2015 Feb 03.
Article in English | MEDLINE | ID: mdl-25549921

ABSTRACT

Welding fumes (WF) are a complex mixture of toxic metals and gases, inhalation of which can lead to adverse health effects among welders. The presence of manganese (Mn) in welding electrodes is cause for concern about the potential development of Parkinson's disease (PD)-like neurological disorder. Consequently, from an occupational safety perspective, there is a critical need to prevent adverse exposures to WF. As the fume generation rate and physicochemical characteristics of welding aerosols are influenced by welding process parameters like voltage, current or shielding gas, we sought to determine if changing such parameters can alter the fume profile and consequently its neurotoxic potential. Specifically, we evaluated the influence of voltage on fume composition and neurotoxic outcome. Rats were exposed by whole-body inhalation (40 mg/m(3); 3h/day × 5 d/week × 2 weeks) to fumes generated by gas-metal arc welding using stainless steel electrodes (GMA-SS) at standard/regular voltage (25 V; RVSS) or high voltage (30 V; HVSS). Fumes generated under these conditions exhibited similar particulate morphology, appearing as chain-like aggregates; however, HVSS fumes comprised of a larger fraction of ultrafine particulates that are generally considered to be more toxic than their fine counterparts. Paradoxically, exposure to HVSS fumes did not elicit dopaminergic neurotoxicity, as monitored by the expression of dopaminergic and PD-related markers. We show that the lack of neurotoxicity is due to reduced solubility of Mn in HVSS fumes. Our findings show promise for process control procedures in developing prevention strategies for Mn-related neurotoxicity during welding; however, it warrants additional investigations to determine if such modifications can be suitably adapted at the workplace to avert or reduce adverse neurological risks.


Subject(s)
Air Pollutants, Occupational/toxicity , Brain/drug effects , Inhalation Exposure/prevention & control , Manganese Poisoning/prevention & control , Manganese/toxicity , Parkinson Disease, Secondary/prevention & control , Welding/methods , Aerosols , Air Pollutants, Occupational/chemistry , Animals , Body Burden , Brain/metabolism , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/metabolism , Equipment Design , Gene Expression Regulation/drug effects , Humans , Inhalation Exposure/adverse effects , Male , Manganese/chemistry , Manganese Poisoning/etiology , Manganese Poisoning/genetics , Manganese Poisoning/metabolism , Parkinson Disease, Secondary/etiology , Parkinson Disease, Secondary/genetics , Parkinson Disease, Secondary/metabolism , Particle Size , Rats, Sprague-Dawley , Risk Assessment , Solubility , Time Factors , Welding/instrumentation
4.
Inhal Toxicol ; 26(12): 720-32, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25265048

ABSTRACT

Welding generates complex metal aerosols, inhalation of which is linked to adverse health effects among welders. An important health concern of welding fume (WF) exposure is neurological dysfunction akin to Parkinson's disease (PD). Some applications in manufacturing industry employ a variant welding technology known as "weld-bonding" that utilizes resistance spot welding, in combination with adhesives, for metal-to-metal welding. The presence of adhesives raises additional concerns about worker exposure to potentially toxic components like Methyl Methacrylate, Bisphenol A and volatile organic compounds (VOCs). Here, we investigated the potential neurotoxicological effects of exposure to welding aerosols generated during weld-bonding. Male Sprague-Dawley rats were exposed (25 mg/m³ targeted concentration; 4 h/day × 13 days) by whole-body inhalation to filtered air or aerosols generated by either weld-bonding with sparking (high metal, low VOCs; HM) or without sparking (low metal; high VOCs; LM). Fumes generated under these conditions exhibited complex aerosols that contained both metal oxide particulates and VOCs. LM aerosols contained a greater fraction of VOCs than HM, which comprised largely metal particulates of ultrafine morphology. Short-term exposure to LM aerosols caused distinct changes in the levels of the neurotransmitters, dopamine (DA) and serotonin (5-HT), in various brain areas examined. LM aerosols also specifically decreased the mRNA expression of the olfactory marker protein (Omp) and tyrosine hydroxylase (Th) in the olfactory bulb. Consistent with the decrease in Th, LM also reduced the expression of dopamine transporter (Slc6a3; Dat), as well as, dopamine D2 receptor (Drd2) in the olfactory bulb. In contrast, HM aerosols induced the expression of Th and dopamine D5 receptor (Drd5) mRNAs, elicited neuroinflammation and blood-brain barrier-related changes in the olfactory bulb, but did not alter the expression of Omp. Our findings divulge the differential effects of LM and HM aerosols in the brain and suggest that exposure to weld-bonding aerosols can potentially elicit neurotoxicity following a short-term exposure. However, further investigations are warranted to determine if the aerosols generated by weld-bonding can contribute to persistent long-term neurological deficits and/or neurodegeneration.


Subject(s)
Air Pollutants, Occupational/toxicity , Brain Chemistry/drug effects , Brain/drug effects , Inhalation Exposure/adverse effects , Neurons/drug effects , Neurotoxicity Syndromes/metabolism , Welding , Adhesives/chemistry , Aerosols , Air Pollutants, Occupational/chemistry , Animals , Biomarkers/metabolism , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/immunology , Blood-Brain Barrier/metabolism , Brain/immunology , Brain/metabolism , Fires , Gene Expression Regulation/drug effects , Male , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/immunology , Neurons/metabolism , Neurotoxicity Syndromes/immunology , Olfactory Bulb/drug effects , Olfactory Bulb/immunology , Olfactory Bulb/metabolism , Oxidation-Reduction , Rats, Sprague-Dawley , Steel/chemistry , Toxicity Tests, Acute , Volatile Organic Compounds/analysis , Volatile Organic Compounds/toxicity , Welding/methods
5.
Toxicology ; 291(1-3): 73-82, 2012 Jan 27.
Article in English | MEDLINE | ID: mdl-22085607

ABSTRACT

Occupational exposure to welding fumes (WF) is thought to cause Parkinson's disease (PD)-like neurological dysfunction. An apprehension that WF may accelerate the onset of PD also exists. Identifying reliable biomarkers of exposure and neurotoxicity are therefore critical for biomonitoring and neurological risk characterization of WF exposure. Manganese (Mn) in welding consumables is considered the causative factor for the neurological deficits seen in welders. Hence, we sought to determine if Mn accumulation in blood or nail clippings can be a marker for adverse exposure and neurotoxicity. To model this, rats were exposed by intratracheal instillation to dissolved or suspended fume components collected from gas metal arc-mild steel (GMA-MS) or manual metal arc-hard surfacing (MMA-HS) welding. Trace element analysis revealed selective Mn accumulation in dopaminergic brain areas, striatum (STR) and midbrain (MB), following exposure to the two fumes. This caused dopaminergic abnormality as evidenced by loss of striatal tyrosine hydroxylase (Th; 25-32% decrease) and Parkinson disease (autosomal recessive, early onset) 7 (Park7; 25-46% decrease) proteins. While blood Mn was not detectable, Mn levels in nails strongly correlated with the pattern of Mn accumulation in the striatum (R(2)=0.9386) and midbrain (R(2)=0.9332). Exposure to manganese chloride (MnCl(2)) caused similar Mn accumulation in STR, MB and nail. Our findings suggest that nail Mn has the potential to be a sensitive and reliable biomarker for long-term Mn exposure and associated neurotoxicity. The non-invasive means by which nail clippings can be collected, stored, and transported with relative ease, make it an attractive surrogate for biomonitoring WF exposures in occupational settings.


Subject(s)
Air Pollutants, Occupational/adverse effects , Air Pollutants, Occupational/toxicity , Biomarkers/analysis , Hoof and Claw/chemistry , Hoof and Claw/metabolism , Inhalation Exposure/adverse effects , Inhalation Exposure/analysis , Manganese Poisoning/metabolism , Manganese/adverse effects , Manganese/metabolism , Nails/chemistry , Nails/metabolism , Occupational Exposure/adverse effects , Occupational Exposure/analysis , Welding , Animals , Atmosphere Exposure Chambers , Blotting, Western , Brain/metabolism , Dopamine/physiology , Environmental Monitoring/methods , Humans , Lung/metabolism , Male , Manganese/pharmacokinetics , Metals/analysis , Metals/pharmacokinetics , Rats , Rats, Sprague-Dawley , Tissue Distribution
6.
J Toxicol Environ Health A ; 74(21): 1405-18, 2011.
Article in English | MEDLINE | ID: mdl-21916746

ABSTRACT

Consequent to the 2010 Deepwater Horizon oil spill in the Gulf of Mexico, there is an emergent concern about the short- and long-term adverse health effects of exposure to crude oil, weathered-oil products, and oil dispersants among the workforce employed to contain and clean up the spill. Oil dispersants typically comprise of a mixture of solvents and surfactants that break down floating oil to micrometer-sized droplets within the water column, thus preventing it from reaching the shorelines. As dispersants are generally sprayed from the air, workers are at risk for exposure primarily via inhalation. Such inhaled fractions might potentially permeate or translocate to the brain via olfactory or systemic circulation, producing central nervous system (CNS) abnormalities. To determine whether oil dispersants pose a neurological risk, male Sprague-Dawley rats were exposed by whole-body inhalation exposure to a model oil dispersant, COREXIT EC9500A (CE; approximately 27 mg/m(3) × 5 h/d × 1 d), and various molecular indices of neural dysfunction were evaluated in discrete brain areas, at 1 or 7 d postexposure. Exposure to CE produced partial loss of olfactory marker protein in the olfactory bulb. CE also reduced tyrosine hydroxylase protein content in the striatum. Further, CE altered the levels of various synaptic and neuronal intermediate filament proteins in specific brain areas. Reactive astrogliosis, as evidenced by increased expression of glial fibrillary acidic protein, was observed in the hippocampus and frontal cortex following exposure to CE. Collectively, these findings are suggestive of disruptions in olfactory signal transduction, axonal function, and synaptic vesicle fusion, events that potentially result in an imbalance in neurotransmitter signaling. Whether such acute molecular aberrations might persist and produce chronic neurological deficits remains to be ascertained.


Subject(s)
Brain/drug effects , Emulsifying Agents/toxicity , Environmental Restoration and Remediation/adverse effects , Inhalation Exposure/adverse effects , Lipids/toxicity , Animals , Brain/metabolism , Glial Fibrillary Acidic Protein/biosynthesis , Male , Models, Animal , Olfactory Marker Protein/biosynthesis , Petroleum Pollution , Rats , Rats, Sprague-Dawley , Toxicity Tests, Acute , Tyrosine 3-Monooxygenase/biosynthesis
7.
FASEB J ; 24(12): 4989-5002, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20798247

ABSTRACT

Welding generates complex metal aerosols, inhalation of which is linked to adverse health effects among welders. An important health concern of welding fume (WF) exposure is neurological dysfunction akin to Parkinson's disease (PD), thought to be mediated by manganese (Mn) in the fumes. Also, there is a proposition that welding might accelerate the onset of PD. Our recent findings link the presence of Mn in the WF with dopaminergic neurotoxicity seen in rats exposed to manual metal arc-hard surfacing (MMA-HS) or gas metal arc-mild steel (GMA-MS) fumes. To elucidate the molecular mechanisms further, we investigated the association of PD-linked (Park) genes and mitochondrial function in causing dopaminergic abnormality. Repeated instillations of the two fumes at doses that mimic ∼1 to 5 yr of worker exposure resulted in selective brain accumulation of Mn. This accumulation caused impairment of mitochondrial function and loss of tyrosine hydroxylase (TH) protein, indicative of dopaminergic injury. A fascinating finding was the altered expression of Parkin (Park2), Uchl1 (Park5), and Dj1 (Park7) proteins in dopaminergic brain areas. A similar regimen of manganese chloride (MnCl(2)) also caused extensive loss of striatal TH, mitochondrial electron transport components, and Park proteins. As mutations in PARK genes have been linked to early-onset PD in humans, and because welding is implicated as a risk factor for parkinsonism, PARK genes might play a critical role in WF-mediated dopaminergic dysfunction. Whether these molecular alterations culminate in neurobehavioral and neuropathological deficits reminiscent of PD remains to be ascertained.


Subject(s)
Manganese/toxicity , Parkinson Disease/metabolism , Welding , Air Pollutants, Occupational/toxicity , Animals , Blotting, Western , Chlorides/toxicity , Inhalation Exposure/adverse effects , Male , Manganese/metabolism , Manganese Compounds , Occupational Exposure/adverse effects , Parkinson Disease/etiology , Polymerase Chain Reaction , Rats , Rats, Sprague-Dawley , Tyrosine 3-Monooxygenase/genetics , Tyrosine 3-Monooxygenase/metabolism , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
8.
Arch Toxicol ; 84(7): 521-40, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20224926

ABSTRACT

The potential for development of Parkinson's disease (PD)-like neurological dysfunction following occupational exposure to aerosolized welding fumes (WF) is an area of emerging concern. Welding consumables contain a complex mixture of metals, including iron (Fe) and manganese (Mn), which are known to be neurotoxic. To determine whether WF exposure poses a neurological risk particularly to the dopaminergic system, we treated Sprague-Dawley rats with WF particulates generated from two different welding processes, gas metal arc-mild steel (GMA-MS; low Mn, less water-soluble) and manual metal arc-hard surfacing (MMA-HS; high Mn, more water-soluble) welding. Following repeated intratracheal instillations (0.5 mg/rat, 1/week x 7 weeks) of GMA-MS or MMA-HS, elemental analysis and various molecular indices of neurotoxicity were measured at 1, 4, 35 or 105 days after last exposure. MMA-HS exposure, in particular, led to increased deposition of Mn in striatum and midbrain. Both fumes also caused loss of tyrosine hydroxylase (TH) protein in the striatum (~20%) and midbrain (~30%) by 1 day post-exposure. While the loss of TH following GMA-MS was transient, a sustained loss (34%) was observed in the midbrain 105 days after cessation of MMA-HS exposure. In addition, both fumes caused persistent down-regulation of dopamine D2 receptor (Drd2; 30-40%) and vesicular monoamine transporter 2 (Vmat2; 30-55%) mRNAs in the midbrain. WF exposure also modulated factors associated with synaptic transmission, oxidative stress, neuroinflammation and gliosis. Collectively, our findings demonstrate that repeated exposure to Mn-containing WF can cause persistent molecular alterations in dopaminergic targets. Whether such perturbations will lead to PD-like neuropathological manifestations remains to be elucidated.


Subject(s)
Manganese/metabolism , Welding , Animals , Dopamine , Down-Regulation , Gases/metabolism , Iron/metabolism , Lung/metabolism , Male , Metals/metabolism , Metals/toxicity , Neurotoxicity Syndromes , Occupational Exposure , Parkinson Disease , Rats , Rats, Sprague-Dawley , Steel/toxicity
9.
Neurotoxicology ; 30(6): 915-25, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19782702

ABSTRACT

Serious questions have been raised by occupational health investigators regarding a possible causal association between neurological effects in welders and the presence of manganese (Mn) in welding fume. Male Sprague-Dawley rats were exposed by inhalation to 40 mg/m(3) of gas metal arc-mild steel (MS) welding fume for 3 h/day for 10 days. Generated fume was collected in the animal chamber during exposure, and particle size, composition, and morphology were characterized. At 1 day after the last exposure, metal deposition in different organ systems and neurological responses in dopaminergic brain regions were assessed in exposed animals. The welding particles were composed primarily of a complex of iron (Fe) and Mn and were arranged as chain-like aggregates with a significant number of particles in the nanometer size range. Mn was observed to translocate from the lungs to the kidney and specific brain regions (olfactory bulb, cortex, and cerebellum) after MS fume inhalation. In terms of neurological responses, short-term MS fume inhalation induced significant elevations in divalent metal ion transporter 1 (Dmt1) expression in striatum and midbrain and significant increases in expression of proinflammatory chemokines (Ccl2, Cxcl2) and cytokines (IL1beta, TNFalpha) in striatum. In addition, mRNA and protein expression of glial fibrillary acidic protein (GFAP) was significantly increased in striatum after MS fume exposure. However, the 10-day MS welding fume inhalation did not cause any changes in dopamine and its metabolites or GABA in dopaminergic brain regions nor did it produce overt neural cell damage as assessed by histopathology. In summary, short-term MS welding fume exposure led to translocation of Mn to specific brain regions and induced subtle changes in cell markers of neuroinflammatory and astrogliosis. The neurofunctional significance of these findings currently is being investigated in longer, more chronic welding fume exposure studies.


Subject(s)
Brain/metabolism , Encephalitis/etiology , Inhalation Exposure/adverse effects , Manganese/metabolism , Steel/toxicity , Welding , Animals , Brain/pathology , Catecholamines/metabolism , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Chromatography, High Pressure Liquid/methods , Cytokines/metabolism , Dopamine/metabolism , Electrochemistry/methods , Encephalitis/metabolism , Encephalitis/pathology , Enzyme-Linked Immunosorbent Assay/methods , Fluoresceins , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Glial Fibrillary Acidic Protein/metabolism , Homovanillic Acid/metabolism , Lung/chemistry , Male , Organic Chemicals , Rats , Rats, Sprague-Dawley
10.
Toxicol Appl Pharmacol ; 233(1): 81-91, 2008 Nov 15.
Article in English | MEDLINE | ID: mdl-18367224

ABSTRACT

The incidence of adenocarcinoma of the lung is increasing in the United States, however, the difficulties in obtaining lung cancer families and representative samples of early to late stages of the disease have lead to the study of mouse models for lung cancer. We used Spectral Karyotyping (SKY), mapping with fluorescently labeled genomic clones (FISH), comparative genomic hybridization (CGH) arrays, gene expression arrays, Western immunoblot and real time polymerase chain reaction (PCR) to analyze nine pairs of high-invasive and low-invasive tumor cell strains derived from early passage mouse lung adenocarcinoma cells to detect molecular changes associated with tumor invasion. The duplication of chromosomes 1 and 15 and deletion of chromosome 8 were significantly associated with a high-invasive phenotype. The duplication of chromosome 1 at band C4 and E1/2-H1 were the most significant chromosomal changes in the high-invasive cell strains. Mapping with FISH and CGH array further narrowed the minimum region of duplication of chromosome 1 to 71-82 centimorgans (cM). Expression array analysis and confirmation by real time PCR demonstrated increased expression of COX-2, Translin (TB-RBP), DYRK3, NUCKS and Tubulin-alpha4 genes in the high-invasive cell strains. Elevated expression and copy number of these genes, which are involved in inflammation, cell movement, proliferation, inhibition of apoptosis and telomere elongation, were associated with an invasive phenotype. Similar linkage groups are altered in invasive human lung adenocarcinoma, implying that the mouse is a valid genetic model for the study of the progression of human lung adenocarcinoma.


Subject(s)
Adenocarcinoma/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Chromosome Aberrations , Lung Neoplasms/genetics , Adenocarcinoma/pathology , Animals , Carcinoma, Non-Small-Cell Lung/pathology , Cell Movement/physiology , Karyotyping/methods , Lung Neoplasms/pathology , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Neoplasm Invasiveness , Species Specificity , Tumor Cells, Cultured
11.
Exp Cell Res ; 313(18): 3868-80, 2007 Nov 01.
Article in English | MEDLINE | ID: mdl-17888903

ABSTRACT

We have previously shown that reactivation of DLC1, a RhoGAP containing tumor suppressor gene, inhibits tumorigenicity of human non-small cell lung carcinoma cells (NSCLC). After transfection of NSCLC cells with wild type (WT) DLC1, changes in cell morphology were observed. To determine whether such changes have functional implications, we generated several DLC1 mutants and examined their effects on cell morphology, proliferation, migration and apoptosis in a DLC1 deficient NSCLC cell line. We show that WT DLC1 caused actin cytoskeleton-based morphological alterations manifested as cytoplasmic extensions and membrane blebbings in most cells. Subsequently, a fraction of cells exhibiting DLC1 protein nuclear translocation (PNT) underwent caspase 3-dependent apoptosis. We also show that the RhoGAP domain is essential for the occurrence of morphological alterations, PNT and apoptosis, and the inhibition of cell migration. DLC1 PNT is dependent on a bipartite nuclear localizing sequence and most likely is regulated by a serine-rich domain at N-terminal part of the DLC1 protein. Also, we found that DLC1 functions in the cytoplasm as an inhibitor of tumor cell proliferation and migration, but in the nucleus as an inducer of apoptosis. Our analyses provide evidence for a possible link between morphological alterations, PNT and proapoptotic and anti-oncogenic activities of DLC1 in lung cancer.


Subject(s)
Apoptosis , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Nucleus/metabolism , Cell Shape , Lung Neoplasms/pathology , Tumor Suppressor Proteins/metabolism , Actins/metabolism , Amino Acid Sequence , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cell Surface Extensions/metabolism , Cytoskeleton/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , GTPase-Activating Proteins , Humans , Lung Neoplasms/metabolism , Molecular Sequence Data , Mutant Proteins/metabolism , Nuclear Localization Signals/metabolism , Protein Structure, Tertiary , Protein Transport , Serine , Structure-Activity Relationship , Tumor Suppressor Proteins/chemistry
12.
Cancer Biol Ther ; 5(4): 407-12, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16481740

ABSTRACT

Translocations and unique chromosome break points in melanoma will aid in the identification of the genes that are important in the neoplastic process. We have previously shown a unique translocation in malignant melanoma cells der(12)t(12;20). The transcription factor E2F1 maps to 20q11. Increased expression of E2F has been associated with the autonomous growth of melanoma cells, however, the molecular basis has not yet been elucidated. To this end, we investigated E2F1 gene copy number and structure in human melanoma cell lines and metastatic melanoma cases. Fluorescent in situ hybridization (FISH) analysis using a specific E2F1 probe indicated increased E2F1 gene copies in melanoma cell lines compared to normal melanocytes. We also observed increased copies of the E2F1 gene in lymph node metastases of melanoma. In addition, Western blot analysis demonstrated increased E2F1 protein levels in 8 out of 9 melanoma cell lines relative to normal melanocytes. Inhibition of E2F1 expression with RNAi also reduced melanoma cell growth. Our results suggest that the release of E2F activity by elevated E2F1 gene copy numbers may play a functional role in melanoma growth.


Subject(s)
E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/metabolism , Genetic Predisposition to Disease , Melanoma/genetics , Melanoma/pathology , Blotting, Western , Cell Line, Tumor , Chromosomes, Human, Pair 12 , Humans , In Situ Hybridization, Fluorescence , Lymphatic Metastasis , Melanocytes/metabolism , Melanoma/metabolism , RNA Interference , Translocation, Genetic
13.
Neoplasia ; 6(4): 412-9, 2004.
Article in English | MEDLINE | ID: mdl-15256063

ABSTRACT

The identification of genes undergoing genetic or epigenetic alterations and contributing to the development of cancer is critical to our understanding of the molecular mechanisms of carcinogenesis. A new approach in identifying alterations of genes that might be relevant to the process of tumor development was used in this study by examining the gene expression profile in human lung cancer cells exposed to 5-aza-2'-deoxycytidine (5-aza-dC). A cDNA array analysis was carried out on 5-aza-dC-treated and untreated non small cell lung cancer (NSCLC) cell line NCI-H522. Sixteen and 14 genes were upregulated and downregulated, respectively, by 5-aza-dC treatment. Among them, downregulation of tyrosine protein kinase ABL2 (ABL2) gene and upregulation of hint/protein kinase C inhibitor 1 (Hint/PKCI-1), DVL1, TIMP-1, and TRP-1 genes were found in expanded observations in two or three of five 5-aza-dC-treated NSCLC cell lines. Among these genes, we found that cDNA transfer of Hint/PKCI-1 resulted in a significant in vitro growth inhibition in two cell lines exhibiting 5-aza-dC-induced upregulation of Hint/PKCI-1 and significantly reduced in vivo tumorigenicity of one NSCLC cell line. Hint/PKCI-1, which is the only other characterized human histidine triad (HIT) nucleotide-binding protein in addition to tumor-suppressor gene FHIT, might be involved in lung carcinogenesis.


Subject(s)
Azacitidine/analogs & derivatives , Azacitidine/toxicity , Gene Expression Regulation, Neoplastic/drug effects , Antimetabolites, Antineoplastic/toxicity , Apoptosis/drug effects , Carcinoma, Non-Small-Cell Lung , Cell Division/drug effects , Cell Line, Tumor , DNA Methylation , Decitabine , Humans , Kinetics , Lung Neoplasms , Oligonucleotide Array Sequence Analysis , Transfection
14.
Oncogene ; 23(7): 1405-11, 2004 Feb 19.
Article in English | MEDLINE | ID: mdl-14661059

ABSTRACT

The deleted in liver cancer (DLC-1) gene at chromosome 8p21-22 is altered mainly by genomic deletion or aberrant promoter methylation in a large number of human cancers such as breast, liver, colon and prostate and is known to have an inhibitory effect on breast and liver tumor cell growth. Given the high frequency of deletion involving region 8p21-22 in human non-small cell lung carcinoma (NSCLC), we examined alterations of DLC-1 in a series of primary tumors and tumor cell lines and tested effects of DLC-1 on tumor cell growth. A significant decrease or absence of the DLC-1 mRNA expression was found in 95% of primary NSCLC (20/21) and 58% of NSCLC cell lines (11/19). Transcriptional silencing of DLC-1 was primarily associated with aberrant DNA methylation, rather than genomic deletion as 5-aza-2'-deoxycytidine induced reactivation of DLC-1 expression in 82% (9/11) NSCLC cell lines showing downregulated DLC-1. It was further evidenced by an aberrant DLC-1 promoter methylation pattern, which was detected by Southern blotting in 73% (8/11) of NSCLC cell lines with downregulation of the gene. The transfer of DLC-1 into three DLC-1 negative cell lines caused a significant inhibition in cell proliferation and/or a decrease in colony formation. Furthermore, stable transfer of DLC-1 abolished tumorigenicity in nude mice of two cell lines, suggesting that DLC-1 plays a role in NSCLC by acting as a bona fide new tumor suppressor gene.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms , Tumor Suppressor Proteins/genetics , Animals , Carcinoma, Non-Small-Cell Lung/metabolism , DNA Methylation , GTPase-Activating Proteins , Humans , Immunoblotting , Mice , Mice, Nude , Promoter Regions, Genetic , Tumor Cells, Cultured/transplantation , Tumor Suppressor Proteins/metabolism
15.
Cancer Res ; 62(4): 1152-7, 2002 Feb 15.
Article in English | MEDLINE | ID: mdl-11861397

ABSTRACT

Although adenocarcinoma is rapidly becoming the most common form of lung cancer in the United States, the difficulty in obtaining lung cancer families and representative samples of the various stages of adenocarcinoma progression has led to intense study of mouse models. As a powerful approach to delineating molecular changes, we have analyzed 15 early-passage mouse cell lines by spectral karyotyping. Entire copies of chromosomes 1, 2, 6, 12, 15, and 19 were gained, and entire copies of chromosomes 4, 7, 8, and 14 were lost. Significant gains of portions of chromosome 1 (93% of the tumor cell lines analyzed), chromosome 2 (53%), chromosome 6 (73%), chromosome 7 (80%), chromosome 12 (47%), and chromosome 15 (73%) and partial loss of chromosome 4 (87%), chromosome 7 (80%), chromosome 8 (53%), chromosome 10 (33%), and chromosome 14 (33%) were observed. Recurrent translocations included 10:del(10)(A1::C1), t(4;8)(C4;A1), and der (1;12)(C2;C2). The minimal regions of chromosomal alteration, 1G1, 2F1, 4C4, 6D, 7F1, 8B3, and 12C2, contain putative susceptibility genes for mouse lung adenocarcinoma. Chromosomal regions containing susceptibility genes linked to tumor size were frequently amplified, whereas regions with susceptibility loci linked to tumor multiplicity were deleted. Similar linkage groups are altered in human lung adenocarcinoma, implying that the mouse is a valid genetic model for the study of human lung adenocarcinoma susceptibility.


Subject(s)
Adenocarcinoma/genetics , Chromosome Aberrations , Lung Neoplasms/genetics , Animals , Gene Amplification , Gene Deletion , Genetic Linkage , Humans , Karyotyping , Mice , Species Specificity , Translocation, Genetic , Tumor Cells, Cultured
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