Begell House Inc.
Journal of Environmental Pathology, Toxicology and Oncology
JEP(T)
0731-8898
28
2
2009
MET, HGF, EGFR, and PXN Gene Copy Number in Lung Cancer Using DNA Extracts from FFPE Archival Samples and Prognostic Significance
89-98
10.1615/JEnvironPatholToxicolOncol.v28.i2.10
Rajani
Kanteti
Department of Medicine, Section of Hematology and Oncology University of Chicago, Chicago, IL
Soheil
Yala
Department of Medicine, Section of Hematology and Oncology University of Chicago, Chicago, IL
Mark K.
Ferguson
Department of Surgery; University of Chicago, Chicago, IL
Ravi
Salgia
Department of Medical Oncology and Therapeutics Research, Comprehensive Cancer Center, City of Hope National Medical Center, Duarte, CA 91010, USA
genomic DNA
FFPE
MET
EGFR
paxillin
gene copy number
Gene copy number analysis for some of the important molecules in lung tumorogenesis, such as MET, hepatocyte growth factor [(HGF), ligand for MET), epidermal growth factor receptor (EGFR), and paxillin (PXN), is likely to determine both the type of treatment and prognosis. Formalin-fixed paraffin-embedded (FFPE) archival tumor tissue samples are an excellent source for determining key molecular changes in the OncoGenome; however, existing extraction procedures yield relatively poor quality genomic DNA fragments. Although FISH is the method of choice for determining amplification of a gene, a more rapid quantitative poly-merase chain reaction (qPCR) technique to determine gene copy number can be used when reasonably good quality genomic DNA is available. We report here a relatively rapid method based on microwave/chelex-100 treatment that gives rise to genomic DNA fragments ranging from 1 to 12 Kb and beyond, thereby attesting to its superior quality. Genomic PCR for β-globin gene gave reliable and reproducible results. The number of steps for extracting the DNA was kept to a minimum, and instead of precipitating the DNA, we preserved the genomic DNA extracts so as to prevent a loss in DNA yield. We found the extracts to be stable and amenable to qPCR and mutational analysis. Using lung adenocarcinoma FFPE samples and cell lines derived from lung adenocarcinomas, we demonstrated that the gene copy number for MET in lung adenocarcinoma tissue samples was preferentially increased over EGFR, HGF, and PXN and that it positively correlated with a better prognosis. In contrast, the genomic DNA extracted from 25 NSCLC cell lines gave a relatively higher gene copy number for all four genes evaluated. Our results indicate that the microwave/chelex-100-based method yields good-quality genomic DNA extracts that can be used for complex DNA analysis, such as determination of gene copy number. In addition, our data demonstrated that the adenocarcinoma cell lines potentially evolved under ex vivo conditions, and therefore, in genetic studies it is imperative to use primary tumors for generalized conclusions about lung tumors.
Wnt Pathway in Pulmonary Fibrosis in the Bleomycin Mouse Model
99-108
10.1615/JEnvironPatholToxicolOncol.v28.i2.20
Li
Liu
Division of Pulmonary and Critical Care Medicine, Department of Medicine, New York University School of Medicine, 550 1st Ave, New York, NY 10016
Benjamin
Carron
Division of Pulmonary and Critical Care Medicine, Department of Medicine, New York University School of Medicine, 550 1st Ave, New York, NY 10016
Herman T.
Yee
Departments of Pathology and Environmental Medicine, and Division of Pulmonary and Critical Care Medicine. NYU School of Medicine, New York, NY 10016
Ting-An
Yie
Division of Pulmonary and Critical Care Medicine, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
Mustapha
Hajjou
Division of Pulmonary and Critical Care Medicine, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
William N.
Rom
Division of Pulmonary and Critical Care Medicine, and Department of Environmental Medicine, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA
pulmonary fibrosis
beta-catenin
Wnt pathway
Background: The Wnt/β-catenin signaling pathway plays an important role in regulating cellular differentiation, proliferation, and polarity. Methods: We used bleomycin to induce lung fibrosis in a transgenic Wnt reporter mouse to characterize the expression pattern of cyclin D1, MMP-7, and TGF-β in conjunction with the Wnt/β-catenin signaling pathway. LacZ expression reveals the Wnt/β-catenin signaling pathway through the activated (nuclear) β-catenin and coactivation of LEF/TCF transcription factors. X-gal staining and immunohistochemical staining of β-catenin, cyclin D1, MMP-7, and TGF-β were assessed after bleomycin administration. Results: We observed LacZ expression in bronchiolar proliferative lesions and the epithelium in remodeled cystic and fibrotic areas at both 1 and 3 weeks. Nuclear β-catenin staining was prominent in epithelial cells of remodeled and fibrotic areas at 3 weeks. MMP-7 was faint in basement membranes of airways and matrix zones in fibrotic areas at 3 weeks. Cyclin D1 was observed in alveolar macrophages (AM), alveolar epithelium, and fibrotic areas consistent with rapid cell turnover in these areas at both 1 and 3 weeks. TGF-β was faintly staining in alveolar macrophages and epithelial cells at 3 weeks. Conclusion: The Wnt/β-catenin pathway is activated in bleomycin-induced lung fibrosis, and downstream genes were localized in AM, alveolar epithelium, and interstitium.
Small Interfering RNAs (siRNAs) Targeting TGF-β1 mRNA Suppress Asbestos-Induced Expression of TGF-β1 and CTGF in Fibroblasts
109-119
10.1615/JEnvironPatholToxicolOncol.v28.i2.30
Tai-Cheng
Lai
Department of Environmental Health Sciences, Tulane University School of Public Health and Tropical Medicine, New Orleans, LA; Kaohsiung
Derek A.
Pociask
Pediatric Pulmonary Division, Children's Hospital of Pittsburgh, Pittsburgh, PA
MaryBeth
Ferris
Department of Microbiology and Immunology, Tulane University Health Sciences Center, New Orleans, LA
Hong T.
Nguyen
Section of Pulmonary Diseases, Tulane University Health Sciences Center, New Orleans, LA
Charles A.
Miller III
Department of Environmental Health Sciences, Tulane University School of Public Health and Tropical Medicine, New Orleans, LA
Arnold
Brody
North Carolina St. University
Deborah
Sullivan
Tulane University School of Medicine
interstitial lung disease
RNA interference
small interfering RNA
transforming growth factor-β1
connective tissue growth factor
asbestos
Interstitial lung disease (ILD) afflicts millions of people worldwide. ILD can be caused by a number of agents, including inhaled asbestos, and may ultimately result in respiratory failure and death. Currently, there are no effective treatments for ILD. Transforming growth factor-β1 (TGF-β1) is thought to play an important role in the development of pulmonary fibrosis, and asbestos has been shown to induce TGF-β1 expression in a murine model of ILD. To better define the role of TGF-β1 in ILD, we developed several small interfering RNAs (siRNAs) that target TGF-β1 mRNA for degradation. To assess the efficacy of each siRNA in reducing asbestos-induced TGF-β1 expression, Swiss 3T3 fibroblasts were transfected with TGF-β1 siRNAs and then treated with chrysotile asbestos for 48 h. Two independent siRNAs targeting TGF-β1 mRNA knocked-down asbestos-induced expression of TGF-β1 mRNA by 72−89% and protein by 70−84%. Interestingly, siRNA knockdown of TGF-β1 also reduced asbestos-induced expression of connective tissue growth factor (CTGF). CTGF can be upregulated by TGF-β1 and appears to play an important role in the development of pulmonary fibrosis. These results suggest that siRNAs could be effective in preventing or possibly arresting the progression of pulmonary fibrosis. Studies are underway in vivo to test this postulate.
Dietary Curcumin Enhances Benzo(a)pyrene-Induced Apoptosis Resulting in a Decrease in BPDE-DNA Adducts in Mice
121-131
10.1615/JEnvironPatholToxicolOncol.v28.i2.40
Rachana
Garg
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai-410 208, India
Girish
Maru
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai-410210, India
apoptosis
benzo(a)pyrene
curcumin
DNA adducts
Curcumin pretreatment has been shown to decrease the formation of B(a)P-derived DNA adducts; however, its effects on disappearance of BPDE-DNA adducts in vivo remain unexplored. We investigate the effect of curcumin on persistence of BPDE-DNA adducts and cell turnover in mouse tissues. Mice administered 1 mg B(a)P (gavage) were randomized after 24 h into group A (sacrificed at zero time), group B (continued on control diet), and group C (shifted to 0.05% curcumin diet), and sacrificed after 24, 48, and 96 h. Compared to group A, a time-dependent decrease in DNA adducts was observed in liver/lungs of group B mice. Mice shifted to curcumin diet showed a relatively higher decrease in DNA adducts when compared to group A/time-matched controls (group B). To investigate if this enhanced decrease was due to dilution by newly synthesized DNA or due to cell turnover; rate of DNA synthesis and apoptosis were evaluated. Similar levels of 3H-thymidine incorporated in tissue DNA from group B/C ruled out the possibility of adduct dilution. Comparative evaluation of apoptosis-related parameters showed increased apoptotic-index, p53, Bax and Caspase-3 protein expression and decreased Bcl2 levels in group C than in group A/B. This implies that curcumin post-treatment augments apoptosis of adducted cells resulting in enhanced decrease in DNA adducts.
Scientific Documentation of the Relationship of Vitamin D Deficiency and the Development of Cancer
133-141
10.1615/JEnvironPatholToxicolOncol.v28.i2.50
Richard
Edlich
Legacy Verified Level I Shock Trauma Center Pediatrics and Adults, Legacy Emanual Hospital; and Plastic Surgery, Biomedical Engineering and Emergency Medicine, University of Virginia Health System, USA
Shelley S.
Mason
Multiple Sclerosis Research Fund, Brush Prairie, WA, USA
Margot E.
Chase
Legacy Emanuel Hospital, Portland, OR, USA
Allyson L.
Fisher
Legacy Emanuel Hospital, Portland, OR, USA
K. Dean
Gubler
Surgical Critical Care, Legacy Verified Level I Shock Trauma Center for Pediatrics and Adults, Legacy Emanuel Hospital, Portland, OR, USA
William B.
Long III
Trauma Specialists LLP, Legacy Verified Level I Shock Trauma Center for Pediatrics and Adults, Legacy Emmanuel Hospital, Portland, OR, USA
Jerry D.
Giesy
The Urology Clinic, PC. Legacy Emanuel Hospital, Portland, OR
Marni L.
Foley
Portland Medical Clinic LLP, Beaverton, Oregon, USA
vitamin D deficiency
breast cancer
colorectal cancer
prostate cancer
multiple myeloma
It is well known that vitamin D plays a key role in calcium homeostasis and is important for optimal skeletal growth. The major function of vitamin D is to enhance the efficiency of calcium absorption from the small intestine. Most physicians relate vitamin D deficiency to disorders of skeletal muscle. Vitamin D deficiency in children can manifest itself as rickets. In adults, vitamin D deficiency results in osteomalacia. Because most physicians do not appreciate the role of vitamin D deficiency in predisposing the development of cancer, we have written this important report as a wake-up call to physicians and other healthcare workers in documenting the relationship of vitamin D deficiency and cancer. Epidemiological data show an inverse relationship between vitamin D levels and breast cancer incidence. In addition, there is a well-documented association between vitamin D intake and the risk of breast cancer. Low vitamin D intake has also been indicated in colorectal carcinogenesis. A vitamin D deficiency has also been documented in patients with prostate cancer, ovarian cancer, as well as multiple myeloma. Larger randomized clinical trials should be undertaken in humans to establish the role of vitamin D supplementation in the prevention of these cancers.
Revolutionary Advances in the Prevention of Demyelinating Diseases
143-152
10.1615/JEnvironPatholToxicolOncol.v28.i2.60
Richard
Edlich
Legacy Verified Level I Shock Trauma Center Pediatrics and Adults, Legacy Emanual Hospital; and Plastic Surgery, Biomedical Engineering and Emergency Medicine, University of Virginia Health System, USA
Shelley S.
Mason
Multiple Sclerosis Research Fund, Brush Prairie, WA, USA
Margot E.
Chase
Legacy Emanuel Hospital, Portland, OR, USA
Allyson L.
Fisher
Legacy Emanuel Hospital, Portland, OR, USA
K. Dean
Gubler
Surgical Critical Care, Legacy Verified Level I Shock Trauma Center for Pediatrics and Adults, Legacy Emanuel Hospital, Portland, OR, USA
William B.
Long III
Trauma Specialists LLP, Legacy Verified Level I Shock Trauma Center for Pediatrics and Adults, Legacy Emmanuel Hospital, Portland, OR, USA
Anthony T.
Newkirk
Synergy Dental, Portland, OR, USA
multiple sclerosis
ultraviolet radiation
vitamin D3
vitamin D receptor gene (VDRG)
silver mercury amalgam
This collective review focuses on three major factors that influence the incidences of multiple sclerosis (MS) to include ultraviolet radiation (UVR), vitamin D3 supplementation, and vitamin D receptor gene (VDRG) polymorphisms. In general, the rate of MS increases with latitude. Individuals tend to carry their original risk with them if they migrate to a different latitude after adolescence. It is important to emphasize that UVR increases the synthesis of vitamin D3, which has a known immune suppressant action via the VDRG. Clinical studies have pointed out that vitamin D deficiency may exacerbate the development of MS. Because vitamin D3 is an inhibitor of MS, providing supplemental D3 for individuals at risk for MS should be mandatory. There is unanimous agreement that exposure to UVR and vitamin D3 supplementation can reduce the incidence of MS. Although there is debate regarding the association of MS with the use of silver mercury fillings, there is general agreement throughout the world that there is a need for dental-patient informed-consent brochures for all dentists who use dental restoration materials. For the dentists who remove silver mercury amalgam from their patients, there is a uniform international agreement that all dental offices should use reliable debris collection devices that prevent pollution of our environment.
Iron Accumulation and Expression of Iron-Related Proteins Following Murine Exposure to Crocidolite
153-162
10.1615/JEnvironPatholToxicolOncol.v28.i2.70
Andrew J.
Ghio
National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Chapel Hill, North Carolina; and Department of Pathology, Duke University Medical Center, Durham, North Carolina
Roderick J.
Tan
Department of Pathology, University of Pittsburgh, PA 15261
Kathleen
Ghio
National Health and Environmental Effects Research Laboratory, Environmental Protection Agency, Research Triangle Park, NC 27711
Cheryl L.
Fattman
Department of Environmental and Occupation Health, University of Pittsburgh, PA 15261
Tim D.
Oury
Department of Pathology, University of Pittsburgh, PA 15261
asbestos
ferritin
metal transport protein 1
divalent metal transporter 1
duodenal cytochrome b
We tested the postulate that asbestos exposure alters iron homeostasis in the mouse lung. Crocidolite asbestos (100 μg intratracheally) was instilled into C57BL/6 mice. TiO2 served as a control exposure. Using iron staining and immunohistochemistry, concentrations of this metal and expression of several iron transport and storage proteins were evaluated at one day and one month following asbestos exposure. Iron was not stainable one day following asbestos instillation but was increased one month later. There was an elevated expression of duodenal cytochrome b (Dcytb), divalent metal transporter 1 (DMT1), and ferritin at both one day and one month after crocidolite exposure. While ferroportin (FPN1) expression was increased one day after asbestos exposure, levels of this metal exporter had returned to baseline at one month. TiO2 did not affect changes in either the iron concentration or the expression of these iron-related proteins at one day and one month. We conclude that asbestos exposure alters lung iron homeostasis with an accumulation of the metal resulting. Elevations in available iron affect changes in the expression of Dcytb, DMT1, ferritin, and FPN1, which further modify metal homeostasis in the lung.
Guilandina bonduc L. Possesses Antioxidant Activity and Precludes Ferric Nitrilotriacetate (Fe-NTA) Induced Renal Toxicity and Tumor Promotion Response
163-175
10.1615/JEnvironPatholToxicolOncol.v28.i2.80
Gurpreet
Kaur
Division of Radiopharmaceuticals and Radiation Biology, Institute of Nuclear Medicine and Allied Sciences, Delhi; Department of Medical Elementology & Toxicology, Jamia Hamdard, Hamdard Nagar, New Delhi-110062, India
Irshad A.
Lone
Department of Chemistry, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi-110062, India
Mohammad
Athar
Department of Medical Elementology & Toxicology, Jamia Hamdard, Hamdard Nagar, New Delhi-110062, India
Sarwar M.
Alam
Department of Chemistry, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi-110062, India
Guilandina bonduc
ferric nitrilotriacetate
tumor promotion
oxidative stress
antioxidant
Guilandina bonduc Linn. (Fabaceae), a prickly shrub that grows abundantly in tropical and subtropical regions of Southeast Asia, possesses an extensive medicinal value. The present communication reports ethanolic extract of G. bonduc fruit pods to possess potent antioxidant activity and significantly protect against Ferric Nitrilotriacetate (Fe-NTA)−induced renal oxi-dative stress, nephrotoxicity, and tumor promotion response. Intraperitoneal administration of Fe-NTA (9 mg Fe/Kg body weight) to Swiss albino mice depleted renal antioxidant armory (glutathione content and activities of antioxidant enzymes), and caused oxidative stress, induced inflammation (inducing the expression of inducible nitric oxide synthase and cyclooxygenase-2 and increasing the secretion of proinflammatory cytokines). It also induced hyperproliferation in kidney-enhancing ornithine decarboxylase (ODC) activity and [3H]-thymidine incorporation into DNA. Histopathological changes and levels of serum creatinine (SCr) and blood urea nitrogen (BUN) suggested Fe-NTA to afflict substantial damage to the kidney. Prophylactic treatment of mice with G. bonduc extract (100−200 mg /Kg body weight) for seven days not only restored renal antioxidant armory close to normal, but also significantly precluded Fe-NTA associated inflammation and hyperproliferation. Furthermore, pretreatment with extract markedly prevented renal oxidative damage and histopathological alterations, and restored the levels of renal damage markers close to control values. The data obtained in the present study indicate G. bonduc fruit pods to possess a potent antioxidant activity and avert chemically inflicted renal oxidative damage and tumor promotion response.