Joseph Pulliam

Association of HFE mutations with neurodegeneration and oxidative stress in Alzheimer's disease and correlation with APOE

Oxidative stress enhanced by transition metals such as iron forms an attractive hypothesis for neurodegeneration in Alzheimers Disease (AD). Iron is increased in the brain in AD, but whether this is a primary abnormality or the result of secondary accumulation is unclear. Among several genetic loci associated with AD, the locus at chromosome 6p21 contains the hereditary hemochromatosis gene HFE. To determine whether a genetic predisposition to iron accumulation is associated with AD, we evaluated three hemochromatosis‐associated HFE mutations and APOE in cognitively and histopathologically evaluated subjects with AD, mild cognitive impairment (MCI), non‐demented controls with AD‐like pathologic changes defined by Braak stage ≥ 3 (high pathology controls (HPC)), and non‐demented controls without significant histologic changes (low‐pathology controls (LPC)). In a subset, we examined ventricular (CSF) fluid F2‐isoprostane (F2‐IsoP) levels, a marker of lipid peroxidation. Seventeen subjects demonstrated homozygous or compound heterozygous HFE mutations, 13 (9.4%) in the AD/MCI group (P = 0.019 vs. LPC) and four (20%) in the HPC group (P = 0.006, P < 0.05 with Bonferroni correction vs. LPC). In contrast, the APOE4 allele frequency was increased only in the AD/MCI patients (P < 10−3 vs. HPC, P < 10−6 vs. LPC). F2‐IsoP levels were increased in AD subjects with any HFE mutation versus wild type HFE (P = 0.027). Although confirmation is required, these findings suggest that HFE mutations are associated with increased oxidative stress and Braak stage, and that HFE and APOE genotypes are different between AD patients, high pathology and low pathology controls.

Uterine sarcomas express KIT protein but lack mutation(s) in exon 11 or 17 of c-KIT.

OBJECTIVE Several tumors express the protein product of the protooncogene c-KIT. Some of these respond to imatinib mesylate, a tyrosine kinase inhibitor. The tumors that respond frequently have mutation(s) in exon 11 of c-KIT that encodes for the regulatory juxtamembrane helix. Some tumors that express KIT protein have mutation(s) in exon 17 of c-KIT; however, these do not respond to imatinib mesylate. This investigation was performed to determine the expression of KIT protein and mutational status of exons 11 and 17 of c-KIT in uterine sarcomas. METHODS Twenty-five uterine sarcomas treated from 1990 to 2002 were evaluated. These included 14 malignant mullerian mixed tumors (MMMT), 7 leiomyosarcomas (LMS), 2 endometrial stromal sarcomas (ESS), and 2 high-grade heterologous sarcomas (HGHS). Formalin-fixed, paraffin-embedded tissue sections were immunostained with anti-KIT antibody (Santa Cruz Biotechnology, Santa Cruz, CA) with a semiquantitative assessment. Normal myometrium when present in the section was used as an internal negative control. Areas of tumor were microdissected followed by DNA extraction, polymerase chain reaction (PCR) amplification of exons 11 and 17, single-strand conformational polymorphism (SSCP), and DNA sequencing to detect the presence of mutation(s). RESULTS All 25 tumors expressed KIT protein at varying levels as assessed by immunohistochemistry. The staining was diffuse and of moderate to strong intensity in 22 tumors. In three tumors (one of each type except MMMT) the staining intensity was weak. In MMMT the epithelial and sarcomatous foci stained similarly. No mutation(s) in exons 11 or 17 of c-KIT were identified in 24/25 tumors. One LMS had deletion of both exons 11 and 17. CONCLUSIONS Although uterine sarcomas express KIT protein, they lack KIT-activating mutation(s) in exon 11 or 17 of c-KIT. Therefore, these tumors are unlikely to respond to imatinib mesylate.

Arsenic and chromium in drinking water promote tumorigenesis in a mouse colitis-associated colorectal cancer model and the potential mechanism is ROS-mediated Wnt/β-catenin signaling pathway

Exposure to carcinogenic metals, such as trivalent arsenic [As(III)] and hexavalent chromium [Cr(VI)], through drinking water is a major global public health problem and is associated with various cancers. However, the mechanism of their carcinogenicity remains unclear. In this study, we used azoxymethane/dextran sodium sulfate (AOM/DSS)-induced mouse colitis-associated colorectal cancer model to investigate their tumorigenesis. Our results demonstrate that exposure to As(III) or Cr(VI), alone or in combination, together with AOM/DSS pretreatment has a promotion effect, increasing the colorectal tumor incidence, multiplicity, size, and grade, as well as cell inflammatory response. Two-dimensional differential gel electrophoresis coupled with mass spectrometry revealed that As(III) or Cr(VI) treatment alone significantly changed the density of proteins. The expression of β-catenin and phospho-GSK was increased by treatment of carcinogenic metals alone. Concomitantly, the expression of NADPH oxidase1 (NOX1) and the level of 8-OHdG were also increased by treatment of carcinogenic metals alone. Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, were decreased. Similarly, in an in vitro system, exposure of CRL-1807 to carcinogenic metals increased reactive oxygen species (ROS) generation, the expression of β-catenin, phospho-GSK, and NOX1. Inhibition of ROS generation by addition of SOD or catalase inhibited β-catenin expression and activity. Our study provides a new animal model to study the carcinogenicity of As(III) and Cr(VI) and suggests that As(III) and Cr(VI) promote colorectal cancer tumorigenesis, at least partly, through ROS-mediated Wnt/β-catenin signaling pathway.

Nicotine Induces the Up-regulation of the α7-Nicotinic Receptor (α7-nAChR) in Human Squamous Cell Lung Cancer Cells via the Sp1/GATA Protein Pathway

Background: Nicotine promotes the proliferation of human squamous cell lung cancer (SCC-L) via the α7-nicotinic receptor (nAChR). Results: Nicotine increases α7-nAChR expression via transcriptional mechanisms involving Sp1 and GATA proteins. Conclusion: Nicotine-induced up-regulation of α7-nAChR accelerates the growth of human SCC-L. Significance: SCC-L patients exposed to nicotine display fast growing lung tumors and worse clinical outcomes. Nicotine, the addictive component of cigarettes, promotes lung cancer proliferation via the α7-nicotinic acetylcholine receptor (α7-nAChR) subtype. The present manuscript explores the effect of nicotine exposure on α7-nAChR levels in squamous cell carcinoma of the lung (SCC-L) in vitro and in vivo. Nicotine (at concentrations present in the plasma of average smokers) increased α7-nAChR levels in human SCC-L cell lines. Nicotine-induced up-regulation of α7-nAChR was confirmed in vivo by chicken chorioallantoic membrane models. We also observed that the levels of α7-nAChR in human SCC-L tumors (isolated from patients who are active smokers) correlated with their smoking history. Nicotine increased the levels of α7-nAChR mRNA and α7-nAChR transcription in human SCC-L cell lines and SCC-L tumors. Nicotine-induced up-regulation of α7-nAChR required GATA4 and GATA6. ChIP assays showed that nicotine induced the binding of GATA4 or GATA6 to Sp1 on the α7-nAChR promoter, thereby inducing its transcription and increasing its levels in human SCC-L. Our data are clinically relevant because SCC-L patients smoked for decades before being diagnosed with cancer. It may be envisaged that continuous exposure to nicotine (in such SCC-L patients) causes up-regulation of α7-nAChRs, which facilitates tumor growth and progression. Our results will also be relevant to many SCC-L patients exposed to nicotine via second-hand smoke, electronic cigarettes, and patches or gums to quit smoking.

Genetic association of low density lipoprotein receptor and Alzheimer's disease

The low density lipoprotein receptor (LDLR) is an attractive candidate gene for genetic association with Alzheimers disease (AD) because: (i) the LDLR is an apolipoprotein E (apoE) receptor, alleles of which have been associated with AD, (ii) LDLR resides at chromosome 19p13.3 within a region linked to AD, and (iii) LDLR modulates the homeostasis of cholesterol, which itself appears associated with AD. Therefore, we evaluated whether LDLR haplotypes alter the odds of AD by performing an association study examining three LDLR single nucleotide polymorphisms (SNPs) in 118 AD patients and 133 non-AD subjects. LDLR genotypes were obtained by TaqMan allelic discrimination assays. Although individual LDLR SNPs were not associated with AD, analyses of unambiguous haplotypes suggested the hypothesis that the 211 LDLR haplotype was associated with reduced odds of AD. We then evaluated this hypothesis in a second study cohort, i.e., the Religious Orders Study. These results supported the hypothesis that the 211 LDLR haplotype is associated with reduced odds of AD. Moreover, these data suggested further associations between LDLR variants and AD. Thus, LDLR variants appear significantly associated with AD and merit additional study.

SPONTANEOUS RESOLUTION OF A SINGLE LESION OF MYELOID LEUKEMIA CUTIS IN AN INFANT: Case Report and Discussion

Though infantile leukemia has a historically poor prognosis, there may be a subset of patients with cutaneous disease whose disease will resolve without therapy. The authors report a case of infantile leukemia cutis who presented with a single subcutaneous chloroma that spontaneously resolved over the course of several weeks and who remains without evidence of disease nearly two years later. After reviewing the literature of congenital leukemia cutis, the authors conclude that withholding chemotherapy in infants with cutaneous myeloid leukemia in the absence of known negative prognostic factors (MLL or BCR-ABL translocations) or progressive disease is clinically indicated.

Peripheral T-Cell Lymphoma with Aberrant Expression of CD19, CD20, and CD79a: Case Report and Literature Review.

A case of lymphoma of T-cell derivation with aberrant expression of three B-cell lineage markers (CD19, CD20, and CD79a), which was diagnosed on a left axillary excision, is described. Immunohistochemical studies and flow cytometry analysis demonstrated neoplastic cells expressing CD3, CD19, CD20, and CD79a with absence of CD4, CD8, CD10, CD30, CD34, CD56, CD68, TDT, MPO, PAX-5, and surface immunoglobulin. Gene rearrangement studies performed on paraffin blocks demonstrated monoclonal T-cell receptor gamma chain rearrangement with no evidence of clonal heavy chain rearrangement. The neoplastic cells were negative for Epstein-Barr virus (EBV) or Human Herpes Virus 8 (HHV-8). At the time of diagnosis, the PET scan demonstrated hypermetabolic neoplastic cells involving the left axilla, bilateral internal jugular areas, mediastinum, right hilum, bilateral lungs, and spleen. However, bone marrow biopsy performed for hemolytic anemia revealed normocellular bone marrow with trilineage maturation. The patient had no evidence of immunodeficiency or infection with EBV or HHV-8. This is the first reported case of a mature T-cell lymphoma with aberrant expression of three B-cell lineage markers. The current report also highlights the need for molecular gene rearrangement studies to determine the precise lineage of ambiguous neoplastic clones.

A cell line selected for resistance to ionizing radiation exhibits cross resistance to other genotoxic agents and a mutator phenotype for loss of heterozygosity events

An ionizing radiation resistant derivative was obtained from the mouse P19H22 (aprt hemizygote) embryonal carcinoma cell line by repeated exposure to137Cs gamma radiation. Ionizing radiation resistance in the 6Gy-R cell line was not correlated with a failure to undergo cell cycle arrest or a loss of the p53 response after exposure to137Cs gamma radiation. Moreover, the cells did not display increased resistance to bleomycin, a double strand break inducing agent. However, the cells did display increased resistance to ultraviolet radiation, ethyl methanesulfonate, and 95% oxygen. A mutational analysis demonstrated a>700 fold-fold increase in the frequency ofaprt mutants for the 6Gy-R cells, but no change in the frequency ofhprt ordhfr mutants. A molecular analysis suggested that theaprt mutations in the 6Gy-R cells arose by recombinational events. A possible association between radiation resistance, DNA repair, and a mutator phenotype for large-scale mutational events is discussed.

Autoimmune Hemolytic Anemia in a 2-Year-Old Child With Pneumococcal Pneumonia

A 2-year-old boy presented to his primary care provider with a 2-week history of daily fevers (to 39°C), malaise, increasing pallor, decreased appetite, and a 3-lb weight loss. Review of systems was otherwise negative (no history of cough, vomiting, diarrhea, or rash). The child was fully immunized and had no relevant past medical problems. His first cousin had been treated for autoimmune hemolytic anemia (AIHA) 3 years prior. An officebased complete blood count found leukocytosis (39 000 cells/mL) and profound anemia (hemoglobin 6.3 g/dL); platelets were read as “clumped” and therefore were uninterpretable. A presumptive diagnosis of acute leukemia was entertained, and the child was transferred to our institution for further management. The child was well developed and in no distress. Height and weight plotted at the 95th and 25th percentiles, respectively. Temperature was 37.2°C, heart rate was 176 beats per minute, and regular and blood pressure, initially obtained at 148/99 mm Hg in the first minutes of hospitalization, later normalized. Respirations were regular and unlabored at 38 per minute, and resting oxygen saturation was 93% as measured by pulse oximetry. HEENT examination was unremarkable and there was no evidence of exanthem, purpurae, or abnormal lymphadenopathy. There was tachycardia without murmur. Auscultation of the chest revealed the presence of breath sounds on both sides and no rales or rhonchi. Only when auscultory findings were directly compared from one side to the other were decreased breath sounds on the left side appreciated. The abdomen was soft and there was no hepatosplenomegaly. Neurologic examination, including gait, was normal for age. A complete blood count confirmed a leukocytosis (40 100 cells/mL), but the differential was inconsistent with acute leukemia; there were only 1% blasts. Rather, neutrophils and bands together accounted for 80% of total white cells. In addition, there were 14% lymphocytes, 3% plasma cells, and 2% nucleated red blood cells. Hemoglobin was 5.1 g/dL, hematocrit 14.8%, and platelet count 657 000/mL. The mean corpuscular volume was 96 fL, and the reticulocyte count was 6.4%. The peripheral blood smear revealed thrombocytosis, a leftshifted leukocytosis including scattered early myeloid forms and blasts, moderate erythrocyte polychromasia, anisocytosis, and slight macrocytosis (Figure 1). Serum electrolytes and uric acid were essentially normal, but lactate dehydrogenase was elevated at 423 U/L. Flow cytometric analysis of the peripheral blood, performed to investigate the possibility of a clonal process such as leukemia, revealed a polyclonal, reactive leukocytosis of normal blood cells. We interpreted the elevated reticulocyte count to be consistent with a destructive red cell process and investigated various causes of hemolysis. A direct Coomb’s test was strongly positive for both IgG and deposition of C3 complement. We next considered the child’s tachypnea, hypoxia, and differential breath sounds. The chest radiograph was markedly abnormal, showing near-complete opacification of the left hemithorax (Figure 2A). To differentiate a primary pneumonic process from a pleural accumulation, a chest computed tomography scan was performed (Figure 2B). The computed tomography scan found evidence of a large left pleural effusion with almost complete collapse of the underlying lung and a slight rightward mediastinal shift. A blood culture rapidly grew gram positive cocci in pairs and chains, later identified as Streptococcus pneumoniae. The child was thus diagnosed with AIHA associated with pneumococcal pneumonia, empyema, and bacteremia. He was transfused with red blood cells and treated with a combination of ceftriaxone and vancomycin. The next day, video-assisted thoracoscopic surgery (VATS) with decortication was performed to remove infectious material surrounding the left lung. A moderate amount of thick fluid within the left chest

Abstract 1032: The acetylcholine signaling pathway: A novel molecular target for lung cancers

Cigarette smoking is a major risk factor for all types of lung cancers. Nicotine, the addictive component of cigarettes, accelerates the growth and angiogenesis of human lung cancers. The biological activity of nicotine is mediated by nicotinic acetylcholine receptors (nAChRs). The endogenous ligand of nAChRs is acetylcholine (ACh). We show that both human SCLCs and NSCLCs contain all proteins of the acetylcholine signaling pathway, namely nAChRs, choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), choline transporter (ChT1) and acetylcholinesterase (AChE). ACh functions as an autocrine growth factor for human lung cancer cells. Lung adenocarcinoma (LAC), squamous cell carcinoma (SCC-L) and invasive mucinous adenocarcinoma (IMA) express a diverse array of nAChRs. In addition, normal human lung cells also express nAChRs and other ACh signaling proteins. Nicotine amplifies the ACh signaling loop in human lung cancer cells. It increases the levels of alpha7-nAChR subunit in human SCC-Ls. The alpha7-nAChR is responsible for the proliferative and pro-angiogenic activity of nicotine in lung cancer. The level of alpha7-nAChR was analyzed in human SCC-L samples isolated from patients. It was found that the level of alpha7-nAChR in SCC-L patients (who are heavy smokers) was much higher than that of moderate smoker suffering from SCC-Ls. Nicotine was also found to elevate the levels of ChAT and VAChT in human lung cancers. The acetylcholine signaling pathway may be a useful molecular target for the diagnosis and therapy of human lung cancers in smokers. Our results are also relevant to lung cancer patients who are exposed to nicotine via secondhand smoke, nicotine patches, gums or electronic cigarettes. Citation Format: Kathleen C. Brown, Jamie K. Lau, Haley E. Perry, Brent A. Thornhill, Cathryn D. Stevenson, William D. Rollyson, Cody A. Stover, Dennie V. Jones, Joseph F. Pulliam, Piyali Dasgupta. The acetylcholine signaling pathway: A novel molecular target for lung cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1032. doi:10.1158/1538-7445.AM2015-1032