Using VBIM technique to identify novel carboplatin resistance gene in ovarian cancer

poster abstractOvarian cancer (OC) is the most lethal gynecology cancer in the world. Although carboplatin is one of the major drugs used to treat OC, resistance to carboplatin remains a major barrier to successful treatment. To date, the mechanisms of carboplatin resistance are still poorly understood. The purpose of this study is to use the novel validation-based insertional mutagenesis (VBIM) technique to identify carboplatin resistance gene in A2780 OC cells. A2780 cells were infected with VBIM virus to cause the overexpression of drug resistance genes, then were further selected under carboplatin treatment. Targeted gene was then identified by using VBIM specific primers. In a preliminary screen, we identified the novel carboplatin resistance gene 1 (NCR1). Overexpression of NCR1 increased carboplatin resistance in A2780 OC cells, while knocking it down with shRNA had the opposite effect. In an attempt to investigate the molecular mechanism that underlying NCR1-mediated carboplatin resistance, we found that NCR1 is a potential NF- B activator. In summary, we conclude that using a novel VBIM technique, we discovered a previously unknown carboplatin resistance gene NCR1, which may mediate drug resistance via NF-B signaling pathway. This study is of extreme importance by identifying a potential novel therapeutic target NCR1 in carboplatin resistance. Development of small chemical inhibitors targeting NCR1 could ultimately lead to novel therapeutic approach for ovarian cancer treatment

STAINING OF OVCA1 ANTIBODY IN HUMAN MALIGNANCIES

poster abstractImmunohistochemistry biomarkers are currently being developed to tar-get specific proteins found in cancer cells. The biomarker and putative tumor suppressor, OvCa1, has a function that is not well characterized. Due to lack of reagents, we developed monoclonal antibodies of OvCa1 to examine mul-tiple human malignancies. Primary cancers with different histologic grades as well as with metastatic lesions were examined with the monoclonal anti-bodies. Ovarian cancer tissue samples from the IU Simon Cancer Center Tis-sue Bank were used for this study. The samples were fixed in neutral buff-ered formalin and processed into a paraffin block. The slides were microtomed, and immunohistochemistry (IHC) with the OvCa1 antibody was performed. Thirty-one low, medium, and high grade tumors as well as meta-static ovarian carcinomas were evaluated. All cases revealed a range of staining intensity with OvCa1. The results indicated that OvCa1 had the highest immunostaining in the high grade, Stage 3 to 4 ovarian carcinomas. Medium grade tumors had less OvCa1 expression, while the metastatic tu-mors had less staining than any of the other three grades. Immunostaining was observed primarily in the cytoplasm and nucleus of the tumor cells. In addition, we evaluated approximately 20 tumors from various different or-gans. These included prostate, breast, spleen, lung, colon, stomach, and kidney tumors, which were positive for immunostaining with the OvCa1 anti-body. In summary, the results indicate that all histologic grades express the biomarker, OvCa1, and the staining intensity was highest in the high grade, Stage 3 and 4 tumors. Our preliminary studies demonstrate a further need to delineate OvCa1 as a potential biomarker, which could be used for early detection and diagnosis of ovarian cancer

DNA/RNA Degradation Rate in Long Term Fixed Museum Specimens

In today’s research driven society, it has become commonplace for institutions to rely upon DNA and RNA extraction techniques to help obtain genomic data from old specimens. Generally, specimens were commonly preserved for future gross examination and/or teaching. Using histological examination of specimens from museum jars from the Pathology Department at the Indiana University School of Medicine, the sequential and chronological degradation of DNA and RNA has been studied. We examined gross specimens from nine decades from 1920 until 2000. We evaluated histologic preservation of kidney, liver, heart, lung, spleen, uterus, and brain for nuclear structure in these samples. Nuclear preservation was based on amount of nuclei per 20x microscopic field and the crispness of the nuclear membrane and internal features. The nuclei in high lipid tissues such as the brain were found to degrade at a quicker rate than dense tissues such as the heart and uterus. Our study has shown specimens preserved beyond fifty years were likely to have little to no nuclei left, thus indicating that there was little to no DNA and RNA remaining. This technique of histologic evaluation first is an important finding and a general guideline which may save research institutions from the expensive process of DNA and RNA extraction

Quantitative Immunohistochemistry Evaluating APE1 Expression in a Mouse Pancreatic Adenocarcinoma Model

poster abstractHigh levels of APE1 expression have been reported in numerous malignant tumors (brain, ovarian, pancreatic, and prostate). APE1 is an emerging target for a variety of novel anticancer drugs. Human apurinic endonuclease/redox factor 1 (APE1/Ref-1) mediates the repair of baseless sites in DNA caused by alkylation and oxidative DNA damage. Compound E3330 targets the redox signaling function of APE1. A pancreatic cancer mouse model was used to evaluate the drug effects of E3330 and Gemcitabine. The following doses were used across eight mice groups: E3330 at 12.5mg/kg, 25mg/kg, and 50mg/kg), Gemcitabine (35mg/kg), a combination of E3330 and Gemcitabine at 12.5mg/kg, 25mg/kg, and 50mg/kg), and an untreated vehicle control group. Mice were dosed i.p. 3 times weekly (MWF) and the study was completed at day 39. At termination, tumors were harvested and cross-sections were processed into a Paraffin block. Tissue sections were prepared and stained for H&E and an immunostain for CD31 (angiogenesis marker). Slides were imaged via Aperio whole slide digital system. The immunostains were evaluated to predict the effectiveness of treatment for pancreatic adenocarcinoma. IHC slides were quantitated using an Aperio positive pixel algorithm to determine the percent of angiogenesis in the various drug treatment groups. A biologically significant correlation was seen amongst the low and middle dose E3330 drug groups in comparison to the vehicle control. The (12.5 & 25) E3330 groups had an anti-angiogenic effect (shown by decreased CD31 positivity). These were slightly lower than the combinations of E3330 and Gemcitabine at the same dose treatment groups (possibly due to blunting of E3330). These results support previous studies demonstrating the antiangiogenic activity of E3330

QUANTITATIVE IMMUNOHISTOCHEMISTRY USING THE APERIO WHOLE SLIDE IMAGING SYSTEM EVALUATING ANGIOGENESIS AND HYPOXIA MARKERS IN PANCREATIC CARCINOMA MOUSE MODEL TREATED WITH VEHICLE CONTROL, E3330, AND A STAT 3 INHIBITOR

poster abstractInvestigation of the signaling pathways and molecular mechanisms that are major contributors to pancreatic tumor progression and its resistance to traditional therapies is lacking. Human apurinic endonuclease/redox factor 1 (APE/Ref-1) mediates repair of radiation-induced DNA lesions and regulates transcription via redox-based activation. Transcriptional factors HIF-1α, NFκB, and AP-1 are regulated by Ref-1 and are implicated in pancreatic tu-mor growth and the response to hypoxia. CD31 and CA IX (carbonic anhy-drase) were biomarkers used in an in vivo study to evaluate the effective-ness of E3330, an APE 1 inhibitor, in a pancreatic mouse model. Immunostained slides were scanned using the Aperio automated whole slide scanning system (Scanscope CS) and were viewed using ImageScopeTM. Single fields of view from each WSDI measuring ∼10,000,000 μm2 and rep-resenting the whole area of the tumor were selected for analysis using the Aperio positive pixel algorithm. The preclinical xenograft model evaluated human pancreatic carcinoma cell lines grown in NOD/SCID mice treated with the E3330 compound, a STAT 3 inhibitor, and an untreated vehicle control group. Immunohisto-chemistry (IHC) was used to predict effectiveness of treatment for pancreat-ic carcinoma based on CD31 and CA IX biomarker expression. IHC slides were quantified using both a traditional pathology hand count and the Aperio Imaging Analysis System. The positive pixel algorithm data closely mirrored the hand count for two biomarkers (CD31 and CA IX). In the E3330 treated group, the data showed CD31 (angiogenesis) was significantly knocked down with increased CA IX expression compared to the vehicle control. Hypoxia of the tumor cells was up in both treated groups. In summary, the Aperio im-aging analysis system matched the hand count pathology data. The drug ef-fects with E3330 exhibited both anti-angiogenesis and tumor hypoxia activi-ty in the tumors. This project was supported by the Center for Research and Learning’s Diversity Scholars Re-search Program

Experimental Cobalt Cardiomyopathy in the Dog.

A technique for production of experimental cobalt cardiomyopathy in the dog is described. A chronic indwelling venous catheter is inserted retrograde into the posterior vena cava via the femoral vein. Colbalt chloride in a solution of sterile saline was infused with a variable speed infusion pump twice weekly over a 24 hour period. The dogs were divided into four groups; Group A (5 dogs) was the normal controls, Group B (5 dogs) was maintained on a protein and thiamine deficient diet, Group C (5 dogs) was on a normal diet with cobalt infusions, and Group D (6 dogs) was on a protein and thiamine deficient diet with cobalt infusions. The primary clinical findings observed in dogs in Group C and D were dyspnea, exercise intolerance, mild systolic murmors with a prominent gallop rhythm, abnormal electrocardiograms illustrating sinus tachycardia and lowered QRS complexes in limb leads, microcardia on thoracic radiographs, elevated left ventricular end diastolic pressure and lowered systolic pressures and angiographic evidence of a small end systolic volume and mildly enlarged left diastolic volume. The principle gross lesions found in dogs in both Group C and D were marked right ventricular dilatation, mild hypertrophy of the left ventricle, uniform paleness of the myocardium and dilatation of both atria. Histologic lesions included vacuolation of myofibers, loss of myofibers with hypertrophy of remaining fibers and randomly scattered areas of coagulation necrosis. Histochemical stains for succinic dehydrogenase and reduced diphosphopyridine nucleotide diaphorase were applied to frozen sections of heart muscle. There was no differences in reduced DPN-diaphorase activity exhibited between groups. Staining for succinic dehydrogenase revealed marked mitochondrial dysfunction and loss of enzyme activity in dogs in the two cobalt treated groups. Ultrastructurally, the treated groups had mitochondria that were extremely swollen with loss of cristae. Electron dense particles were seen in some damaged mitochondria. The sarcoplasmic reticulum and the T system contained numerous dilated vesicles in cells with mitochondrial and myofibrillar damage. Loss of cross striations and Z-band thickening were present in affected fibers. Lipid droplets of varying sizes were observed, some containing dense osmiophilic particles at the periphery

Natural Cardiac Deaths in Central Indiana

poster abstractCardiovascular disease is still the major cause of death in the USA for the past 50 to 60 years. Within cardiovascular disease there are many subtypes that cause death including hypertensive heart disease, atherosclerosis, coronary heart disease (CAD), myocardial infarction (MI), dilated cardiomyopathy, hypertrophic cardiomyopathy, cardiomegaly, and misc.). In this review study we examined the Marion County, Indianapolis, Indiana Morgue, Indiana database for the total of deaths that occurred between 2004 through 2012 and evaluated the number of cardiovascular deaths including the various CV subtypes mentioned above. There were approximately 13,000 deaths examined that were sent to the Marion County Morgue during that time frame in Central Indiana. Approximately 2950 deaths were due to CV disease (22.6%). Total ischemia (coronary artery disease) was 1939 made up the majority of the CV related deaths. This was followed by hypertensive heart disease (571) and congestive heart failure (189). Hypertrophic cardiomyopathy (89), cardiomegaly (16), and cardiac tamponade (11) made up the rest. Cardiac arrhythmias and myocarditis made up the remaining CV causes of death (131). In a previous study done at the Marion County Morgue from 1987 to 2003 focused on hypertensive CV disease and hypertrophic cardiomyopathy found 165 deaths and 134 deaths respectively. Compared to the previous local study in the same population the incidence of hypertensive heart disease was moderately increased. There was not much difference between hypertrophic cardiomyopathy between the two studies. Both studies are fairly consistent when compared to national statistics on cardiovascular death in the country

Increased Ischemic Cardiac Deaths in Central Indiana in Summer Months Compared to Winter Months

poster abstractCardiovascular diseases have been the leading cause of death in the United States for several decades. Despite sustained declines in the mortality rates from these diseases, the magnitude of the disease is still staggering. One large recent study, using data on hundreds of heart attacks documented in the National Registry of Myocardial Infarction, found that 53 percent more cases in winter than in summer. The primary culprit, many believe, is temperature. Cold weather narrows coronary arteries and raises blood pressure, stressing the heart. Physical strain and ruptured plaques caused by shoveling snow are also commonly cited. But in a recent study, two researchers, found that the risk increases even in warm climates. Analyzing death certificates in seven regions with different climates, Los Angeles, Texas, Arizona, Pennsylvania, Massachusetts and others found that cardiovascular deaths rose up to 36 percent between summer and winter, regardless of climate and temperatures In this study we evaluated the incidence of ischemic cardiomyopathy in the Central Indiana area in the winter months compared to the summer months for the years 1998 to 2002. Approximately 5325 deaths were seen in the Marion County Morgue in central Indiana in this time period. There were 609 ischemic cardiac deaths seen in the summer (March 15th through October 15th) compared to 434 ischemic cardiac deaths seen in the winter (October 15th through March 15th). The deaths by years in the summer were 129, 131, 92, 127, and 130 and in the winter were 95, 96, 90, 96, and 57 respectively. In conclusion, this study was consistent with the outcome as the previous study done in multiple northern and southern cities in the country

XPC protects against smoking- and carcinogen-induced lung adenocarcinoma

Cigarette smoke (CS) contains hundreds of carcinogens and is a potent inducer of oxidative and bulky DNA damage, which when insufficiently repaired leads to activation of DNA damage response and possibly mutations. The DNA repair protein xeroderma pigmentosum group C (XPC) is primed to play an important role in CS-induced DNA damage because of its function in initiating repair of both bulky oxidative DNA damage. We hypothesized that loss of XPC function will increase susceptibility to developing CS- and carcinogen-induced lung cancer through impaired repair of oxidative DNA damage. Mice deficient in XPC (XPC-/-) exposed to chronic CS developed lung tumors whereas their wild-type littermates (XPC+/+) did not. XPC-/- mice treated with the CS-carcinogen urethane developed lung adenocarcinomas representing progressive stages of tumor development, with lung tumor number increased 17-fold compared with XPC+/+ mice. Mice heterozygous for XPC (XPC+/-) demonstrated a gene-dose effect, developing an intermediate number of lung tumors with urethane treatment. Treatment of XPC-/- mice with the carcinogen 3-methylcholanthrene followed by the proliferative agent butylated hydroxytoluene resulted in a 2-fold increase in lung adenocarcinoma development. Finally, tumor number decreased 7-fold in the lungs of XPC-/- mice by concurrent treatment with the antioxidant, N-acetylcysteine. Altogether, this supports a mechanism by which decreased XPC expression promotes lung adenocarcinoma development in response to CS-carcinogen exposure, due in part to impaired oxidative DNA damage repair

Using TMAs (Tissue MicroArrays) to Evaluate GLS, GLUL, and CAV 1 Immunostaining in Breast Cancer

poster abstractApproximately 1 out of 8 women in the United States will develop invasive breast cancer over the course of their lifetime. Breast cancer has a greater potential of being cured if diagnosed in the earlier phases. We evaluated three well-recognized biomarkers, GLUL, GLS, and Cav 1 (glutamine synthetase, glutaminase, caveolin-1) in 14 TMA (tissue Microarrays). The tissues were normal breast and various subtypes of breast carcinoma by immunohistochemistry (IHC) to determine expression and localization in cancerous tissues in breast carcinoma cases. Approximately 80 to 90 breast biopsies in each of the 14 breast TMA immunostaining were evaluated with the GLUL, GLS, and Cav 1 antibodies. With GLS, immunostaining was seen in most tumor cells (mainly cytoplasm and nucleus) and the stain was clean with no background except in cases that had lymphocytes in the core along with the tumor cells. With GLUL, immunostaining was seen in most tumor cells (mainly cytoplasm and nucleus) and the stain was clean with no background except in cases that had lymphocytes in the core along with the tumor cells. Cav1 was seen only in the endothelial cells in blood vessel walls and some smooth muscle cells in small arterioles in the stroma and surrounding normal ducts, DCIS, and some invasive carcinoma tumor clusters. This information from the immunostains was obtained after analyzing 14 tissue microarrays which is not only time effective but cost effective when analyzing multiple research cases from cancer patients. The data for the three antibodies are currently being analyzed by the biostatistics core group and correlated with the severity of the breast cancer disease with multiple patient demographics