WIHIIHIIW Will 1 l LIBRARY L Michigan State \ University QJQ-tf' _ 9799 9 I <95 This is to certify that the thesis entitled COMPARATIVE ETIOLOGY OF HUMAN AND CANINE NASAL CARCINOMAS presented by MlNG—YU LIN has been accepted towards fulfillment of the requirements for the MASTER OF degree in COMPARATIVE MEDICINE SCIENCE AND INTEGRATIVE BIOLOGY W/// Major Pr6fessor’s Signature J“ 9 6/ x w; / Date MSU is an Affinnatlw Action/Equal Opportunlty Institution PLACE IN RETURN Box to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 2/05 c:/CIRC/DateDm.lEp.Is COMPARATIVE ETIOLOGY OF HUMAN AND CANINE NASAL CARCINOMAS By Ming-Yu Lin A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Comparative Medicine and Integrative Biology 2005 ABSHUHHT COMPARATIVE ETIOLOGY OF HUMAN AND CANINE NASAL CARCINOMAS By Ming-Yu Lin Canine nasal carcinomas (CNCs) are aggressive neoplasms with poor prognosis. Treatment approaches include surgical excision and radiation therapy. But, mortality remains high and the etiology of this malignancy is poorly understood. On the other hand, many genetic and environmental factors including inactivation of tumorsuppresser gene p16, a susceptibility locus on human chromosome 4p15.1-q12, and Epstain-Barr virus (a human herpesvirus) have been found to play important roles in the etiology of human nasopharyngeal carcinoma (NPC). Based on the knowledge of NPC, this study was undertaken to determine the roles of these factors of NPC in CNCs. Our results ruled out the involvement of any member of the herpesvirus family and the canine chromosomal region orthologous to human Ch 4p15.1-q12 in the etiology of CNCs. However, frequent loss of p16 expression was observed in the presence of low frequency of loss of heterozygosity (LOH). These result point to the involvement of p16 inactivation, most likely through promoter hypermethylation, as a major contributor to tumorigenesis in CNCS. Copyright by NfingJYuIJn 2005 ACKNOWLEDGMENTS I want to thank many people for their kind advice and help then I can accomplish my research and this thesis. My wonderful mentor Dr. Vilma Yuzbasiyan-Gurkan was always very patient and kind in guiding me on my research and studies. I also want to thank her for easing my transition to life at MSU. My committee members Dr. Matti Kiupel, Dr. Chia-Cheng Chang and Dr. Barbara Kitchell gave me excellent advice and helped me a lot in my thesis. The members of the canine genetics lab, Donna Housley, Lee Alexander, and Joshua Webster, were always very nice to me and held me when I encountered some difficulties in the experiments. I also want to thank Dr. John Kruger, Dr. Roger Macs and Dr. Annabel Wise for their generous ofi‘ering positive controls for the herpesvirus primers. I would also like to acknowledge the Companion Animal Fund of the College of Veterinary Medicine for fimding most of my research. Finally I want to thank my family, my mother, my father, my sister, my boyfriend, and my fiiends in Taiwan and in the USA. With their support, I overcame many obstacles and reached my goals. Now I can share my happiness with them. iv TABLE OF CONTENTS LIST OF TABLES .......................................................................................................... vi LIST OF FIGURES ................................................................ I ....................................... vii CHAPTER 1 INTRODUCTION ..................................................................................... 1 CHAPTER 2 MATERIALS AND METHODS ............................................................... 5 Identification of cases of canine nasal carcinomas ....................................................... 5 Isolation of DNA from formalin fixed paraflin embedded tissues ................................. 5 Investigation of viral presence ..................................................................................... 6 Identification of two SNPs around canine chromosomal region that is orthologous to human Ch4p15.1-q12 .................................................................................................. 8 Identification of microsatellite markers around p16 ..................................................... 8 Loss of heterozygosity (LOH) studies .......................................................................... 9 Identification ofcaninep16 exon 1 by 5’ RACE ....................... 10 Immunohistochemical staining for p16 and p53 ......................................................... 15 CHAPTER 3 RESULTS ................................................................................................ l7 Investigation of viral presence ................................................................................... 17 LOH on canine chromosomal region orthologous to human Ch4p15.1-q12 ................ l7 LOH around canine p16 region .................................................................................. 18 Identification of canine p16 exon 1 by S’RACE ........................................................ 18 IHC staining with anti p16 antibody and anti p53 antibodies ...................................... 19 CHAPTER 4 DISCUSSION .......................................................................................... 20 APPENDICES .............................................................................................................. 24 REFERENCE ................................................................................................................ 39 LIST OF TABLES TABLE 1 Signalments of 43 cases studied ..................................................................... 24 TABLE 2 Species of herpesviruses (21) detected by PCR with universal herpes primers ...................................................................................................................... 26 TABLE 3 ....................................................................................................................... 27 (1) Primer sequences for PCR reactions for investigation of viral presence and LOH (2) Cycling conditions for PCR reactions for investigation of viral presence and LOH TABLE 4 ....................................................................................................................... 28 (1) The number of cases and percentage for each tumor type for positive or negative p16 nuclei staining (2) The number of cases and percentage for each tumor type for positive or negative p53 nuclei staining (3) The number of cases and percentage for each tumor type of different p53 and p16 staining level combination LIST OF FIGURES FIGURE 1 Tumor-suppressive pathway of p16 ............................................................. 29 FIGURE 2 The sequences of intron 3 of cCNCGI (X99913) and SCGB (AF427093) with SNPs .................................................................................................................. 30 FIGURE 3 Structure of INK4A/ARF locus .................................................................... 32 FIGURE 4 Detection of presence of herpesvirus ............................................................ 33 (1) Universal herpesvirus primers (2) Primers for BHV-4 FIGURE 5 LOH study on canine chromosomal region orthologous to human Ch 4p15.1-q12 ............................................................................................................... 34 (1) SNP marker cCNCG] (2) SNP marker SCGB FIGURE 6 LOH study on canine p16 with microsatellite markers (TTTC)n and (CCA)n .................................................................................................. f ....................... 35 (l) Microsatellite marker (TTTC)n (2) Microsatellite marker (CCA)n FIGURE 7 a. Immunohistochemical staining of canine nasal carcinoma for p16 ...................... 36 (1) Nasal carcinoma with high percentage of p16 positive in nuclei (2) Nasal carcinoma with low percentage of p16 positive nuclei (3) Nasal carcinoma with no p16 staining in nuclei b. Immunohistochemical staining of canine nasal carcinoma for p53 ..................... 37 (I) Nasal carcinoma with high percentage of p53 staining in nuclei (2) Nasal carcinoma with low percentage of p53 staining in nuclei vii (3) Nasal carcinoma with no p53 staining in nuclei viii CHAPTER 1 INTRODUCTION Nasal tumors account for 1-2% of all neoplasms in dogs.4 The vast majority of these 4’28 and have an overall poor prognosis1 due to their tumors (80-90%) are malignant, locally aggressive, commonly infiltrative behavior, but they rarely metastasize. Nasal tumors of epithelial origin are more prevalent than those of mesenchymal origin. Of these, carcinomas are the most common and comprise 60—70% of all nasal tumors}3o including adenocarcinomas, transitional carcinomas and squamous cell carcinomas. Nasal carcinomas occur most frequently in middle age or older dogs; the median age of onset is 8-10 years but cases have been reported from 1-15 years old.28 There is no sex predilection?“30 although one paper reported an excess risk in males.15 Currently, breed predilection is still not very clear, but several papers suggested that dolichocephalic breeds are more likely to develop nasal carcinomasé’15 This is possibly due to an increased chance of exposure and trapping of carcinogens in long-length noses. Only few papers have discussed the etiology of canine nasal carcinomas. Some have suggested that environmental pollutants might contribute to tumorigenesis.10 Indoor kerosene or coal combustion were demonstrated as risk factors in one case—controlled study.6 No genetic factors were investigated in past studies. Nasopharyngeal carcinoma (NPC) is a human malignancy with a high incidence in southern China and South East Asia. They occur more ofien in men than in women; the ratio is about 2 or 3 to 1. The age distribution is younger than other human cancers, usually occurring at 50-60 years of age.34 Several factors, including genetic, environmental and viral infections, have been demonstrated to contribute to the etiology of NPC. Genetic factors that are associated with an increased risk of NPC in the Chinese population include some MHC profiles, HLA alleles A2, B14 and B46.13 Deletion on chromosome 3p and 9p are the most frequent genetic changes observed in NPC,24 suggesting tumor suppressor genes in these regions. The most probable candidate genes for these regions are RASSFI and p] 6.5126 p16, also known as cyclin-dependent kinase inhibitor 2A, is a tumor suppressor gene located at human chromosome 9p21. In cell cycle, cyclin dependent kinase 4-6 (cdk4-6)/cyclin D complex phosphorylates Rb, which leads to the release of transcriptional factor E2F and promotes the cell from G1 to S phase. p16 protein inhibits the formation of cdk4-6/cyclin D complex and results in G1 arrest (Figure l).23’33 Loss of p16 was first found in several human tumor cell lines derived from non-small cell lung cancer, melanoma, and leukemia.17 Later p16 was shown to be inactivated through deletion, point mutation or/and promoter methylation in a high percentage of human cancers, including pancreatic adenocarcinomas, head and nasal squamous cell carcinomas, melanomas, leukemia, and gliomas.2t1’32 Deletion, point mutation or promoter hypermethylation of p16 are also found in 60-80% of primary tumors in NPC cases.24 Another important tumor suppressor gene p53, which plays a role in DNA repair, cell cycle arrest, and induction of apoptosis of DNA damaged cells, does not mutate as frequently in NPCs as in other human tumors and only some point mutations were reported in NPCs.8’36 However, an increased amount of the p53 protein was observed by immunohistochemistry in one study of NPCs.2 As wild-type p53 turns over rapidly, normal tissues do not show significant amounts of p53 staining by IHC and the presence of p53 staining is generally taken as evidence of a mutation in p53, which stabilizes the protein resulting in increased cell staining.29 In addition, linkage to a region of chromosome 4 (4p15.1-q12) has been demonstrated in certain families from Guangdong province, China, with LCD scores 3.54 and 4.2 for human chromosome 4 markers D4S405 and D483002,ll but a specific susceptibility gene has not yet been identified. I Another important characteristic of NPCs is a strong association with Epstein-Barr virus (EBV) infection.40 This virus belongs to the gammaherpesviridae subfamily and is a human herpesvirus found in association with several human malignancies including Burkitt’s lymphoma and gastric adenocarcinoma.37 Several genes encoded in its genome including Epstein-Barr nuclear antigen 1 (EBNAl), latent membrane protein 1 (LMPl), and EBV-encoded RNA 1 and 2 (EBERs 1 and 2) are consistently transcribed in malignant cells and their products can be detected in NPC cells}4 The oncogenic activity of these products has been demonstratedw’m’42 Several environmental factors are suspected to play a role in the tumorogenesis of NPCs, including the intake of preserved food at an early age, use of salt cured food, which includes the traditional salty diet in South China, occupational exposure to formaldehyde and wood dust, and tobacco smoke and alcohol abuse.43 Based on the knowledge of human NPCs, this study was undertaken to determine if any of the genetic or infectious factors identified in human NPCs are involved in the etiology of canine nasal carcinomas. We selected p16, human chromosome 4p15.1-q12 region, herpesviruses, in particular EBV and p53 protein expression as the targets of our investigation. We hypothesized that: 1. p16 inactivation is a frequent event in canine nasal carcinomas. 1.a. Nasal carcinomas show loss of heterozygosity (LOH) around canine p16 gene. 1.b. 1.c. Nasal carcinomas show hypermethylation in canine p16 promoter region. Nasal carcinomas show reduced p16 expression as assayed by immunohistochemistry (IHC) with an anti-p16 antibody. Losses on canine chromosomal regions orthologous to human Ch 4p15.1-q12 can be found in canine nasal carcinomas. A herpesvirus can be found in tumor cells in canine nasal carcinomas. Canine nasal carcinomas express p53 protein as assayed by IHC with an anti-p53 antibody. CHAPTER 2 MATERIALS AND METHODS Identification of cases of canine nasal carcinomas The surgical pathology reports of all canine nasal carcinomas that had been submitted to the Diagnostic Center for Population and Animal Health (DCPAH), Michigan State University over a 5 year period (1999 to 2003) were retrieved. The formalin fixed, paraffin embedded tissue blocks of these cases were retrieved from DCPAH archives. These blocks were recut and stained with hematoxilin and eosin. Tumor pathology was reevaluated. In total, 43 cases of nasal carcinomas were identified that included the following 3 tumor entities: adenocarcinomas, squamous cell carcinomas and transitional carcinomas. Of these, 30 cases including 14 adenocarcinomas, 10 squamous cell carcinomas and 6 transitional carcinomas had sufficient amounts of normal and tumor tissue and were selected for LOH studies on canine p16 and the canine chromosomal region that is orthologous to human Ch 4p15.1-q12. All 43 tumor sammes were studied for the presence of herpesvirus. The Signalments of these 43 cases are listed in Table 1. The average age of affected dogs was 10 years and ranged from 4 to 16 years. Eighteen cases were male and 10 cases were female. Most of the cases were mixed breed dogs, followed by Labrador retriever. Isolation of DNA from formalin fixed paraffin embedded tissues The slides of each case were reviewed and the normal and tumor tissue where definite diagnosis could be made were marked for the following DNA extraction. DNA was extracted based on a method described by Banerjee et a1., 1995.3 A small section of normal and tumor tissue, each around 2 x 2 x 2 mm, was excised from each block using a scalpel blade and placed into separate 1.5 mL microcentrifuge tube. To each tube, 400 uL of digestion buffer (50 mM Tris pH 8.5, 1 mM EDTA, 0.5% Tween) was added. The sample was heated at 95°C for 10 minutes and was microwaved for 30 seconds twice at full power. The sample was vortexed thoroughly at each stop point during this period. The sample was then allowed to cool down to room temperature and 5 uL of 15 mg/mL proteinase K was added and the sample was incubated at 42°C overnight. The next day, the sample was heated at 95 °C for 10 minutes to inactivate proteinase K and was centrifuged at 12,000 rpm for 10 minutes at room temperature, and a 150 uL of aliquot was transferred to a fresh microcentrifuge tube. Negative controls without tissue samples were placed every three samples. Original concentration, 10, 25, and 50-fold dilution of this preparation were used as templates in PCR reactions. Investigation of viral presence Investigation of the presence of 21 species of herpesviruses by universal herpes primers and PCR A set of degenerate PCR primers that amplify 21 species of herpesviruses (8 htunan and 13 animal viruses)39 (Table 2) was used to detect the presence of herpesvirus in canine nasal carcinoma samples. Twenty five microliters of PCR mixture contained 5.0 uL (1-750 ng) of template DNA, 1 uM of each primer (5’- TGTAACTCGGTGTAYGGNTTYACNGGNGT-3’ and 5’-CACAGAGTCC GTRTCNCCRTADAT-3’), 80 uM (each) of deoxynucleoside triphosphate, 0.5 U of T aq polymerase (Invitrogen, Carlsbad, CA), 2.5 uL of 10x PCR buffer (200 mM Tris HCl (pH 8.4) and 500 mM KCl, Invitrogen, Carlsbad, CA) and 2 mM MgC12_ PCR reaction was performed under mineral oil and cycled 45 times with 30 seconds of denaturation at 94°C, 1 minute of annealing at 46°C and 1 minute of extension at 72°C. Negative controls were performed every 5 samples. Viral isolates were used as positive controls. PCR products were analyzed on a 2% agarose gel stained with ethidium bromide and detected under UV light. Investigation of presence of bovine herpesvirus 4 (BHV-4) by PCR Bovine herpesvirus 4 is another member of the garnmaherpesvirinae subfamily. It is found ubiquitously in healthy cattle and cattle with several clinical signs.14 BHV-4 was also isolated from cats and shown to replicate in a wide variety of animal hosts including dogs.20 The 21 species of herpesviruses that the universal herpes primers detect do not include BHV-4. For detecting the presence of bovine herpesvirus 4 (BHV-4), a heminested PCR20 was performed as described. The first stage PCR was performed in a 25 uL PCR mixture that contained 1-2.5 ug of template DNA, 0.2 uM of primer 1 (5’- CATGACACACTATTTTAAGTACTA-3’) and primer 2 (5’- ATCTTTCT'I‘TCAGTCTCACTGTA-3’), 80 uM (each) of deoxynucleoside triphosphate, 0.5 U of Taq DNA polymerase (Invitrogen, Carlsbad, CA), 2.5 uL of 10x PCR buffer (200 mM Tris HCl (pH 8.4) and 500 mM KCl, Invitrogen, Carlsbad, CA) and 2 mM MgC12 under mineral oil, The reaction was cycled for 35 times with 1 minute of denaturation at 94°C, 2 minutes of annealing at 59°C and 3 minutes of extension at 72°C. One microliter of the product from the first stage PCR was taken as the DNA template in the second stage PCR. The second stage PCR was performed under the same condition as the first stage PCR, but primer 2 and 3 (5’-ATAAATTTGTGAAGACAATGGGTA-3’) were used. The viral DNA positive controls as well as negative water blank were used. PCR products were analyzed on a 2% agarose gel stained with ethidium bromide and detected under UV light. Identification of two SNPs around canine chromosomal region that is orthologous to human Ch 4p15.1-q12 ' All of the linkage markers around human Ch 4p15.1-q12 were obtained from the MyScience webpage of Applied Biosystems website (https://myscience.appliedbiosystems.com/navigation/mysciMain.jsp). Each genomic sequence in the region between 46 Mb and 55 Mb on chromosome 4 was blasted against NCBI databases for canine genome (http://wwwncbi.nlm.nih.gov/genome/seq/CfaBlast.html) and two canine orthologous markers with single nucleotide polymorphisms (SNPs) were identified; a C/T variant at position 266 (in the corresponding GenBank entry) in intron 3 (GenBank accession no. X99913) of the canine rod photorecepter cGNP-gated cation channel alpha-subunit (cCNCGI) (GenBank accession no. X99914), which results in a change of anI digestion site,41 and a G/A variant at position 265 (in the corresponding GenBank entry) of beta sarcoglycan (SCGB) gene (GenBank accession no. AF427093), which results in a change of Hinfl digestion site (Figure 2).5 Identification of microsatellite markers around canine p16 The human p16 sequence (GenBank accession no. AB060808) obtained from NCBI website was blasted against the NCBI databases for canine genome (http://www.ncbi.nlm.nih.gov/genome/seq/CfaBlast.htm1) to identify canine p16 ortholog and its genomic neighborhood. The orthologous region was found on canine chromosome 11. All the repeat sequences around canine p16 on canine chromosome 11: between nucleotide 42581011 and 42617539 were acquired from the website of Ensembl Genome Browser (http://www.ensembl.org/Canis_familiaris/exportview) and three microsatellite polymorphisms: (TTTC)n, (CCA)n, and (CAAAA)n (canine Ch] 1: nucleotide 42599640- 42599717, 42606047-42606122, and 42608679-42608710, respectively) were selected for LOH study. Loss of heterozygosity (LOH) studies DNA of normal and tumor tissues were isolated from the block of each case. Genotyping at the above loci were carried out using the optimum method for detection for each marker identified. For human Ch 4p15.1-q12 orthologous region, a PCR amplification fragment length polymorphism test was carried out for each SNP. For cCNCGI, primers were designed for a 300 bp region that contains the SNP in intron 3. PCR was performed under the same condition as described in BHV-4 section. The sequences of primers and cycling condition were summarized in Table 3. Five microliters of the PCR product was analyzed on a 2% agarose gel stained with ethidium bromide and detected under UV light. Three point five microliters of 50 mM MgC12 and 4 U of anI (New England Biolabs, Ipswich, MA) were added to 20 uL of the PCR product for digestion, which was carried out at 37°C overnight. The digested sample was analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide and the bands detected under UV light. For SCGB gene, primers (5’-AACTGCATACCAATGTGACT and 5’-ATGTTCTTGATGAATTTTGGC-3’) were used to amplify a 148 bp region containing the SNP site according to Brouillette JA et al., 2002.5 PCR was performed in the same condition as for cCNCGI followed by digestion with 4 U of Hinfl (New England Biolabs, Ipswich, MA). PCR reactions for the three microsatellite markers identified around canine p16 were performed under the same condition as described above. The sequences of three sets of primers and the cycling conditions were summarized in Table 3. PCR products were analyzed on a 2% agarose gel stained with ethidium bromide and detected by UV light. For those samples that needed higher resolution, 6% polyacrylamide gel was used to increase separation. Six percent polyacrylamide gel contained 6% acrylamide/bis (19:1) in 1x TBE with 10% ammonium persulfate and TEMED as catalysts. The electrophoresis was performed in a vertical gel electrophoresis device (Bethesda research laboratories) powered by 200 volts for 1 hour. The gel was stained with ethidium bromide and detected under UV light. Identification of canine p16 exon 1 by 5’RACE Human and mouse INK4A/ARF loci give rise to two different transcripts, p16 (CDKN2A) and p14 (p19 in the mouse). These transcripts have the common exon 2 and exon 3 but different in the first exon with p16 having exon 1a and p] 4 having exon 15 (Fig. 3).”’35 Only a portion of the sequence of p16 exon 2 was identified in dog (GenBank accession No. AF234176).18 In order to identify the sequence of canine p16 exon 1 for promoter methylation study, the sequence of human p16 exon 1 was blasted against NCBI databases for canine genome (http://www.ncbi.nlm.nih.gov/genome/seq/CfaBlast.html) but no significant hit was found. Therefore 5’ rapid amplification of cDNA ends (S’RACE) was performed to identify canine p16 exon 1 region. RNA extraction 10 RNA was extracted from dog brain tissue using the TRIzol method (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Four hundred milligram of brain tissue was homogenized in 4 mL of TRIzol reagent using a glass tissue grinder. Because brain tissue has a high fat content, the sample was centrifuged at 12,000xg at 4°C for 10 minutes to separate RNA-containing supernatant from bottom extracellular material and high molecular weight DNA. The clear homogenate phase was transferred to a fresh tube and incubated at room temperature for 5 minutes. Eight hundred microliters of chloroform was added followed by vigorous mixing by hand for 15 seconds. The sample was then incubated at room temperature for 2-3 minutes and centrifuged at 4°C, 12,000xg for 15 minutes. After the centrifugation the mixtures separated into three phases. The RNA containing upper aqueous phase was transferred to a fresh tube, mixed with 2 mL of isopropanol and incubated at room temperature for 10 minutes. The sample was centrifuged at 4°C, 12,000xg for 10 minutes. After centrifugation, the RNA pellet was observed. The supernatant was removed carefully and the RNA pellet was washed with 4 mL of 75% ethanol, air-dried and redissolved in 120 uL DEPC water. The final RNA sample was analyzed to check for integrity of the ribosomal bands on a 2% agarose gel stained with ethidium bromide and detected by UV light. DNase treatment RNA extracted from brain tissue was treated with DNase to remove DNA contamination. The TURBO DNA-free kit (Ambion, Inc. Austin, TX) was used according to the manufacturer’s instructions. Five microliters of 10x DNase buffer, 2 U of DNase and 39 uL of DEPC water were added to 10 ug of RNA to make 50 uL of final volume. The mixture was incubated at 37°C for 30 minutes. Five microliters of DNase ll inactivation reagent was then added and the mixture was incubated at room temperature for two minutes. During the incubation, the mixture was vortexed for 2-3 times. The mixture was then centrifuged at 10,000xg for 1.5 minutes. The DNase inactivation reagent precipitated at the bottom of the tube. The supernatant, which contained RNA, was transferred to a fresh tube. RNA was analyzed on a 2% agarose gel stained with ethidium bromide and detected by UV light. 5’ Rapid amplification of cDNA ends (5’RACE) The 5’RACE system by Invitrogen was used according to manufacturer’s instructions. Two gene specific primers GSPl (5’-ACCAGCGTGTCCAGGAA-3’) and GSP2 (5’-TGGCGGGGTCGGCACAGTT-3’) were designed for this system. Both of these two primers were designed from the published sequence of canine p16 exon 2. GSP2 is located upstream of GSPl to increase the specificity in the final step PCR reaction. First strand cDNA synthesis from RNA Five microliters of DNase-treated RNA and 2.5 pmol of GSPl in a total 15.5 uL volume was heated at 70°C for 10 minutes and was then placed on ice for 1 minute. Then 2.5 uL of 10x PCR buffer, 2.5 mM of MgClz, 0.4 mM of deoxynucleoside triphosphate and 10 mM of DTT were added. The mixture was incubated at 42°C for 1 minute and 1 uL of SuperScriptTM II reverse transcriptase was added. The reaction was carried out at 42°C for 50 minutes followed by heating at 70°C for 15 minutes to stop the reaction. The sample was centrifuged for 10 to 20 seconds and was placed at 37°C. One microliter of RNase was added and the sample was incubated at 37°C for 30 minutes to digest RNA. 12 Purification of cDNA using S.N.A.P. column One hundred and twenty microliters of binding solution (NaI) was added to the sample. The binding solution/cDNA mixture was transferred to a S.N.A.P. column, centrifuged at 13,000xg for 20 seconds, and the flowthrough was removed. Four hundred microliters of cold (4°C) 1x wash buffer was added to the column, centrifuged at 13,000xg for 20 seconds, and the flowthrough was removed. This wash step was repeated three more times. Then the column was washed with 400 uL of cold 70% ethanol for two times. After the wash, the column was centrifuged one more time at 13,000xg for 1 minute. The cartridge insert was transferred into a fresh 1.5 mL microcentrifuge tube. Fifty microliters of sterilized, distilled water, heated to 65°C was added to the cartridge and centrifuged at 13,000xg for 20 seconds to elute the cDNA. TdT tailing of cDNA A poly C tail was added to the 5’ end of the purified cDNA. Ten microliters of purified cDNA, 5 uL of 5x tailing buffer and 20 uM of dCTP were added to a 0.5 uL microcentrifuge tube to make 24 uL of final volume. The mixture was heated at 94°C for 2-3 minutes and was put on ice for 1 minute. One microliter of TdT was added to each reaction and incubated at 37°C for 20 minutes. After the incubation, the reaction was inactivated by heating at 65 °C for 10 minutes. PCR of dC -tailed cDNA A PCR reaction was performed with our GSP2 and the abridged anchor primer (with multi G, provided from the system) to amplify the upstream region of canine p16 exon 2. The 50 uL PCR reaction contained 5 uL of 10x PCR buffer, 1.5 mM of MgClz, 0.2 mM of deoxynucleoside triphosphate, 0.4 uM of each primer, 2.5 U of Taq DNA polymerase 13 and 5 uL of dC-tailed cDNA under mineral oil. The cycling conditions were: denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute and elongation at 72°C for 2 minutes for 35 cycles. The product was analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide and detected by UV light. DNA extraction from agarose Gel The PCR product from the 5’RACE reaction was extracted from agarose gel using QIAEX 11 gel extraction kit (Qiaex Inc. Valencia, CA) based on manufacturer’s instructions. The DNA band was cut from the agarose gel with a clean scalpel and was placed in a 1.5 mL microcentrifuge tube. The gel piece was weighed on a scale. Three volumes of buffer QXl to the gel piece were added to the tube and vortexed for 30 seconds. Ten microliters of Qiaex II was added to the sample. The sample was then incubated at 50°C for 10 minutes and was vortexed every 2 minutes. After the incubation, the sample was centrifuged for 30 seconds and the supernatant was removed. The pellet remained in the tube was resuspended in 500 uL of buffer QXl by vortexing, centrifuged for 30 seconds, and the supernatant was removed. Then the pellet was washed twice with 500 uL of buffer PE. After the final removal of the supernatant, the pellet was air dried for 10-15 minutes and redissolved in 20 uL of sterile, distilled H20. The sample was vortexed, incubated at room temperature for 5 minutes, centrifuged for 30 seconds, and the clear supernatant was transferred to a fresh tube for use. Sequencing Sequencing reactions were performed by using thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit (U SB, Cleveland, OH). For each sample, four reactions were carried out. Each reaction mixture contained 2 uL of dGTP master mix, 0.5 uL of 14 [a-33P]dd GTP, [a-33P]dd ATP, [d-33P]dd TTP or [a-33P]dd CTP, 0.5 uL of reaction buffer, 2.5 uL of DNA, 0.5 pmol of primer and 2 U of thermo sequenaseTM DNA polymerase in 7 uL of final volume under light mineral oil. Sequencing reactions were cycled 40 times at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute. Four microliters of stop solution was added to each tube after the last cycle. Samples were heated at 70°C for 2-3 minutes before loading to the sequencing gel. The sequencing gel contained 6% acrylamide (19:1 acrylamide: bis) and 7 M of urea in 1x TBE with 10% ammonium persulfate and TEMED as catalysts and was run in a vertical sequencing gel electrophoresis apparatus (Bethesda Research Laboratory, Life Technology Inc. Model 82). After the electrophoresis, the sequencing gel was transferred to a chromatography paper (Whatrnan, Florham Park, NJ) and dried by a gel dryer for two hours. Then a Kodak Biomax MR film (Kodak, Rochester, NY) was exposed to the radioactive gel for 3 days and developed. Immunohistochemical staining for p16 and p53 Tissue sections of canine nasal carcinomas were used for immunohistochemical evaluation of the expression of p16 and p53 protein. Deparaffinization, antigen retrieval and immunostaining of formalin-fixed paraffin embedded tissues were performed on automated immunostainers. Immunohistochemical staining for p16 was performed on the Bond maXTM Automated Staining System (Vision BioSystemsTM) using the BondTM Polymer Detection System (Vision BioSystemsTM) and a mouse monoclonal antibody against p16 (clone 6H12, NovoCastraTM) at a dilution of 1:20. Antigen retrieval was achieved using the Bond Epitope Retrieval Solution 2 (Vision BioSystemsTM) for 20 min. The immunoreaction was visualized with 3,3-diaminobenzidine substrate (Vision 15 BioSystemsTM) and sections were counterstained with haematoxylin. Immunohistochemical staining for p53 was performed on the Bench Mark Automated Staining System (V entana Medical Systems, Inc.) using the Enhanced V-Red Detection (Alk. Phos. Red) Detection System (V entana Medical Systems, Inc.) and a rabbit polyclonal antibody against p53 (Signet Laboratories) at a dilution of 1:100. Antigen retrieval was achieved using the Ventana Medical Systems Retrieval Solution CCl (V entana Medical Systems) for 60 min. Sections were counterstained with haematoxylin. Positive immunohistochemical controls included a canine soft tissue sarcoma with strong p53 expression and normal canine nasal turbinates and lymphoid tissue to which the apprOpriate antisera were added. For negative controls the primary antibodies were replaced with homologous non-immune sera. Only nuclear staining was evaluated as positive staining for p16 and p53. Nasal carcinomas were divided into‘ positive or negative staining tissues based on the absence or expression of p16 and p53, respectively. Neoplasms with rare single positive staining cells were considered negative for statistical evaluation. 16 CHAPTER 3 RESULTS Investigation of viral presence Forty-three tumor cases including 23 adenocarcinomas, 7 transitional carcinomas, and 13 squamous cell carcinomas were investigated by using PCR with universal herpes primers and the primers for BHV-4. Positive control for universal herpes primers had a predicted 207 bp band on a 2% agarose gel, but no viral amplicons were amplified from the 43 tumor samples. Positive control for BHV-4 had a predicted 153 bp band on a 2% agarose gel, but no viral amplicons were detected from the 43 samples, either (Figure 4). LOH on canine chromosomal region orthologous to human Ch4p15.1-q12 The polymorphisms of both SNP markers in normal dog population were investigated in 19 DNA samples from normal dogs, including 9 of mixed breed and 9 huskies. For cCNCGI, a 301 bp fragment was amplified after PCR. A T at position 266 (in the corresponding GenBank entry) of the gene gave an extra an1 digestion site. Therefore, samples of 266T homozygous DNA would give three fragments of different sizes (55 bp, 172 bp and 74 bp) afier digestion by restriction enzyme an1. If there was a C at position 266, one anI digestion site would exist to give two fragments of 227 bp and 74 bp sizes. Heterozygous samples with one allele of 266T and one allele of 266C would display four fragments of 227 bp, 172 bp, 74 bp and 55 bp after anI digestion. A 148 bp fragment representing SCGB was amplified from sample DNA by PCR. An A/G variant at position 132 (in the corresponding GenBank entry) gave a Hian digestion site on the reverse primer, resulting in one 128 bp fragment and one 20 bp fragment after Hinfl digestion. Seven of 19 and 6 of 19 normal samples were heterozygous for the 17 cCNCGI and SCGB markers, respectively. This showed that polymorphisms could be observed for these two SNP markers. Thirty cases with pairs of normal and neoplastic tissue were then studied for LOH with both SNP markers. For cCNCGI, 12 of the 30 cases studied were heterozygous in the normal tissue and thus informative for LOH studies. However, no LOH was found among them. Sixteen of the 30 cases studied were informative for SCGB, but no tumor sample showed LOH compared to normal samples. In conclusion, 19 of the 30 cases studied were informative either for SNP marker cCNCGI or for SCGB on canine chromosomal region orthologous to human Ch 4p15.1- q12, but no LOH was found in these 19 informative samples (Figure 5). LOH around Canine p16 region PCR products of 159 bp, 248 bp and 244 bp were amplified for TTTC, CCA, and CAAAA repeats around the canine p16 region, respectively. For (TTTC)n, 8 cases were informative, and 2 tumor samples among them showed LOH (Figure 6.1). For (CCA)n, 15 cases were informative but no LOH was found (Figure 6.2). Only 'few samples showed polymorphisms on (CAAAA)n, so this locus was not studied further. In conclusion, 19 of the 30 cases studied were informative for either (TTTC)n or (CCA)n, and two cases showed LOH for (TTTC)n. Identification of canine p16 exon 1 by 5’RACE The last step of the S’RACE system is a PCR reaction amplifying the dC-tailed cDNA with one gene specific primer and the abridged anchor primer, which has multiple G to pair with the dC-tail of the cDNA. From this last PCR of 5’RACE, some very faint PCR products were detected on the 2% agarose gel stained with ethidium bromide under UV light. The same PCR was performed again but with gradient annealing temperatures from 18 54°C to 62°C, increasing 2°C each reaction, to try to optimize amplification. From this repeat PCR reaction, multiple bands were consistently observed on the 2% agarose gel from the five reactions with different annealing temperatures. A clear band of reasonable size (around 400 bp) was purified from the gel and sequenced. Unfortunately, the result of sequencing was unreadable due to too many noises. The sequence will be identified by cloning in the future; this part of the experiments is ongoing. IHC staining for p16 and p53 The results of IHC staining for p16 and p53 expression in the nucleus were summarized in Table 4. Figure 7 shows the tumor tissue with different levels of p16 and p53 staining. Among the three histopathological types of tumors, squamous cell carcinomas showed most reduction of p16 expression, 72.73% of which had negative p16 staining; followed by adenocarcinomas, 46.15% of which had negative p16 staining. Most of the three types of tumors had negative p53 staining. Positive p53 staining happened most in the cases of adenocarcinomas, which accounted for 38.46% of all adenocarcinoma cases. 19 CHAPTER 4 DISCUSSION Canine nasal carcinomas are locally aggressive and typically have poor prognosis. As described previously, very few studies investigated the tumorigenesis of this malignancy and the etiology remains poorly understood. The current study investigated the etiology of canine nasal carcinomas from genetic and virological aspects. A herpesvirus was first found in association with cancer in the renal adenocarcinomas in leopard frogs in 1934.31 Other herpesviruses were then found to contribute to the tumorigenesis of several animal and human cancers including Burkitt’s lymphoma, Marek’s disease, Kaposi’s sarcoma and NPC. In this study, the universal herpes primers used can amplify 21 types of herpesviruses (8 human and 13 animal viruses) including EBV and canine herpesvirus; PCR for detection of BHV-4 was also performed because BHV-4 is shown to replicate in many animal hosts including dogs. Among 43 tumor cases we investigated, no viral amplicons were amplified from the sammes. According to this result, a significant contribution of herpesvirus to canine nasal carcinomas can be ruled out. Linkage to the human Ch 4p15.1-q12 region was demonstrated in association with NPC in certain families from Guangdong province in China.11 We planned to study this region in young onset cases of canine nasal carcinomas, but unfortunately only two cases, one 4 year old and the other 5year old, can be considered “young onset” in the 30 cases we studied. LOH was found in neither of the two young onset cases, nor in the other 28 cases, which were of middle to advanced age. Although these were not all young onset cases and the contribution of this region to the risk of nasal carcinomas in dogs cannot be 20 completely excluded, LOH at the canine equivalent to human Ch 4p15.1-q12 involvement would seem very rare. Another study in human NPC conducted in another patient population found a susceptibility locus on Ch3p21.31-21.2,44 which is consistent with the demonstration that deletion of Ch3p is a frequent genetic change in NPC.1(”27 But this study did not identify any linkage to Ch 4p15.1-q12 nor 9p21. The divergent results between different studies may be due to the different patient populations studied. Considering canine nasal carcinomas, if more young onset cases can be obtained for study, the relevance of the human 4p15.1-q12 orthologous region and onset of canine nasal carcinomas can be addressed more definitely. We can also investigate the region orthologous to human Ch3p21.31-21.2, which might possibly harbor a susceptibility gene. As an important tumor suppressor gene, inactivation of pl 6 has been found in many human cancers.'633'26 Lack of p16 expression either at the mRNA or protein level was also reported in tumors including melanomas and osteosarcomas in dogsum2 In our LOH study, 2 of 19 informative cases showed losses with either microsatellite marker around p16 region in tumor samples. The percentage is 10.53%. The other marker, (CAAAA)n repeat, is located closer to canine p16 gene than the (TTTC)n repeat and (CCA)n repeat. However, when testing the (CAAAA)n repeat in regular control dog DNA from our lab, it did not show much heterozygosity. Although the ratio of loss in our tumor samples was not very high, it demonstrates the occurrence of loss of pl 6 in canine nasal carcinomas. The results of IHC staining confirmed reduced p16 expression, revealing that 72.73% of squamous cell carcinomas and 46.15% of adenocarcinomas lacked p16 protein expression. 21 The low frequency of LOH around p16 suggested that p16 inactivation might be occurring through other mechanisms. Promoter hypermethylation is another important mechanism, which has been reported in NPC.25 Future study will focus on the identification of the p16 promoter region, using cloning to identify the correct sequence of the product from 5’RACE, and performing bisulfite conversion reactions and sequencing to investigate the methylation status of the p16 promoter region in canine nasal carcinomas. Detection of overexpression of p53 protein in nasal adenocarcinomas by IHC was reported previously.‘2 According to this paper, 11 of 19 (57.89%) cases of canine nasal adenocarcinomas showed more than 10% stained nuclei for p53, which indicated the possible p53 mutation or defects in p53 degradation. In our study, only 38.46% of cases of adenocarcinomas had positive p53 staining. The percentage of p53 positive cases in transitional carcinomas and squamous cell carcinomas were even lower. The disagreement between our findings and that of Gamblin RM et al., 199712 may be due to the different sample population and detection methods. It is also possible that the mutation of p53 in our samples resulted in the complete loss of p53 protein or in a mutant form that cannot be detected by this IHC protocol. While herpesvirus and the canine chromosomal region orthologous to human Ch4p15.1—q12 were not shown to play a role in the etiology of canine nasal carcinoma, inactivation of tumor suppressor gene p16 seems to be associated. Future studies will focus on promoter hypermethylation of the canine p16 gene and collecting more cases for IHC to clarify the expression of p16 and p53 protein in this tumor. Moreover, since no cell line derived from canine nasal carcinomas is available currently, establishing tumor 22 cell lines and normal nasal cell culture for relevant study will help elucidate the etiology of canine nasal carcinomas. 23 APPENDICES Table l Signalments of 43 cases studied No. 5147 127976 163839 185356 11611 8389 40298 10045 20270 57329 19203 59269 59432 58427 58428 670468 3343 713913 44741 764707 88446 14870 0353 1587 9596 446947 461986 reed chow tervuren llie ullmastiff e hetland hetland hetland ack Russell terrier hound chow hound hih tzu lden retriever Table continued on next page 24 castrated castrated castrated castrated castrated castrated castrated castrated castrated castrated castrated castrated A castrated castrated castrated castrated Table 1 continued 2513346 Golden retriever 1 1y 1M castrated SCC 2555434 Mixed 12y 1M castrated SCC 2709556 Chow chow 9y6m IM castrated SCC 2759984 Labrador retriever 6y7m F SCC 2764845 'Labrador retriever 8y [M castrated SCC 2811976 Labrador retriever 9y lM castrated SCC 2834357 abrador retriever 9y NA SCC 2179157 Mixed 14y10m castrated TC 2198895 German shorthaired pointer 11y NA TC 2542573 Brittany spaniel 13y FM castrated TC 2609483 NA 7y F spayed TC 2685932 Airedale terrier 11y F spayed TC 2840551 German shepherd 9y10m F spayed TC 2854419 Golden retriever 8y11m F spayed TC M=Male F=Female SCC=Squamous cell carcinoma TC=Transitional carcinoma 25 Table 2 Species of herpesviruses (21) detected by PCR with universal herpes primers Virus Common Name Subfamily Strain Aotine herpesvirus 1 Herpesvirus aotus type 1 B S43E Ateline herpesvirus 2 Herpesvirus ateles y 810 Callitrichine herpesvirus 1 Herpesvirus sanguinus y S-388D Canid herpesvirus 1 Canine herpesvirus a D004 Cercopithecine African green monkey B CSG herpesvirus 5 cytomegalovirus Equid herpesvirus 2 Equine cytomegalovirus y 82-A Feline herpesvirus 1 Feline herpesvirus 1, Feline a C-27 rhinotracheitis virus Gallid herpesvirus 1 Infectious laryngotracheitis virus a N-71851 Gallid herpesvirus 3 Marek’s disease herpesvirus type a GAS 2 Human herpesvirus 1 HSV-l a Maclntyre Human herpesvirus 2 HSV-2 a G Human herpesvirus 3 VZV 0 Ellen Human herpesvirus 4 EBV y B95-8 Human herpesvirus 5 Human CMV B AD169 Human herpesvirus 6 HI-IV-6B B Z-29 Human herpesvirus 7 Human herpesvirus 7 B SA Human herpesvirus 8 Kaposi’s sarcoma-associated virus y Leporid herpesvirus 2 Rabbit herpesvirus; Herpesvirus v 923.1 cuniculi Psittacid herpesvirus 1 Parrot herpesvirus (1 RSL-l Saimirine herpesvirus 1 Herpes platyrrhinae a MV-5-4 Saimirine herpesvirus 2 Herpesvirus saimiri y S 295C 26 Table 3 (1) Primer sequences for PCR reactions for investigation of viral presence and LOH Forward/Reverse Primer Universal Herpes F-TGTAACTCGGTGTAYGGNTTYACNGGNGT R-CACAGAGTCCGTRTCNCCRTADAT BHV-4 l-CATGACACACTATTTTAAGTACTA 2-ATC'I'ITCTTTCAGTCTCACTGTA 3-ATAAATTTGTGAAGACAATGGGTA CNCG-l F-CACAAACACCCTTGCTGGTC R-GACAACCATATTCCCCTCAC SCGB F - ATGTTCTTGATGAATTTTGGC R- AACTGCATACCAATGTGACT (TTTC)n F -CTGGCAGGCTCAGAGATTCAGAG R-ACCCACTTTGCTGGC GAATTAGA (CCA)n F -TACGGTAAGGAGTGAGGGCTGAC R-TAAC GCCACCTTAGAAGTCAGTC (CAAAA)n F-TTGCCGAACGCTGTGTTCCGTG R-GGGGCCGGGGGAAGGGTC (2) Cycling conditions for PCR reactions for investigation of viral presence and LOH Cycle Condition Product Size Universal Herpes 94°C 303, 46°C 1min, 72°C 1min for 45 cycles 207bp BHV-4 94°C 1min, 59°C 2min, 72°C 3min for 35 cycles 153bp CNCG-l 94°C 1min, 54°C 1min, 72°C 1min for 45 cycles 301bp SCGB 94°C 1min, 54°C 1min, 72°C 1min for 45 cycles 148bp (FITCH 94°C 1min, 62°C 1min, 72°C 1min for 50 cycles 159bp (CCA)n 94°C 1min, 62°C 1min, 72°C 1min for 50 cycles 248bp (CAAAA)n 94°C 1min, 60°C 1min, 72°C 1min for 50 cycles 244bp 27 Table 4 (1) The number of cases and percentage for each tumor type for positive or negative p16 nuclei staining Negative Positive Adenocarcinoma 6 (46.15%) 7 (53.85%) Transitional carcinoma 2 (33.33%) 4 (66.67%) S uamous cell carcinoma 8 (72.73%) 3 (27.27%) (2) The number of cases and percentage for each tumor type for positive or negative p53 nuclei staining Negative Positive Adenocarcinoma 8 (61 .54%L 5 (38.46%) Transitional carcinoma 4 (66.67%) 2 (33.33%) Squamous cell carcinoma 8 (72.73%) 3 (27.27%) (3) The number of cases and percentage for each tumor type of different p53 and p16 staining level combination p53+ p16+ p53+ p16- p53- p16+ p53- p16- Adenocarcinoma 3 (23.08%) 2 (15.38%) 4 (30.77%) 4 (30.77%) Transitional carcinoma 1 (16.67%) 1 (16.67%) 3 (50%) 1 (16.67%) Squamous cell carcinoma 2 (18.18%) 1 (9.09%) 1 (9.09%) _ 7 (63.63%) 28 Figure 1 Tumor-suppressive pathway of p16. During the cell cycle, the cyclin D and CDK4 complex phosphorylates pRB, which leads to the release of transcriptional factor E2F to promote cell cycle. p16 competes with CDK4 to bind to cyclin D, which leaves pRB unphosphorylated and bound to E2F, resulting in cell cycle arrest at G1 stage. M - . . V A 62 CELL CYCLE (31/ .7. u k 29 Figure 2 The sequences of intron 3 of cCNCGI (X99913) and SCGB (AF427093) with SNPs. The SNPs result in the change of a anI and Hinfl cutting site. anI recognizes the sequence 5’-GAANNNNTTC-3’ and cuts between GAANN and NNT'I‘C resulting in a blunt end. Hinfl recognizes the sequence 5’-GANTC-3’ and cuts between G and ANT C. The reverse primer designed for SCGB has one G altered from the correct sequence to create a diagnostic site for SNP. The SNPs are in red and the cutting sites are in gray. The primers designed for PCR are underlined. >X99913 cCNCGI GTGAGCAGTATGAGCTACTCCTCCATAGCTCTI‘TI'I'AAACTCT'I‘A’I'I‘TAGATG AAATATAGGTATTCTACTCCTCTAT'I'I‘AGTGGGCTTGACAAATGCTCCTGCTA CCCCCACTTC AGAGGATC'I'I‘GGGAGTCACTTGCTACTCAC C'I‘T'I'I‘CCTCTC’I'I‘G GAAAACTGCCACAGCCTGGTGACTATI‘CATGTCCATGGGGCCTCAAGTQAQA AACACCCT'I‘GCTGGTCATTTTCAGAGGCCTGTC'ITTCTTTGTCCCTGAAGAAC C TCATGCCCCAGTCCCCTATGACTTCTTTCTGACCC'I'I'CTTTCAGGGTCCTGAA GCAA'ITGTACGTGGGAGGGGTTGGCTGTGCTCCTCTAAAAAGCTGTGTCCTAT 'I'I‘GACCCTAC’I'I'I‘TAGGGAAGTCGAGCACTAGTGTGAGCAGCTAGAAAAGAA AAGCTAGAAGTCCTTCATAGGAAATGAAACACT C ATGAAAACTTCCTACC CG TGACTCTGAGGGTGTGAGGGGAATATGGTTGTCGGTGTGGATCAGGGGCTCC CATCCTGTGGAACATA'I'I'I‘AC'I'I'I'I‘ACT'I‘TGCTI‘TI‘GCTCCCTCCCT'I‘T’I'I‘CCCC TCTC'I'I‘AATCCTTTTCCCACTGACC ACCTI‘CAGT'I'I'I‘CCCACTGTTI'I'I’ATI'I‘AT C'I'I'I'I‘C'I'I‘CTC'I'I‘CCCATC’I'I‘CCCTGACAATCTAAGTCTTAGGTCTC'I'I’GCTGG CAGTTGCAGCATCCAAGGTGGCTCACTTGAGGCCACGGAGGATGCTAAGCTT 30 GGGTGTTAGCATTGA'I'I'AATAGGT'I'I'ITACTAATTACTTTGGTTCATAGCACT GATACTCAGCAACTCATGGAGGCTCTATA'I'I'ITCAAAAA’ITGTCTAGGCAGTA GTTAGAACAAAGAGT'I'TG'I'I'CTTI‘AAGACTG'I'I‘GCTTGCCATGAACATTATAC AGATGGTGACGATTATGATT'I'CACTGTACATC'I'I‘CAT’I'I'ITCAG C/TSNP >AF 427093 SC GB T'I'TGTGCAGAGC AGTGTAAGCAAACC AGCATATAAGCCCTCT'ITTTAAAGAA TTCTTCCCAAGTGGATAGAGAGGGGAC AGACCAATGC AGTTAAAA’ITCTT'I‘G AAGTTAGTCTA'I'I‘ATI'ITAAAGTG'I'I'I‘T'I‘GATAATGTTCTTGATGAATTTTGGC TGCAGTCTCCTTI'GAAAACATACTC ATGATAAAGTGTT'I'ITCTGAAGATTGTC 'I'I'TCAGC AAGGGAC AAC AAAGCTC AGTGTAGAAAAGAAC AAAACTI‘CTATTA CGAQI GAC ATTQTATGCAQTT G/ A SNP G altered base used to create diagnostic restriction site for SNP 31 Figure 3 Structure of INK4A/ARF locus. INK4A/ARF locus in human and mouse gives rise to two distinct transcripts: p16 with exonl a , exon2 and exon3, and p14 with exon] 3, exon2 and exon3. However, these two proteins are translated in alternative reading frames and share no amino acid homology. [tum I?" [ELI a .I KEEP? I'VE-'3 .] mom 32 Figure4Detcctionofpresenceofherpesvirus M e v th' MP 1 n 1 In E . . . i—i . ..‘ - a .. 4D " '9 w ,tfz.t2a V k M M - ‘ H. :1 - ‘ ii: '9 .. {- " L . ,., ”a In '50; i“ 3 ~- ~ .. A” at. i 1. Universal herpes primers 2. Primers for BHV-4 P=Positive control, P1=Positive control: FHV-l, P2=Positive control: BHV-4 strain DN599, M=100 bp DNA ladder (Invitrogen, Carlsbad, CA) 33 Figure 5 LOH study on canine chromosomal region orthologous to human Ch 4p15.1-q12 l. The first two samples were homozygous for C and the third sample was heterozygous for C T. 2. From left to right: homozygous for G homozygous for A, heterozygous for G A, heterozygous for G A NTNTNTNT UwWH "'1 13“ '1 1.5., , l. SNP marker cCNCGI 2. SNP marker SCGB N=normal DNA, T=tumor DNA, M(l)=100 bp DNA ladder (Invitrogen, Carlsbad, CA), M(2)=pBR322 DNA-Msp I digest (New England Biolabs, Ipswich, MA) as molecular weight standard 34 Figure 6 LOH study on canine p16 with microsatellite markers (TTTC)n and (CCA)n. The two losses in 8 informative cases for (TTTC)n are shown (1), but no loss was observed in the 15 informative cases for (CCA)n (2). M N 'l' N T N '1' Different alleles amtmd 1501p 1. Microsatellite marker (TTTC)n NTNTNTNTMNTNTNT Difluentallcles murdzsobp 2. 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