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Analysis of Mitogen-Activated Protein Kinase (MAP2K2) Gene Polymorphisms in Relation to the Occurrence of Adenocarcinoma During Aging in Dogs1)

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Analysis of Mitogen-Activated Protein Kinase (MAP2K2) Gene Polymorphisms in Relation to the Occurrence of Adenocarcinoma During Aging in Dogs1) 46 Med. Weter. 2016, 72 (1), 46-52 Praca oryginalna Original paper Analysis of mitogen-activated protein kinase (MAP2K2) gene polymorphisms in relation to the occurrence of adenocarcinoma during aging in dogs1) BARTOSZ KEMPISTY*, **, KATARZYNA ZAORSKA*, DOROTA BUKOWSKA***, MARCIN NOWAK****, KATARZYNA WOJTANOWICZ-MARKIEWICZ***, SZYMON POROWSKI*, EDYTA OCIEPA*, PAWEŁ ANTOSIK***, KLAUS-PETER BRÜSSOW*****, MAŁGORZATA BRUSKA**, MICHAŁ NOWICKI*, MACIEJ ZABEL*, ****** *Department of Histology and Embryology, **Department of Anatomy, Medicine Faculty I, Poznan University of Medical Sciences, Święcickiego 6 St., 60-781 Poznan, Poland ***Institute of Veterinary, Faculty of Animal Breeding and Biology, Poznan University of Life Sciences, Wojska Polskiego 52 St., 60-637 Poznan, Poland ****Department of Pathology, Faculty of Veterinary Medicine, Wroclaw University of Life Sciences, C. K. Norwida 31 St., 50-375 Wrocław, Poland *****Institute of Reproductive Biology, Department of Experimental Reproductive Biology, Leibniz Institute for Farm Animal Biology, Dummerstorf, Germany ******Department of Histology and Embryology, Wroclaw Medical University, Chalubinskiego 6a St., 50-368 Wrocław, Poland Received 09.04.2014 Accepted 14.11.2014 Kempisty B., Zaorska K., Bukowska D., Nowak M., Wojtanowicz-Markiewicz K., Porowski S., Ociepa E., Antosik P., Brüssow K.-P., Bruska M., Nowicki M., Zabel M. Analysis of mitogen-activated protein kinase (MAP2K2) gene polymorphisms in relation to the occurrence of adenocarcinoma during aging in dogs Summary In the most recent years adenocarcinoma has been found to be the most common cancer that occurs in both humans and animals. It is well recognized that the induction of carcinogenesis and/or cancer growth and development is often associated with aging. Moreover, the molecular basis of carcinogenesis involves disturbances in expression of genes or mutation in genes that play a crucial role in regulating cell division cycle and inter- or intracellular signaling pathways. MAP2K2 belongs to the large family of kinases that are described as “checkpoints” of cell division and signaling, and therefore may be involved in carcinogenesis. In this study, blood samples were obtained from 22 female dogs diagnosed with mammary tumors. Moreover, blood samples were obtained from geriatric (> 5 to 10 years old; n = 15), mature adult (> 2 to 5 years old; n = 10) and young (from 1 to 2 years old; n = 11) individuals. 36 bitches diagnosed because of other reasons served as controls. After Sanger sequencing analysis we found 17 single nucleotide variations, of which 3 were situated in exons (exon 2, 3 and 11), 2 other in 5’UTR non-coding region and the remaining 12 in splice regions of introns. Some of the polymorphisms, such as g.C-81T, could have higher probability of being involved in tumor development, also in correlation with aging. Furthermore, both variants c.A384G and g.T9144C were associated with strong risk factors of tumor occurrence and aging. In conclusion, it may be suggested that some of the MAP2K2 gene polymorphisms may be recognized as markers for occurrence of adenocarcinoma in dogs. This also showed that possible disruption in expression of MAP2K2 protein kinase may lead to the induction of carcinogenesis, since it plays a crucial role in regulating the cell division cycle and cell signaling. Keywords: mutation, cancer, Canis familiaris 1) Supported by the Polish National Centre of Science (Grant No. 5279/B/P01/2011/40). Med. Weter. 2016, 72 (1), 46-52 47 The development, growth and progression of cancer pathway. This pathway is responsible for the regula- are associated with dysfunction of the cell division tion of normal cell division through a series of protein cycle that is regulated by multiple genes and by phosphorylations, transition of signals into the nucleus expression of proteins involved in many biochemical and induction of transcription of target genes (5, 9). and metabolic pathways (8, 15, 18). Recent findings The second one involves phosphatidyl inositol-3 kinase have demonstrated that proteins belonging to protein (PI3K) and its pathway that regulates cell survival, kinases family are in most cases responsible for pro- cycling and growth (6, 7). cesses related to cells growth and development (1, 4, The MAP2K2 protein belongs to the first group 17). Moreover, protein kinases are crucial regulators of signal transduction kinases that regulate crucial in normal cell signaling, since they up-regulate key steps for cell cycle control. Therefore, dysfunction of processes such as cell proliferation, differentiation this pathway often leads to the induction of carcino- and migration. It has been clearly demonstrated in the genesis. Moreover, it was found that disturbances in case of several proteins kinases dysregulation of their signal transduction pathways may be a direct reason expression through mutation, polymorphisms and/or of induction/inhibition of apoptosis, which is sig- chromosomal translocation results in the activation nificantly associated with aging. The aim of this study of several improper pathways and subsequent uncon- was to analyze the MAP2K2 gene sequence in order trolled transfer of cell signals from extracellular matrix. to find the mutation and/or polymorphisms that may It was also found that dysfunction of protein kinases be responsible for adenocarcinoma in domestic dogs expression is associated with induction of carcinogen- (Canis familiaris) during aging. esis in humans; however most recent findings have Material and methods also shown a similar level of dysfunction in develop- ment of canine and feline cancers (13, 16, 19). If the Subjects and samples collection. Blood samples were mutation occurs in the protein kinase gene sequence, obtained from 22 female dogs diagnosed with mammary it often leads to alterations in the protein structure. As tumors during surgery in the Small Animal Clinic, Univer- a result, phosphorylation occurs in the absence of an sity of Life Sciences, Poznan, Poland. Blood was collected appropriate stimulus. during standard surgery procedures. The 36 bitches diag- nosed for other reasons (routine control of health-blood col- The signal from the extracellular matrix is trans- lection, following ovariohysterectomy) served as controls. ferred via receptors for tyrosine kinase (RTKs) and All examined dogs were mongrels. They were divided into is passed through the cytoplasm into the cell nucleus 3 subgroups differing in age, according to the classification with the activation of series of intermediates that are by Jugdutt et al. (10): geriatric (> 5 to 10 years old; n = 15), also phosphorylated. It was found that two specific mature adult (> 2 to 5 years old; n = 10) and young (from cytoplasmic pathways are dysregulated in cancer 1 to 2 years old; n = 11) ones. Blood was collected from cells, which leads to cancer progression. The first one the jugular vein into vials with EDTA and frozen at –80°C includes the RAS-RAF-MEK-ERK-p38-JNK signaling until further analyses. Tab. 1. PCR primer pairs used for the amplification of exons of MAP2K2 gene Exon Sequences Annealing temp. Length of the amplicon Length of noncoding sequences exon 1 F 5’-TAGGTCTCCGCCCCTTTC-3’ 58°C 580 bp (394 bp of 5’UTR/92 bp of exon 1/ 488 bp R 5’- TGCATCTTCTCCTCTCAGGTG-3’ 94 bp of intron 1 exon 2 F 5’- GTCCTTCCAGCCACACATCT-3’ 58°C 458 bp (123 bp of intron 1/211 bp of exon 2/ 247 bp R 5’- AGCCAATCAGAAAGCACAGG-3’ 124 bp of intron 2) exon 3 F 5’- TTGGTTGCTGTCTTGAGCAC-3’ 58°C 345 bp (108 bp of intron 2/147 bp of exon 3/ 198 bp R 5’- TTGGAAATAAGCGTGCTGTG-3’ 90 bp of intron 3) exon 4 F 5’- GCTTCGCTCTCATACCTGCT-3’ 59°C 297 bp (116 bp of intron 3/78 bp of exon 4/ 219 bp R 5’- GGGGGCAGGTTAAGGATAAA-3’ 103 bp of intron 4) exon 5, exon 6 F 5’-ACGCTATGCTCACCTCACCT-3’ 60°C 527 bp (127 bp of intron 4/52 bp of exon 5/ 350 bp R 5’- AACCGCTGCTAGAATTTGGA-3’ 89 bp of intron 5/125 bp of exon 6/134 bp of intron 6) exon 7 F 5’- GAACAGTGTCGGTGACAGGA-3’ 58°C 434 bp (106 bp of intron 6/214 bp of exon 7/ 220 bp R 5’- GACAGGTGGTGCTGAGAGG-3’ 114 bp of intron 7) exon 8 F 5’- TGGTTTCCCAAGAAAGCAAG-3’ 58°C 291 bp (122 bp of intron 7/74 bp of exon 8/ 217 bp R 5’- CCATGTGGAATTCCCTTCC-3’ 95 bp of intron 8) exon 9 F 5’- AAAGTGCTGTGCTGTCCTCA-3’ 59°C 266 bp (108 bp of intron 8/62 bp of exon 9/ 204 bp R 5’- GTGGGCAACTATCCCAGGAG-3’ 96 bp of intron 9) exon 10 F 5’- CTGGCCTATTACGGCTCAAA-3’ 59°C 268 bp (89 bp of intron 9/46 bp of exon 10/ 222 bp R 5’- CCCCACTAATGGAGTCAGGA-3’ 133 bp of intron 10) exon 11 F 5’- CTCTGGCTCTGGCTTCCTGT-3’ 62°C 350 bp (99 bp of intron 10/111 bp of exon 11/ 239 bp R 5’- AGTGTCCACTGCGGGGTAT-3’ 140 bp of 3’UTR) 48 Med. Weter. 2016, 72 (1), 46-52 Molecular analyses. Genomic DNA was extracted from Tab. 2. Single nucleotide variations identified in MAP2K2 whole peripheral blood using QIAamp DNA Blood Mini gene in studied subjects Kit (Qiagen), according to the manufacturer’s instructions. Name of Localization nt change aa change DNA was suspended in 100 µl of Qiagen elution buffer and variation stored at –20°C. Polymerase chain reaction (PCR) was used 1 g.G-236C 5’UTR G > C – to amplify the MAP2K2 gene. All 11 exons were amplified, 2 g.C-81T 5’UTR C > T – including approximately 60-bp flanking regions of each exon. The sequences of primers used are listed in Table 1. 3 c.C210T exon 2 C > T syn: D70D (GAC > GAT) The reactions were carried out in a total volume of 4 g.T9144C intron 2 T > C – 12.5 µl, containing: 10 × Taq DNA Polymerase buffer with 5 g.G9145T intron 2 G > T – MgCl2, 5 × GC-rich solution, 0.24 mM dNTPs, 0.5 µM of 6 g.C9176T intron 2 C > T – the primers, 1 unit of Taq Polymerase (Roche) and 40-60 ng of genomic DNA.
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