Association between Polymorphic Variants of <em><em>RAF1</em></em> Gene with Occurrence of Mammary Tumor and Aging in Canines

Research Article

J Drug Discov Develop and Deliv. 2014;1(2): 8.

Association between Polymorphic Variants of RAF1 Gene with Occurrence of Mammary Tumor and Aging in Canines

Bartosz Kempisty1,2*, Katarzyna Zaorska1, Dorota Bukowska3, Marcin Nowak4, Sylwia Ciesiólka1, Katarzyna Wojtanowicz-Markiewicz3, Karol Jopek1, Artur Bryja1, Pawel Antosik3, Klaus-Peter Brüssow5, Malgorzata Bruska2, Michal Nowicki1 and Maciej Zabel6

1Department of Histology and Embryology, Medicine Faculty I, Poznan University of Medical Sciences, Swiecickiego 6 St., 60-781, Poznan, Poland

2Department of Anatomy, Medicine Faculty I, Poznan University of Medical Sciences, Swiecickiego 6 St., 60-781 Poznan, Poland

3Institute of Veterinary Sciences, Faculty of Animal Breeding and Biology, Poznan University of Life Sciences, WojskaPolskiego 52 St.

4Department of Pathology, Faculty of Veterinary Medicine, Wroclaw University of Life Sciences, 50-375 Wroclaw, C.K. Norwida

5Institute of Reproductive Biology, Department of Experimental Reproductive Biology, Leibniz Institute for Farm Animal Biology, Dummerstorf, Germany

6Department of Histology and Embryology, Wroclaw Medical University, 6a Chalubinskiego St., 50-368, Wroclaw, Poland

*Corresponding author: Bartosz Kempisty, Department of Histology and Embryology, Medicine Faculty I, Poznan University of Medical Sciences, Swiecickiego 6 St., 60-781, Poznan, Poland

Received: Aug 12, 2014; Accepted: Sep 29, 2014; Published: Oct 02, 2014


The induction of carcinogenesis as well as cancer growth and development are often associated with an altered expression of genes encoding protooncogenes and proteins responsible for regulation of cell division cycle. Although the role of RAF1 protein in development of apoptosis resistance mechanisms is well known in many types of human cancer, the association between changes in the structure of RAF1 gene and induction of canine carcinogenesis and/or aging remains still poorly recognized. Therefore, the goal of this study was focused on new mutations in the RAF1 gene as well as on association between frequency of RAF1 gene polymorphisms and the occurrence of mammary tumor in groups of domestic bitches of various ages.

In this study, blood samples were obtained from 22 female dogs diagnosed with mammary tumors. Moreover, blood samples were also collected 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) dogs. Thirty six bitches examined for other reasons served as controls.

After Sanger sequencing analysis, 13 single nucleotide variations were identified, of which two were localized in coding regions (exons 3 and 9) and the rest 11 - in introns (introns 3, 4, 5, 7, 8, 10 and 16). We observed differences in prevalence of heterozygotes and alternative alleles between study groups in 4 polymorphisms, two of which could indicate the putative protective variants (c.T237C, c.A918C) and the other two could indicate the putative risk variants (g.A4805C, g.A6902C) with reference to the cancer occurrence in dogs.

In conclusion, we demonstrated that RAF1 polymorphisms may serve as a protective and/or risk factor for the occurrence of mammary tumor in domestic bitches, manifesting a type- and localization of mutation-dependent manner. Although the frequency of occurrence of these polymorphisms was often not statistically significant, haplotype blocks revealed strong linkage between investigated polymorphisms.

Keywords: Mammary Tumor; RAF1 Polymorphisms; Aging; Canine


The carcinogenesis is a complex process, involving many molecular, biochemical and morphological changes, which finally lead to growth and development of cancer [1,2,3]. It has been suggested that various types of cancer reflect type of affected tissue, tissue morphology as well as organ origin [4,5,6]. Moreover, several lines of experiments indicated that canine cancers display many similarities to human cancers, especially in respect to morphology, growth of cancer, clinical features and clinical prognosis. Therefore, some of the authors regarded canine cancers to represent a model of human carcinogenesis [7,8,9,10].

Induction of carcinogenesis was demonstrated to be strongly associated with molecular changes and disruptions. Furthermore, in several studies changes in gene sequences caused by spontaneous or heritable mutations/polymorphisms were found to lead in a simple way to disruptions in amino acid sequence and/orto formation of improper protein [7,11]. Induction of carcinogenesis is often caused by mutation in the genes encoding proteins responsible for cell cycle control, formation of free radical and oxygen species, apoptosis or uncontrolled prolongation of cell life as well as for formation of fibrous collagen and cell cytoskeleton [12,13,14].

The RAF1 is known as an oncogene that can be targeted to the mitochondria by BCL2 as well as it is known to belong to the main regulators of apoptotic cell death [15]. Active RAF1enhanced BCL2-mediated resistance to apoptosis and may be involved in phosphorylation of BAD. Alavi et al [16]. Demonstrated that FGFB and VEGF may differentially activate RAF1, which leads to protection through distinct pathways of apoptosis in human endothelial cells and chick embryo vasculature. The two studies showed that activation of RAF1 is strongly associated with resistance to apoptosis, which may provide the main reason for uncontrolled cell cycle divisions leading finally to induction of carcinogenesis.

The problem of mammary tumor of non-identified origin in aging bitches is increasing. Therefore, the aim of the study was searching for new molecular markers that would predict the occurrence of tumor in bitches as well as determining the correlation between frequency of RAF1 gene polymorphisms and the occurrence of mammary tumor in domestic bitchesin relation to aging.

Materials & Methods

Subjects and samples collection

Blood samples were obtained from 22 female dogs of mongrel dogs diagnosed with mammary tumors, histologically classified as malignant tumors, during surgery in the Small Animal Clinic, University of Life Sciences, Poznan, Poland. Blood was collected during standard surgery procedures. Thirty six bitches served as controls. The affected bitches were divided into 3 subgroups of different age, according to the classification by Jugdutt et al. [17]: geriatric (>5 to 10 years old; n=15), mature adult (>2 to 5 years old; n=10) and young ones (from 1 to 2 years old; n=11). Blood was collected from from the jugular vein into vials with EDTA and frozen at-80 °C until further analyses.

Molecular analyses

Genomic DNA was extracted from whole peripheral blood using QIAamp DNA Blood Mini Kit (Qiagen), according to the manufacturer’s instructions. DNA was re-suspended in 100 μl of Qiagen elution buffer and stored at -20 °C. Sixteens exons of the RAF1 gene (exon 2 - exon 17) were amplified in Polymerase Chain Reaction (PCR), including up to 70-bp flanking regions of every exon. The sequences of the primers used with thermal and time conditions are listed in Table 1. The reactions were carried out in a total volume of 12.5 μl containing: 10 x Taq DNA Polymerase buffer with MgCl2, 5 x GC-rich solution, 0.24 mMdNTPs, 0.5 μM of the primers, 1 unit of Taq Polymerase (Roche) and 40-60 ng of genomic DNA. The PCR cycle conditions were: an initial denaturation at 94 °C for 4 min, followed by 35 cycles of denaturation at 95 °C for 30 sec, annealing at temperatures shown in Table 1 for 1 min and elongation at 72 °C for 30/60 sec, with a final extension at 72 °C for 7 min. PCR products were purified using membrane plates (Millipore) and used as templates in PCR-sequencing reamplification. The latter reaction was performed in Veriti 96 well Thermal Cycler using BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) and one of the specific primers (Forward or Reverse). Reamplification products were purified with EDTA and ethanol precipitation and separated by electrophoresis using ABI 3130 sequencer (Applied Biosystems).