Sequence Analysis of P53 Gene in Different Mammary Neoplasms of Brazilian Dogs
S.C. Fernandes; C.R. Daleck; J.A.D. Sena; S.G. Calazans; A.B. De Nardi; S.M. Rodigheri; J.R.F. César; I.L. Abreu; M.T. Costa
Via de Acesso Prof. Paulo Donato Castellane s/n, Jaboticabal, SP, Brazil
After a year of the surgical intervention, 48% of the dogs die or are submitted to euthanasia because of recidivation or metastasis (Graham & Myers 1999). The prognostic for canine mammary neoplasm is variable. The median survival after the mammary carcinoma surgical excision was 7 to 16 months. The prognostic is particularly unfavorable for sarcoma and inflammatory carcinoma (Knapp et al. 2004). Despite the increasing clinicopathological investigation, little is known about the canine mammary tumors prognostic (Benjamin et al. 1999). The tumor suppressor genes perform a fundamental role in the determination of mammary oncogenesis, and the increase of this knowledge related to molecular biology will allow a better proceeding in the fields of prevention, diagnostic, therapeutic and definition of prognostic factors (Camargo et al. 1999, Lopes et al. 1999). The p53 tumor suppressor gene function is blocking cellular division in case the cell detected some damage in the genetic material. This pause on the cellular cycle allow that the cell use repair mechanisms for the flaw to be corrected, blocking the transcription from phase G1 to phase S and not allowing propagation to the next generation. In case this repair attempt doesn't succeed, the p53 conducts the cell to a process of apoptosis (Lopes et al. 1999, Silva 2004). The loss of p53 function during the oncogenesis, after DNA damage, can determine an inappropriate progression on the cellular cycle and allow the survival of the cells that, in normal conditions, would be destined to die (Lopes et al. 1999). Cancer cells with p53 mutations enter on phase S, duplicate the DNA damage and separate the chromosomes abnormally (Levine et al. 1994). The p53 gene mutation has been described on the pathogenesis of numerous human and canine neoplasms (Lee & Kweon 2002). This gene is the one that most often shows alteration in human cancer, and it is mutated in almost 50% of neoplasms (Lana et al. 2007, Lopes et al. 1999, Silva 2004). It is believed that part of the remaining 50% has a p53 signalization pathway that is compromised by others mechanisms (Silva 2004). Studies suggest that mutation of p53 gene is associated to mammary tumor progression (Lee & Kweon 2002) and show increase on the maligned potential and prognostic worsening (Lee et al. 2004, Muto et al. 2000). Most human mammary tumor researches mention both pre-recidivation and survival periods are significantly reduced on patients that showed mutations on the p53 gene (Silva 2004). This present study had as its objective to detect possible alterations on exons 5 to 8 in the p53 tumor suppressor gene in canine mammary tumors and in common mammary gland samples.
Materials and Methods
Thirty bitches of different ages and breeds, all with mammary tumor diagnostic, were examined on Veterinary Oncology Service (SOV) in the Veterinary Hospital of São Paulo State University (UNESP/Jaboticabal). This research used also samples of mammary glands without pathologic alterations from five bitches with ages between seven and ten years, mongrel and clinically healthy, from the Sertãozinho Municipal Kennel (São Paulo), as the control group. The animals were submitted to regional or radical mastectomy, according to the location of the node(s) for surgical excision. The tumors were divided in two parts. First one was stored on freezer at -24°C, for DNA extraction. The other one was fixed in 10% formalin for histopathologic examination.
The genomic DNA was extracted from a portion of the frozen node, based on protocol already described by Pearson & Stirling (2003). The samples that presented RNA were treated with RNAse (Ribonuclease A--Sigma®) and quantified in Beckman--DU® spectrophotometer, for the relation between the absorbance reading made by 260 and 280nm (Sambrook & Russel 2001). After this quantification, samples were stored in -24°C until the moment of the PCR technique procedure.
The PCR technique was performed for amplification of exons 5 to 8, with the initiator oligonucleotides (primers) based on description by Chu et al. (1998). For this, 10 pM of each primer (Operon) were mixed with almost 100 ng of genomic DNA, 2 mM of Magnesium Sulfate (Invitrogen), 0,2 mM of each dNTP (Invitrogen), 1 U of Taq DNA Polymerase high fidelity (Invitrogen) and 1X PCR buffer (Invitrogen), in a final volume of 25 µL. The PCR program was conducted in 40 cycles.
The samples were purified by a protocol based on the enzymes Exonuclease I (EXO-USB®) and Shrimp Alkaline Phosphatase (SAP-USB®). A solution of 5 µL of PCR product, 0.5 µL of EXO (10 U/µL), 1 µL of SAP (1U/µL), 0.5 µL of SAP dilution buffer and 3 µL of Milli Q water were mixed for a 10 µL final reaction.
To verify the possible occurrence of mutations on this gene, the amplified products were sequenced with the primers P5F, P5R, P6F, P6R, P7F, P7R, P8F and P8R. The reaction was elaborated in an amount of 10 µL consisting of: 3 µL of 5X buffer (400 mmoL/L Tris-HCl, pH 9.0; 10 mmoL/L MgCl2), 0.25 µL of Dynamic Terminator (GE), 0.5 µL of each primer (10 pmol/µL), 10 ng of the DNA fragment and sterile Milli-Q water. The samples were bring to the thermocycler using the same program used on the PCR technique.
After the PCR technique, the samples were submitted to DNA automatic sequencing using ABI 3700 DNA Analyzer-Applied Biosystems on capillary system. The sequences obtained were analyzed by Sequencing Analysis 3.4, and the sequences assembling, the nucleotide quality verifying on the chromatogram and the files created on the FASTA format were executed by the Phred/Phrap/Consed program pack. The DNA/protein sequence translation was made by the Swiss-Prot translation tool (http://br.expasy.org/tools/dna.html). The Clustal W alignment program (http://clustalw.genome.jp/) was used to compare the sequences generated on this experiment with the ones deposited on GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html).
According to the histopathological exam, a higher incidence of malign tumors (76%) was noted, compared to benign tumors (24%). Adenomas (17%), benign mixed tumors (7%), carcinomas (33%), sarcomas (13%) and malign mixed tumors (30%) could be found. In the control group, all five mammary gland samples didn't show any alterations on histopathological exam. A consensus between the five controls was obtained for each exon, and the consensus-sequence was analyzed with the normal sequence (Chu et al. 1998). For a better visualization, sequences were aligned in the Clustal W 1.83 program (http://align.genome.jp). Six mutations were found in 30 neoplasms, five in 23 malign tumors and one in seven benign tumors. Still on this study, there was a T-to-C transition on codon 156 (exon 5) in all sequences of control group and tumoral samples. These mutations can be classified as silenced, because there was no alteration on the amino acid.
Discussion and Conclusions
The mutational frequency found in this study is consistent with the ones found in other researches (Chu et al. 1998, Lee & Kweon 2002, Mayr et al. 1999, Muto et al. 2000, Van Leeuewn et al. 1996, Wakui et al. 2001). On those, the mutational percentage in malign tumors is higher if compared to benign tumors, exception being Wakui et al. (2001) which studies carcinomas exclusively. As a T-to-C transition on codon 156 (exon 5) could be noticed in all sequences of control group and neoplastic mammary glands, one can conclude that the normal sequence is the TGC codon, and not the TGT codon mentioned by Chu and collaborators (1998). This polymorphism can be explained by an evaluative characteristic, since the research which the sequence is based on was conducted in Canada and the Netherlands (Chu et al. 1998), and not in Brazil. Unfortunately, to this moment, there is no publication about the normal sequence of canine p53 gene in Brazil.
1. Benjamin SA, Lee AC, Saunders WJ. 1999. Classification and behavior of canine mammary epithelial neoplasms based on life-span observations in beagles. Veterinary Pathology. 36:423-436.
2. Inoue M, Shiramizu K. 1999. Immunohistochemical detection of p53 and c-myc proteins in canine mammary tumours. Journal of Comparative Pathology. 120:169-175.
3. Kitchell BE. 1995. Mammary tumors, p.1098-1103. In: Bonagura J.D. Kirk´s Current Veterinary Therapy XII Small Animal Practice. 12th ed. W.B. Saunders, Philadelphia.
4. Misdorp W. 2002. Tumors of the mammary gland, p.575-606. In: Meuten DJ. Tumors in Domestic Animals. 4a. ed. Blackwell Publishing Company, Iowa.
5. De Nardi AB, Rodaski S, Sousa RS, Costa TA, Macedo TR, Rodigheri SM, Rios A, Piekarz CH. 2002. Prevalência de neoplasias e modalidades de tratamentos em cães, atendidos no Hospital Veterinário da Universidade Federal do Paraná. Archives of Veterinary Science. 7:15-26.
6. Graham JC, Myers RK. 1999. The prognostic significance of angiogenesis in canine mammary tumors. Journal of Veterinary Internal Medicine. 13:416-418.
7. Knapp DW, Waters DJ, Schmidt BR. 2004. Tumores do sistema urogenital e das glândulas mamárias, p.574-580. In: Ettinger, S.J.; Feldman, E.C. Tratado de Medicina Interna Veterinária. 5th ed. Guanabara Koogan S.A., Rio de Janeiro.
8. Camargo AA, Neto ED, Simpson AJG. 1999. Mutação e câncer, p.111-123In: Rossi BM & Pinho M. Genética e Biologia Molecular para o Cirurgião. 1a.ed. Lemar, São Paulo.
9. Lopes A, Nakagawa WT, Mello CAL. 1999. Oncogenes e genes supressores de tumor: um equilíbrio necessário, p.125-142. In: Rossi BM & Pinho M. Genética e Biologia Molecular para o Cirurgião. 1a.ed. Lemar, São Paulo.
10. Silva RLA. 2004. Oncogenes e genes supressores de tumor, p.29-42 In: Ferreira CG & Rocha JC. Oncologia Molecular. 1a.ed. Editora Atheneu, São Paulo.
11. Levine AJ, Perry ME, Chang A, Silver A, Dittmer D, Wu M, Welsh D. 1994. The 1993 Walter Hubert Lecture: The role of the p53 tumor suppressor gene in tumorigenesis. British Journal of Cancer. 69:409-416.
12. Lee CH, Kweon OK. 2002. Mutations of p53 tumor suppressor gene in spontaneous canine mammary tumors. Journal of Veterinary Science. 3:321-325.
13. Lana SE, Rutteman GR, Withrow SJ. 2007. Tumors of the mammary gland, p. 619-636. In: Withrow SJ & Vail DM. Withrow & MacEwwen´s Small Animal Clinical Oncology. 4th ed. Saunders Elsevier, Philadelphia.
14. Lee CH, Kim WH, Lim JH, Kang MS, Kim DY, Kweon OK. 2004. Mutation and overexpression of p53 as a prognostic factor in canine mammary tumors. Journal of Veterinary Science. 5:63-69.
15. Muto T, Wakui H, Takahashi S, Maekawa T, Masaoka T, Ushigome S, Furusato M. 2000. P53 gene mutations occurring in spontaneous benign and malignant mammary tumors of the dog. Veterinary Pathology. 37:248-253.
16. Pearson H, Stirling D. 2003. DNA extraction from tissue, p. 33-34. In: Bartlett JMS & Stirling D. PCR Protocols. 2nd ed. Humana Press, Totowa.
17. Sambrook J, Russel DW. 2001. Molecular Cloning: A Laboratory Manual, 720p . 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
18. Chu LL, Rutteman GR, Kong JM, Ghahremani M, Schmeing M, Misdorp W, Van Garderen E, Pelletier J. 1998. Genomic organization of the canine p53 gene and its mutational status in canine mammary neoplasia. Breast Cancer Research and Treatment. 50:11-25.
19. Mayr B, Reifinger M, Brem G, Feil C, Schleger W. 1999. Cytogenetic, ras, and p53: studies in cases of canine neoplasms (hemangiopericytoma, mastocytoma, histiocytoma, chloroma). The Journal of Heredity. 90:124-128.
20. Van Leeuwen IS, Hellmén E, Cornelisse CJ, Burgh BVD, Rutteman GR. 1996. p53 mutations in mammary tumor cell lines and corresponding tumor tissues in the dog. Cancer Research. 16:3737-3744.
21. Wakui S, Muto T, Yokoo K, Yokoo R, Takahashi H, Masaoka T, Hano H, Furusato M. 2001. Prognostic status of p53 gene mutation in canine mammary carcinoma. Anticancer Research. 21:611-616.