Identification of Canine Transmissible Venereal Tumor Cells Using in Situ Polymerase Chain Reaction and the Stable Sequence of the Long Interspersed Nuclear Element

Abstract

Abstract. Canine transmissible venereal tumor (CTVT) is a unique tumor that can be transplanted across the major histocompatibility complex (MHC) barrier by viable tumor cells. In dogs, CTVT grows progressively for a few months and then usually regresses spontaneously. A long interspersed nuclear element (LINE) insertion is found specifically and constantly in the 5Ј end of the CTVT cell c-myc gene, outside the first exon. The rearranged LINE-c-myc gene sequence has been used with polymerase chain reaction (PCR) to diagnose CTVT. However, in CTVT cells, the total length of the inserted LINE gene is not constant. In this experiment, variation in the inserted LINE gene was studied to determine which parts of the LINE sequence can be used as primers to identify CTVT cells with in situ PCR (IS PCR). The LINE gene was inserted between the TATA boxes in the promoter region of c-myc. In CTVT cells, deletions of different lengths are frequent in this gene. However, the 550-bp segment at the 5Ј end of the LINE-c-myc gene was stable. Thus, primers were designed to cover the stable 0.55-kb segment from the 5Ј end outside the first exon of the c-myc gene to the 5Ј end of LINE gene stable segment. With these primers and IS PCR, individual CTVT cells in formalin-fixed tissue sections and CTVT cultures were identified. Cells from other canine tumors were negative for this gene. In addition, the CTVT-specific, 0.55-kb segment was not found in any spindle-shaped cells from progressive or regressive phase CTVT. The IS PCR technique also did not detect any positive spindle-shaped cells in CTVT cell cultures. Thus, fibroblastic terminal differentiation is less likely to be a mechanism for spontaneous regression of CTVT cells.Canine transmissible venereal tumor (CTVT) is a naturally occurring tumor transmitted by viable tumor cells. CTVT cells are small and round, with a large nucleolus and prominent cytoplasm in which cytoplasmic vacuolization is common. Although the origin of CTVT cells has not been well defined, studies using immunohistochemical staining indicate that the tumor origin is histiocytic. 25,27 The normal chromosome number for dogs is 78, and only the sex chromosomes are metacentric. In CTVT cells, the number of chromosomes ranges from 57 to 59, and 16 to 18 chromosomes are metacentric. 28,31 Transposable elements (TE), or transposons, are a group of discrete genes that transport themselves directly from one site to another within the genome. 26 Transposon activity has been associated with chromosomal rearrangements, such as deletions, duplications, inversions, and translocations, and the recombination of host genomes. A 1.6-kb-long interspersed nuclear element (LINE), 20 a TE sequence or retroposon, was found in CTVT cells, inserted in the 5Ј end of the c-myc gene, outside the first exon. The truncated LINE-1 element has been sequenced, 7 and it contains a 1,378-bp insert flanked by a 10-bp direct repeat upstream of the c-myc gene. The DNA sequence of the

Clonally transmissible cancers in dogs and Tasmanian devils

“…This genomic rearrangement has been identified in a large set of globally distributed CTVT tumors, but not in any other canine tissue, and is now considered diagnostic evidence for CTVT (Katzir et al, 1987;Amariglio et al, 1991;Choi et al, 1999;Chu et al, 2001b;Choi and Kim, 2002;Liao et al, 2003b;Murgia et al, 2006;Park et al, 2006;Vazquez-Mota et al, 2008;Rebbeck et al, 2009). It is possible that this rearrangement was present in the germ line of the CTVT founder, that it occurred somatically during the development of the founding CTVT tumor or that it occurred somatically in a CTVT clone that has subsequently achieved global distribution.…”

Concise Review: Transmissible Animal Tumors as Models of the Cancer Stem-Cell Process

“…For CTVT, cytogenetic analysis of tumors from various geographical locations all show a similar tumor karyotype (2n ¼ 57-59) distinct from the constitutional canine karyotype (2n ¼ 78) [1,7]. Furthermore, in all cases of CTVT analyzed, the tumor cells harbor a unique LINE element insertion, near the cmyc locus, absent in the natural canine germline genome [3,27]. This is a strong diagnostic marker in pathology practice [18].…”

Canine transmissible venereal tumor. The oldest clonal lineage known in nature

“…Na terceira linha de observação, Katzir et al (1985) utilizando o método de Southern Bloting (no qual se verifica se uma determinada sequência de DNA está ou não presente em uma amostra de DNA analisada), examinaram o estado do oncogene cmyc em amostras de TVTC, demonstrando uma longa inserção de um elemento nuclear intercalado (Long interspersed nuclear elements -LINE-1), próximo ao c-myc do tumor. Corroborando com este trabalho, Liao et al (2003) Na edição da Science, os pesquisadores descreveram a primeira sequência do genoma completo da TVTC, obtida a partir dos tumores de um cão de campo aborígene australiano de raça aleatória e um cão Cocker Americano de raça pura no Brasil. A análise de sequência genômica revelou aproximadamente 1,7 milhão de variantes somáticas compartilhadas entre os dois tumores, que se supõe ter surgido antes da separação dos tumores por limites geográficos.…”