研究犬傳染病性花柳病瘤規避同種移植排斥之機制(3/3)

Canine transmissible venereal tumor (CTVT) grows progressively (P-phase) in the host and then spontaneously regresses (R-phase). The mechanisms behind the transition from the P-to R-phases ow not well understood. In this study in order to determine the prol(feration characteristics of CTVE we evaluated telomerase activity and enumerated nuclear organizing regions (AgNOR) and proliferating cell nuclear antigen (PCNA). It was found that CTV7¡¦ cells from the P-and R-phases were both positive for telonserase activity, although it was lower in the R-phase. Evaluations of telomerase activity should take into account the stage of misosis. Although, in the majority of cases, telomerase activity can be used to d(fferentiare between benign and malignant tumors in dogs, other factors or markers should also be used to obtain accurate diagnoses. The PCNA -positive rate and the number and area of AgNOR per cell increased much more in the F-phase than the R-phase. However, the AgNOR value.c were always higher. Thus, the AgNOR count can he used to distinguish the P-and R-phssses of CTVT In addition, mitotic figures were much higher in number in the F-phase as compared to the R-phase. We believe that, during spontaneous regression of CTVT cells, slow tumor cell proliferation must contribute to the decrease in rumor size. However, shortening of inmor cell telomcres is not directly involved in this process, Other factors, such as expression of MHC antigens on CTVT cells, humoral immunity, cytokines released by the inflammatory cells an4 especially, tumor infiltrating lymphocytes may contribute to CTVT regression. Canine transmissible venereal tumor (CTVT) is transmitted naturally to susceptible animals by a rare mechanism, the transplantation of viable tumor cells. Although CTVT grows aggressively in dogs, it may undergo spontaneous regression (1-4). CTVT seldom metastasizes. However, metastasis to other tissues and organs, especially regional lymph nodes, has Con-espondence to: Rea-Min Chu, Department of Veterinary Medicine, National Taiwan University, 142 Chou-San Road, Taipei. Taiwan 106, ROC. e-mail; redman@ns.cc.ntu.edu.tw 11ev Words: Transmissible venereal tumor, telomerase activity, AgNOR, PCNA, TUNEL stain, proliferation. been observed in animals (4-7). In CTVT cells, a transposable genetic element sequence, known as the long interspersed DNA element (LINE) (8), has been found inserted in the 5’ end, near the first exon of the e-myc gene. Choi et at. (9) sequenced truneated LINE-f elements and found that CTVT cells contain a 1378 bp insert flanked by a 10 bp direct repeat upstream of the c-mye gene. This unique insertion has been useful for diagnosing CTVT (10). Telomeres are specialized structures at the end of eukaryotic chromosomes. Telomere shortening may he the mitotic clock that determines the number of divisions normal cells can un&rgo (11) and may be a signal for replicative senescence (12). In contrast, the telomeres of immortal cells exhibit no net decrease in length, suggesting that maintenance of the telomeres is essential for cells to avoid replicative senescence (13). However, in tibroblast-origin tumors, telomerase is insufficient for malignant transformation (14). In humans, telomerase has been studied for early cancer detection (15) and for monitoring the effect of chemotherapy on cancer (16). There are only a few reports of telomerase activity in vertebrates other than mice and humans, though recenily telomerase activity was reported in some neoplastic tissues of dogs (17,18). The great majority of malignant tumors arc positive for telomerase activity, while benign tumors and normal tissues are negative. Thus, telomerase activity can be used to diagnose malignant tumors in dogs. In this study, we used the telomerase activity repeat amplification protocol (TRAP) assay to evaluate telomerase activity in the P-and R-phase CTVT. Argyrophilic nucleolar organizer regions (AgNORs), highly- specialized loops of ribosomal DNA, are found in the telomeric regions of chromosomes 7, 8, 27 and, sometimes, 38 of dogs (19). AgNORs are involved in the transcription of ribosemal RNA and the assembly of the ribosomal subunit precursors (20). They are widely used to estimate the level of cellular and nuclear activity (21,22) and have been used to evaluate the prognosis of CTVT (23). Proliferating cell nuclear antigen (PCNA) is an evolutionarily highlyconserved, 36kDa, non-histone nuclear protein. FCNA is a reliable marker that is commonly used to evaluate S-phase activity during mitosis (22,24,25). In this study, we characterized the proliferation pattern of CTVT in terms of PCNA and AgNOR counts and area, mitotic and telomerase activities in the tumor cells