IMPROVED DIAGNOSIS OF TUMORS IN COMPANION ANIMALS USING PCR METHOD

本研究检测了erbB 1、B2、B3、B4和c-kit原癌基因在犬和猫乳腺肿瘤标本中的表达，以评价其作为肿瘤标志物的潜在价值。通过比较几种哺乳动物cDNA核苷酸序列的同源性，合成了用于逆转录-聚合酶链反应（RT-PCR）的引物。检测11例乳腺肿瘤（腺癌、良恶性混合瘤）中c-kit转录本的表达。除肥大细胞瘤外，12例不同组织来源的肿瘤中，5例仅检测到低水平的RT-PCR产物，7例无明显扩增。正常乳腺组织中不表达erbB家族的任何一个成员，而肿瘤组织中常观察到erbB家族成员的多重表达。在3例猫卵巢癌中，也观察到erbB基因的频繁和多重表达。虽然在两个样本中检测到c-kit转录本，但在一个观察到所有erbB基因表达的样本中c-kit转录本不明显。这些结果表明，erbB家族和c-kit转录本的表达是诊断犬和猫乳腺肿瘤的有用标记，对猫乳腺癌细胞系的染色体分析表明，FMT-3和FMT-4细胞表现出特异性标记染色体中特定染色体的单体性或完全等位基因缺失。在所检查的猫肿瘤中，均未检测到抑制素的RT-PCR产物。应用荧光原位杂交技术将猫prohibitin基因定位于染色体B4长臂上。由于两种细胞系的核型均显示B4三体，因此研究了该基因在FMT-3细胞中的染色体定位。细胞中的一对B4显示出明显的双重杂交信号。

The expression of erbBl, B2, B3, B4 and c-kit proto-oncogene in canine and feline mammary tumor specimens was investigated to evaluate its potential usefulness as tumor markers. By comparing the homology among the nucleotide sequences of cDNA reported for several mammalian species, pairs of primers were synthesized for the reverse transcriptase-polymerase chain reaction (RT-PCR) method. The expression of c-kit transcript was detected in 11 mammary tumors (adenocarcinomas, benign and malignant mixed tumors). Of the 12 tumors originated from various tissues except mastocytomas, only low level of RT-PCR products iwre detected in 5 samples, whereas no apparent amplification was observed in 7 tumors. Any of erbB family was not expressed in normal mammary tissue, while the multiple expression was frequently observed in tumor samples. In three feline mammAry tumors, the frequent and multiple expression of erbB genes was also observed. Although c-kit transcript was detected in two samples, it was not apparent in one specimen in which the expressions of all of erbB genes were observed. These results indicate that the expression of erbB family and c-kit transcript is useful markers for the diagnosis of both canine and feline mammary tumors.The chromosome analysis of cell lines derived from feline mammary adenocarcinoma demonstrated that FMT-3 and FMT4 cells showed monosomy or complete alleic deletion of particular chromosomes which were included in specific marker chromosomes. In none of the feline tumors examined, RT-PCR product of prohibitin was detected. Feline prohibitin gene was mapped at the long arm of chromosome B4 by fluorescence in situ hybridization method. Because the karyotypes of both cell lines showed B4 trisomy, the chromosomal localization of the gene in FMT-3 cells was investigated. A couple of B4 in the cells was shown to have apparent doublet signals of hybridization.