Detection of neoplastic cells in blood of miniature pigs with hereditary melanoma

Abstract

Tyrosine, a key enzyme of melanin biosynthesis, is widely used as a specific marker for the detection of dissemination or metastatic melanoma cells in peripheral blood and other tissues like lymph node or bone marrow, which are normally tyrosinase negative. The amplification of tyrosinase-specific mRNA by means of RT-PCR is a sensitive technique capable of detecting a single tumour cell in 5-10 ml of whole blood. We have utilised this method to analyse the peripheral blood of laboratory miniature pigs with advanced cutaneous melanoma for the presence of tumor cells. This highly invasive hereditary malignancy can serve as an experimental model for the study of melanoma development and dissemination. For amplification of porcine gene, oligonucleotide primers derived from the sequence of human tyrosinase were used. These primers amplified fragment of the predicted length and restriction enzyme digestion confirmed their homology with the sequences of human tyrosinase gene. After the second round of amplification, tyrosinase could be detected up to the amount of 1 × 10-5 µg of total RNA isolated from porcine melanoma per 1 µg of control RNA. Blood samples from eight animals with advanced melanoma and from five non-melanoma control animals were examined for tyrosinase expression. Tyrosinase mRNA was detected in five samples from animals with malignant melanoma. Non-melanoma control animals gave negative results.

Keywords

• melim strain
• melanoblastoma
• circulating tumour cells
• tyrosinase
• tyrosinase-specific mrna