Isolation of genes related to malignant transformation of thyroid tumors and gene therapy for undifferentiated carcinoma of the thyroid

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To elucidate the molecular basis for malignant transformation of thyroid tumors, changes in mRNA and genome were analyzed with the differential display method and arbitrarily primed (AP)-PCR method.1. Differential display methodRNA was extracted from a thyroid follicular adenoma cell line, three thyroid papillary carcinoma cell lines, a thyroid follicular carcinoma cell line, and two undifferentiated thyroid carcinoma cell lines. Bands with different strength of signals among cell lines in the differential display method were marked in the autoradiography and cut out from the gels. They were amplified, cloned, and sequenced. Half of the clones were the known genes such as ribosome RNA,mitochondrial gene, fibronectin, R-ras, a subunit of proteasome, 23 kD highly basic protein. Half of the clones were unknown genes. Northern blot analysis using these clones as probes showed that any clones have the expression levels less than 2-fold among cell lines.2. AP-PCR methodGenomic DNAs were extracted from 12 thyroid tumor cell lines. DNAs were amplified in conditions that an arbitrarily oligonucleotide with 20 uncleotides was used as a primer and DNA templates were amplified with the low annealing temperature in the first 5 cycles. Patterns of bands among cell lines in the autoradiograph were compared. A loss of signal suggesting deletion was found in a thyroid papillary carcinoma cell line, however, an apparent deletion was not detected in southern blot analysis using the cloned DNA as a probe. In addition, no signals suggesting gene amplification in cell lines were not found.