INTRANUCLEAR POSTTRANSCRIPTIONAL DOWN-REGULATION RESPONSIBLE FOR LOSSOF A KERATIN DIFFERENTIATION MARKER IN TUMOR PROGRESSION

Apparent loss of differentiation markers characterizes advanced malign ant neoplasms. Post-transcriptional down-regulation of keratin message to levels undetectable with a partial cDNA probe to rat keratin K5 ha d been observed in anaplastic cells (T952/F7) derived from benign kera tin-producing cells (A5P/B10) (1). The entire fifth introns of both th e K5 and K6 genes were generated from rat genomic DNA by PCR to define expression of these closely related proteins. Sequencing of the PCR p roducts revealed 84% homology in the K5 and K6 exon regions included, but absence of any homology in the introns. Active transcription of K5 could be demonstrated in the anaplastic cells with reverse transcript ion of nuclear RNA (RTn-PCR) by the presence of PCR-generated products confirmed by sequencing as unspliced and spliced transcripts of rat K 5. In situ hybridization with ssDNA probes for the spliced message fro m this region of the K5 gene demonstrated a punctate distribution in t he cytoplasm of the benign cells and absence of any detectable message in the anaplastic derivatives. ssDNA probes for the unspliced transcr ipt containing intron 5 and the same flanking exon sequences as the sp liced probe detected transcription of hnRNA in the anaplastic cells as discrete signals confined to the nuclear compartment. These results s how that failure to express mRNA for a differentiation marker in the c ytoplasm of anaplastic cells can be due to a mechanism operating in th e nuclear compartment after gene transcription and indicate that the m echanism functions shortly after splicing of the transcript.