Intranuclear post-transcriptional down-regulation responsible for loss of a keratin differentiation marker in tumour progression

Apparent loss of differentiation markers characterizes advanced malignant neoplasms. Post-transcriptional downregulation of keratin message to levels undetectable with a partial cDNA probe to rat keratin K5 had been observed in anaplastic cells (T952/F7) derived from benign keratin-producing cells (A5P/B10) (1). The entire fifth introns of both the K5 and K6 genes were generated from rat genomic DNA by PCR to define expression of these closely related proteins. Sequencing of the PCR products 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 transcription of nuclear RNA (RTn-PCR) by the presence of PCR-generated products confirmed by sequencing as unspliced and spliced transcripts of rat K5. In situ hybridization with ssDNA probes for the spliced message from this region of the K5 gene demonstrated a punctate distribution in the cytoplasm of the benign cells and absence of any detectable message in the anaplastic derivatives. ssDNA probes for the unspliced transcript containing intron 5 and the same flanking exon sequences as the spliced probe detected transcription of hnRNA in the anaplastic cells as discrete signals confined to the nuclear compartment. These results show that failure to express mRNA for a differentiation marker in the cytoplasm of anaplastic cells can be due to a mechanism operating in the nuclear compartment after gene transcription and indicate that the mechanism functions shortly after splicing of the transcript.