Isolation of differentially expressed genes in carcinoma of the esophagus

The genetic alterations that occur in the transformation of normal esophageal mucosa (NEM) to carcinoma of the esophagus (CAE) are not well understood. Differential display of mRNA is a recently described technique that uses reverse transcription and PCR to compare cDNA from paired normal and malignant tissue to determine whether there is either genetic loss (putative tumor suppressor gene) or overexpression (putative oncogene) in malignant cells. Our goal was to identify some of these genes from patients with CAE.

Specimens of NEM and corresponding CAE were obtained from patients at endoscopy or surgical resection and immediately snap frozen. Total RNA was isolated, reverse transcribed to cDNA, and PCR amplified with a predefined 10-mer oligonucleotide. The products were displayed on a polyacrylamide gel. Differential bands were isolated and sequenced and/or used as probes for Northern analysis.

Application of the differential display method resulted in the isolation of 49 cDNA clones from three patients with CAE. Sequencing of the clones has revealed five unique sequences not previously reported and one that has been identified as histone H3.3. Northern analysis of histone H3.3 has revealed overexpression in four of six CAEs but not the paired NEM. In addition, whereas only 5 of 13 normal human cell lines of various origins overexpressed this gene, 11 of 12 human cancer cell lines (9 of 9 adenocarcinomas) overexpressed it.

Differential display can be used to isolate potential oncogenes and tumor suppressor genes. We have identified five unique sequences and one known gene that may contribute to the development of CAE.