MOLECULAR ANALYSIS OF THE HUD GENE ENCODING A PARANEOPLASTIC ENCEPHALOMYELITIS ANTIGEN IN HUMAN LUNG-CANCER CELL-LINES

Small cell lung cancer (SCLC) is known to express the HuD protein, the neuronal antigen homologous to Drosophila Elav and Sxl genes involved in neuronal and sex development. HuD is the target of an immune respo nse including high titered antibodies causing paraneoplastic encephalo myelitis and sensory neuropathy. Because the p53 recessive oncogene is mutated and anti-p53 antibodies frequently occur in cancer patients, we wondered if the development of anti-HuD antibodies signaled the pre sence of HuD mutations in lung cancer. The HuD gene was mapped to chro mosome region 1p using a human-mouse hybrid cell panel. We confirmed t hat 26 of 46 cancer (43 lung cancer and 3 mesothelioma) cell lines exp ressed HuD mRNA and that this expression, as well as protein expressio n by Western blot, correlated strongly with the SCLC neuroendocrine ph enotype. Southern blot and single-strand conformation polymorphism ana lyses showed that HuD was not mutated in 78 lung cancers, including pa tients with the severe paraneoplastic syndrome. Northern blot analysis showed that lung cancer cell lines expressed two major mRNAs (43 and 4.0 kilobases) of HuD. We found the three previously described alterna tive spliced mRNA forms (HuDpro, HuD, and HuDmex). In addition, we als o found HuD mRNA had an alternative splicing form in its 5'-coding reg ion This alternative splice introduced 87 base pairs of sequence and a termination codon resulting in a predicted small, truncated protein ( 11 amino acids) reminiscent of the male-specific truncated protein in the Sex-lethal (Sxl) gene of Drosophila. However, mRNAs encoding both full-length and truncated proteins were expressed in all SCLCs. These results show that the HuD gene is not mutated in lung cancer, includin g tumors from patients producing anti-HuD antibodies, but HuD expressi on is an independent marker or determinant of the neuroendocrine diffe rentiation seen in SCLC.