Alternative and aberrant messenger RNA splicing of the mdm2 oncogene in invasive breast cancer.

mdm2 is part of a complex mechanism that regulates the expression of p53 as well as the function of Rb, p19ARF, and other genes. In humans, mdm2 dysregulation is associated with gene amplification. This study was undertaken to characterize altered mdm2 expression in a cohort of 38 invasive breast cancers and 9 normal breast specimens. Reverse-transcription PCR with primers spanning the entire open reading frame of the mdm2 gene in breast tissue RNA samples generated PCR products of full-length mdm2 (1526 bp) as well as smaller products (653, 281, 254, and 219 bp). Sequence analysis demonstrated that the 653-bp product was an alternatively spliced product (defined as splicing at the exon/intron boundary consensus sites), whereas the 281, 254, and 219 bp mdm2 products were aberrantly spliced products (splicing at sites not considered to be exon/intron boundary sites). Reverse-transcription-PCR with normal breast tissue RNA samples yielded only the 1526-bp product in five samples and the 1526-bp product and the 653-bp product in four samples. The 653-bp alternatively spliced product was expressed in 21% of breast cancers, and the smaller, aberrantly spliced mRNA products (281 bp, 254 bp, and/or 219 bp) were expressed in 16% of breast cancers. The protein products predicted by the alternatively spliced mRNAs and the aberrantly spliced mRNAs lacked either the entire binding domain for p53 or the majority of the binding domain for p53. Immunohistochemical analysis of HER2/neu (c-erbB2), estrogen receptor, progesterone receptor, epidermal growth factor receptor, and p53 protein was performed. p53 sequence alterations were identified by mismatch detection and confirmed by p53 oligonucleotide microarray technology. An association was demonstrated between the expression of aberrantly and/or alternatively spliced mdm2 mRNAs and a lack of progesterone receptor. An association was also demonstrated between mdm2 aberrantly and/or alternatively expression products and the presence of p53 tumor suppressor gene mutations. mdm2 is transcribed from two different promoters: one, p53-dependent, and the other, p53-independent. The 5' untranslated region of the transcripts was evaluated to determine the promoter usage in each breast cancer specimen. No correlation was observed between mdm2 splice products and promoter usage. The presence of aberrant expression products of mdm2 in breast cancer specimens was correlated with a shortened overall patient survival. These observations suggest that mdm2 expression is altered in invasive breast cancer and is associated with more aggressive disease.

faint sausage encodes a novel extracellular protein of the immunoglobulin superfamily required for cell migration and the establishment of normal axonal pathways in the Drosophila nervous system.

We examined the structure of the nervous system in Drosophila embryos homozygous for a null mutation in the faint sausage (fas) gene. In the peripheral nervous system (PNS) of fas mutants, neurons fail to delaminate from the ectodermal epithelium; in the central nervous system (CNS), the positions of neuronal cell bodies and glial cells are abnormal and normal axonal pathways do not form. Sequence analysis of fas cDNAs revealed that the fas protein product has characteristics of an extracellular protein and that it is a novel member of the immunoglobulin (Ig) superfamily. In situ hybridization demonstrated that fas transcripts are expressed throughout the embryo but they are in relatively high concentrations in the lateral ectoderm, from which the peripheral nervous system delaminates and in the CNS. Antiserum directed against Fas protein was found to stain neurons but not glia in the CNS. We conclude that fas encodes a protein that, in the developing nervous system, is present on the surface of neurons and is essential for nerve cell migration and the establishment of axonal pathways.