Alternative splicing of neural-cell-adhesion molecule mRNA in human small-cell lung-cancer cell line H69.
The neural-cell-adhesion molecule (NCAM) is expressed in all small-cell lung cancers (SCLC) and in approximately 20% of non-small-cell lung tumors (non-SCLC). These NCAM-positive lung tumors have a poor prognosis compared with NCAM-negative tumors. Multiple NCAM protein isoforms are expressed from a single-copy gene as a result of alternative splicing and/or post-translational modifications. Therefore, we studied the NCAM isoforms expressed in a human small-cell lung-cancer cell line, H69. NCAM mRNA transcripts of 7.2, 6.7, 4.3 and 4.0 were detected in these cells on Northern blots. Since the various NCAM isoforms may have different biological properties, we performed a more precise examination of NCAM mRNAs, using polymerase chain reactions (PCR) with primers flanking the various NCAM exon boundaries. The shortest alternatively spliced sequence that we found was the trinucleotide AAG located between exon 12 and 13 in the so-called hinge region of the NCAM protein. This AAG trinucleotide was present in the majority of the NCAM mRNAs. A second alternatively spliced 30 nt-exon VASE (immunoglobulin-variable domain-like alternatively spliced exon) was present in all NCAM transcript isoforms at the exon 7/exon 8 junction. VASE resulted in the insertion of 10 amino acids into the 4th immunoglobulin-like loop of the NCAM protein. Within the limits of the PCR methodology, no evidence for the presence of mRNA containing exon 15, encoding the glycosyl-phosphatidylinositol-linked (GPI-linked) NCAM isoform in H69 cells was obtained. Considering that H69 cells express 2 major NCAM protein classes (NCAM-180 and NCAM-140), and that the VASE and AAG alternative mRNA splice variants result in minor differences in protein sizes, at least 8 polypeptide isoforms of NCAM might be expressed in H69 cells that contribute to the binding interactions of NCAM.
Moolenaar
C E
CE
Division of Tumor Biology, The Netherlands Cancer Institute, Amsterdam.
Pieneman
C
C
Walsh
F S
FS
Mooi
W J
WJ
Michalides
R J
RJ
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D018288:Carcinoma, Small Cell / Q000235:genetics*; D015816:Cell Adhesion Molecules, Neuronal / Q000235:genetics*; D005091:Exons; D052058:Hinge Exons; D006801:Humans; D007438:Introns; D008175:Lung Neoplasms / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D012334:RNA, Neoplasm / Q000737:chemistry* / Q000235:genetics; D014407:Tumor Cells, Cultured
D015816:Cell Adhesion Molecules, Neuronal; D012333:RNA, Messenger; D012334:RNA, Neoplasm