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Updated: Aug 12, 2026
Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line
Published on: February 17, 2016
Characterization of ret proto-oncogene mRNAs encoding two isoforms of the protein product in a human neuroblastoma
1Carcinogenesis Division, National Cancer Center Research Institute, Tokyo, Japan.
Abstract:
The ret proto-oncogene expresses four major mRNA species of different lengths in human malignant cell lines and rat tissues. We isolated ret proto-oncogene cDNA clones from a cDNA library of a human neuroblastoma line, Nagai, which over-expressed these mRNAs. Four cDNA clones differing from each other in their 3' portions were analysed. The sequence of the region common to the cDNA clones is essentially identical to a reported cDNA sequence derived from THP-1 monocytic leukemia cells, that encodes a protein with characteristic features of receptor-type tyrosine kinase. From the 3' heterogeneity, two isoforms of the ret proto-oncogene product of 1072 and 1114 residues that differed from each other in their 9 and 51 C-terminal amino acids are predicted. Comparison of the structures of cDNA clones with that of the genomic clone showed that the 3' heterogeneity is produced by alternative polyadenylation and splicing of mRNA. Northern blot analysis using various fragments of cDNA indicated that the 4.5 kb, 3.9 kb and possibly 7.0 kb transcripts may encode a protein of 1072 residues, while the 6.0 kb transcript and a (minor) 4.6 kb transcript may encode a protein of 1114 residues.
Insights
The ret proto-oncogene produces multiple mRNA variants, leading to two distinct protein isoforms. This 3' mRNA heterogeneity arises from alternative polyadenylation and splicing, impacting receptor-type tyrosine kinase expression.
Area of Science:
- Molecular Biology
- Genetics
- Oncology
Background:
- The ret proto-oncogene is known to express multiple mRNA species.
- Overexpression of these mRNAs is observed in human malignant cell lines and rat tissues.
Purpose of the Study:
- To isolate and analyze ret proto-oncogene cDNA clones from a human neuroblastoma cell line.
- To understand the structural basis and functional implications of ret mRNA heterogeneity.
Main Methods:
- Isolation of cDNA clones from a human neuroblastoma cell line (Nagai).
- Sequence analysis of cDNA clones and comparison with genomic sequences.
- Northern blot analysis to examine transcript sizes and their correlation with protein isoforms.
Main Results:
- Four cDNA clones with differing 3' portions were identified, sharing a common region encoding a receptor-type tyrosine kinase.
- Two distinct ret proto-oncogene product isoforms (1072 and 1114 amino acids) are predicted due to 3' heterogeneity.
- Alternative polyadenylation and splicing of mRNA were identified as mechanisms generating this heterogeneity.
- Specific mRNA transcript sizes (4.5, 3.9, 7.0 kb) correlate with the 1072-residue isoform, while (6.0, 4.6 kb) correlate with the 1114-residue isoform.
Conclusions:
- The 3' heterogeneity in ret proto-oncogene mRNA results in the production of two distinct protein isoforms.
- Alternative polyadenylation and splicing are key regulatory mechanisms controlling ret proto-oncogene expression.
- Understanding these isoforms and their regulation is crucial for comprehending ret proto-oncogene function in malignancy.
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