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Chain-terminating mutations in the APC gene lead to alterations in APC RNA and protein concentration
V Gismondi1, P Stagnaro, S Pedemonte
1Laboratorio di Oncologia Sperimentale, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy.
Abstract:
APC mutations introduce premature stop codons into the open reading frame of the gene, leading to the formation of truncated tumor suppressor proteins. Both RNA and protein levels are likely to be profoundly altered by such nonsense mutations. To test this hypothesis, Western blotting and RT-PCR strategies were used to characterize mutant and normal APC protein and APC RNA concentrations in lymphoblastoid cell lines from 22 unrelated polyposis patients carrying different APC mutations. Variable levels of truncated APC peptides were observed in 14 of 14 cell lines with APC mutations within exon 15. No truncated APC protein was detected in six of eight cell lines with APC mutations located 5' of exon 15. Mutations located in exon 15 showed mutant RNA underrepresentation in four of eight cell lines, whereas mutations located 5' of exon 15 showed RNA reduction in five of six cell lines. These findings indicate that a two- to threefold decrease in RNA concentration is common when APC alleles carry chain-terminating mutations. They also suggest that the severe decrease of truncated APC protein observed in some cell lines is due to mechanisms acting at the protein level.
Insights
APC mutations cause truncated tumor suppressor proteins, altering RNA and protein levels. Research shows decreased RNA is common, with protein level changes suggesting post-transcriptional regulation.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Adenomatous polyposis coli (APC) gene mutations, specifically nonsense mutations, lead to truncated tumor suppressor proteins.
- These mutations are implicated in various cancers, including polyposis syndromes.
- The impact of nonsense mutations on both RNA and protein levels of APC requires detailed investigation.
Purpose of the Study:
- To investigate the effects of APC nonsense mutations on APC RNA and protein concentrations.
- To correlate mutation location within the APC gene with observed RNA and protein alterations.
- To elucidate the mechanisms underlying altered protein expression in APC-mutated cell lines.
Main Methods:
- Western blotting was employed to quantify mutant and normal APC protein levels.
- Reverse Transcription Polymerase Chain Reaction (RT-PCR) was utilized to assess APC RNA concentrations.
- Analysis was performed on lymphoblastoid cell lines from 22 unrelated polyposis patients with diverse APC mutations.
Main Results:
- Truncated APC peptides were detected in all 14 cell lines with mutations in exon 15.
- No truncated APC protein was found in 6 of 8 cell lines with mutations upstream of exon 15.
- RNA reduction (two- to threefold decrease) was observed in multiple cell lines, particularly with mutations upstream of exon 15.
Conclusions:
- APC nonsense mutations commonly lead to a decrease in APC RNA levels.
- The absence of truncated APC protein in some cases suggests post-transcriptional regulatory mechanisms.
- Mutation location within the APC gene influences both RNA stability and protein expression.