Related Experiment Videos
Low frequency of ras gene mutations in neuroblastomas, pheochromocytomas, and medullary thyroid cancers
J F Moley1, M B Brother, S A Wells
1Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110.
Abstract:
Little is known about the prevalence and significance of ras gene activation in neural crest tumors such as neuroblastomas, pheochromocytomas, and medullary thyroid cancers (MTCs). Therefore, we analyzed DNA from 10 human neuroblastoma cell lines and 10 primary human pheochromocytomas for activating mutations in N-ras, H-ras, and K-ras. We also studied DNA from 24 primary neuroblastomas and 10 MTCs for N-ras mutations. ras genes were analyzed by direct sequencing of specific DNA fragments amplified by the polymerase chain reaction. With the exception of the SK-N-SH cell line, the examined ras gene sequences were normal in all the neuroblastomas, pheochromocytomas, and MTCs tested. A single point mutation was identified at codon 59 (GCT(ala)----ACT(thr)) in one N-ras allele in an SK-N-SH subline. Interestingly, this mutation is different from the activating codon 61 mutation which resulted in the initial identification of N-ras from SK-N-SH DNA. Therefore, we analyzed the sequences of earlier passages and sublines of the SK-N-SH cell line, but mutations at codon 59 or 61 were not detected, suggesting that neither mutation was present in the primary tumor. Our results indicate that N-ras mutations may occur spontaneously during in vitro passage of cell lines but rarely, if ever, occur in primary neuroblastomas, pheochromocytomas, and MTCs. In addition, we have not found H-ras or K-ras mutations in any neuroblastoma cell line or primary pheochromocytoma.
Insights
Ras gene mutations are rare in primary neural crest tumors like neuroblastomas and pheochromocytomas. Activating ras mutations were not found in these tumors, suggesting they occur spontaneously in cell lines during in vitro passage.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Ras gene family (N-ras, H-ras, K-ras) mutations are implicated in various cancers.
- The role of ras gene activation in neural crest tumors, including neuroblastomas, pheochromocytomas, and medullary thyroid cancers (MTCs), is not well understood.
Purpose of the Study:
- To investigate the prevalence and significance of ras gene mutations in primary neuroblastomas, pheochromocytomas, and MTCs.
- To analyze N-ras, H-ras, and K-ras gene sequences for activating mutations in these tumor types.
Main Methods:
- DNA was extracted from 10 human neuroblastoma cell lines, 10 primary human pheochromocytomas, 24 primary neuroblastomas, and 10 MTCs.
- Polymerase chain reaction (PCR) was used to amplify specific ras gene fragments.
- Direct sequencing was performed on amplified DNA fragments to detect mutations.
Main Results:
- Activating ras gene mutations were not detected in any of the tested primary neuroblastomas, pheochromocytomas, or MTCs.
- A single N-ras mutation at codon 59 was found in one SK-N-SH neuroblastoma cell line subline, which was absent in earlier passages, suggesting in vitro artifact.
- No H-ras or K-ras mutations were found in neuroblastoma cell lines or primary pheochromocytomas.
Conclusions:
- Ras gene mutations are uncommon in primary neuroblastomas, pheochromocytomas, and MTCs.
- Observed mutations in cell lines likely arise spontaneously during in vitro culture rather than being present in the original tumors.
- This study highlights the importance of distinguishing between mutations in primary tumors and those acquired during cell line propagation.