Studies on the Molecular Mechanisms of Tumorigenesis in Neurofibromatosis Type 1

Neurofibromatosis type 1 (NF1) is one of the most common inherited autosomal dominant disorders, with an estimated incidence of 1 per 3,500 births. NF1 is characterized by a high incidence of benign and malignant tumors attributed to loss of function of NF1. NF1 gene encodes neurofibromin, a tumor suppressor with Ras-GAP activity that negatively regulates p21-RAS signaling. Neurofibromin deficiency typically causes chronic activation of Ras, which is thought to be a major contributor to manifestation of NF1. The cardinal features of NF1 are cafe-au-lait (CAL) spots, neurofibromas, freckling of the axillary or inguinal region, Lisch nodules, optic nerve glioma, and bone dysplasias. NF1 is notable for the existence of a gradual malignant degeneration of normal and/or benign tumor cells. Although TP53 mutation and chromosome 17p deletions have been detected in malignant tumors, the mechanism of progression to malignancy has not yet been clarified. In this study, we have studied the characterization of tumor cells derived from a Korean patient with NF1. Among three major RASs, K-RAS, H-RAS and N-RAS, H- and K-RAS expression were detected by Western blotting. Particularly, significant up-regulation of K-RAS was detected in the malignant NF1 cells. RAS activation test revealed a large amount of activated form (GTP-RAS) of K- and H-RAS in the malignant NF1 cells. Mutation analysis showed that the recurrent nonsense mutation, Y2264X, was identified in the NF1 gene, but no mutations were detected in three RAS and TP53 genes. Next, we carried out Western blot analysis of the proteins related Ras pathway and apoptosis using the isolated normal, benign and malignant NF1 cells. Expression of the phosphorylated-ERK and Bcl-xL were increased in the malignant NF1 cells. Interestingly, we observed lower RASA1 gene expression in the malignant cells compared to normal and benign cells in both mRNA and protein levels by RT-PCR and Western blot analysis. RASA1 gene encodes p120RasGAP, which is first described as a negative regulator of Ras in 1988. To find out the reason why RASA1 gene was down-regulated in the malignant cells, we had checked methylation status in CpG island on the promotor region of RASA1. But, we could not detect methylation of RASA1. To clarify the cellular functions of p120RasGAP in the malignant NF1 cells, we have used vector-mediated RASA1 gene expression. Western blot analysis after RASA1 over-expression in the NF1 malignant cells revealed the decreased expression of H- and K-Ras and p-ERK protein. In addition, high level expression of cleavaged-caspase 3 and following high percentage of apoptotic cell death of the NF1 malignant cells were detected, suggesting that RASA1 is a strong candidate gene involved in the tumorigenesis and malignant transformation of NF1. A further functional analysis of p120RasGAP may lead to not only a better understanding the mechanisms of tumorigenesis and malignant transformation but also an effective application to the therapeutic treatment of NF1.

Author dc:creator

• 이, 수진

Contributors dc:contributor

• 정, 선용
• 김, 현주
• 대학원 의학과
• 200624307

• 신경섬유종증
• 세포자멸사
• 뉴로파이브로민
• GTPase activating protein(GAP)
• p120RasGAP
• Ras
• RASA1
• Apoptosis
• neurofibromin
• NF1

Language dc:language

ko

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000006846
000000006846

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/1836