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Updated in 2020/8/12 下午 12:04:33      Viewed: 160 times      (Journal Article)
Clinical cancer research : an official journal of the American Association for Cancer Research 9 (17): 6419-25 (2003)

Detection of mutations in the mitogen-activated protein kinase pathway in human melanoma.

Janivette Alsina , David H Gorsk , F Joseph Germino , Weichung Shih , Shou-En Lu , Zhi-Gang Zhang , Jin-Ming Yang , William N Hait , James S Goydos
ABSTRACT
Recent studies suggest that activating point mutations in B-RAF may commonly occur in melanoma. We devised a method to detect point mutations in heterogeneous tissues containing both wild-type and mutant B-RAF and N-RAS genes by using site-directed mutagenesis to introduce new restrictions sites in the cDNA sequence when the specific point mutations are present. We used this technique to determine the incidence of mitogen-activated protein kinase (MAPK) mutations in human melanoma.We screened 85 melanoma samples for the most common B-RAF and N-RAS mutations found in melanoma using a site-directed mutagenesis-based detection technique. Western blotting was used to evaluate downstream up-regulation of the mitogen-activated protein kinase pathway in these tissues.Thirty-three samples (7 of 25 primaries, 15 of 25 regional metastases, 5 of 25 nodal metastases, and 6 of 10 distant metastases) harbored the V599E B-RAF mutation (39%), 12 contained a Q61R N-RAS mutation and 5 a Q61K N-RAS mutation. Western blotting with antiphosphorylated extracellular signal-regulated kinase 1/2 antibodies demonstrated up-regulation of the MAPK pathway in samples containing activating B-RAF or N-RAS mutations compared with wild-type samples. This method of detection was sensitive and specific with no false positives.Activating mutations of the MAPK pathway were present in approximately 60% of samples tested and caused activation of this cellular pathway that appears to be important in the pathogenesis of melanoma.
ISSN: 1078-0432