RESUMO
Mouse Kit L575P, the ortholog of human KIT L576P, a common KIT mutation found in human melanoma was expressed in an immortalized but non-transformed mouse Ink4a-Arf-deficient melanocyte cell line. The resultant Ink4a-Arf-deficient Kit L575P-expressing melanocytes exhibited increased proliferation, the ability to grow in soft agar, and increased migration. When these cells were injected subcutaneously into NOD/SCID/gamma(c) mice, melanomas arose in 5 of 7 (71%) mice. One of seven mice (14%) injected with these cells developed metastatic disease. Evaluation of signal transduction pathways downstream of constitutively activated Kit L575P revealed striking activation of the phosphatidyl inositol 3-kinase (PI3K) pathway. Inhibition of the PI3K pathway pharmacologically or genetically abolished the transformation phenotypes gained by the L575P single mutant. These studies validate this Kit L575P-activated model of melanoma and establish the PI3K pathway as a dominant signaling pathway downstream of Kit in melanoma.
Assuntos
Melanoma/enzimologia , Melanoma/etiologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Animais , Benzamidas , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Dasatinibe , Ativação Enzimática/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Melanócitos/efeitos dos fármacos , Melanócitos/enzimologia , Melanócitos/patologia , Melanoma/patologia , Camundongos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologiaRESUMO
INTRODUCTION: Participant's adherence to use of study product is a major concern in microbicide clinical trials, which can impact on proving product efficacy. In a previously described assay, single-use microbicide applicators exposed to the vagina were tested by spraying the applicator with trypan blue dye, resulting in vaginal mucus staining on inserted applicators. As subjects in our Phase 3 trials return applicators only at quarterly visits, often mixing inserted and not-inserted applicators together in the same bag, cross-contamination could confound results. In addition, trypan blue is carcinogenic and thus potentially hazardous to technicians spraying daily. METHODS: Applicators that were exposed to the vagina were placed in the same bag as unexposed applicators and shaken daily for up to 4 months. Validation was carried out in three clinical sites in South Africa. RESULTS: Trypan blue was replaced with FD&C Blue #1 granular food dye. Cross-contamination did not occur, nor did the length of time affect reaction to dye. In South Africa, the assay was validated with an accuracy of over 95%. CONCLUSION: Applicator assay modifications render the test safe and suitable for use in clinical trials.
