Protein kinase C δ is a potential therapeutic target in malignant melanoma with NRAS mutation or B-RAF inhibitor-resistance

Thesis (Ph.D.)--Boston UniversityMetastatic melanoma is the major cause of skin cancer death, and the annual incidence of melanoma continues to increase. Despite the impressively high rates of response to BRAF inhibitors in patients with melanomas harboring BRAF mutations, most of these patients eventually relapse after developing resistance to the drug, due in part to secondary mutations in NRAS. Although NRAS mutation is the second most common genetic mutation in melanoma patients (after BRAF mutation), there is currently no treatment option that targets NRAS-mutated melanomas. Previous reports have demonstrated the sensitivity of cancer cell lines carrying RAS mutations to apoptosis initiated by inhibition of protein kinase C delta (PKCδ), suggesting the possible association between RAS mutational status and sensitivity to PKCδ inhibition. I therefore hypothesized that PKCδ inhibitors might also be cytotoxic in melanomas with primary or acquired NRAS mutations. In this project, the effect of PKCδ inhibition, and the efficacy of a new PKCcS inhibitor, BJE6-106 (B106), in melanoma were investigated. Inhibition of PKCδ inhibited the growth of multiple human melanoma cell lines carrying NRAS mutations, and induced apoptosis mediated by terminal caspase activation. Analysis of the molecular mechanisms demonstrated activation of the JNK pathway after PKCδ inhibition, leading to the activation (phosphorylation) of H2AX, a histone H2A variant. Activation of H2AX was attenuated when JNK1/2 levels were repressed, indicating that H2AX activation is mediated by the JNK pathway in response to PKCδ inhibition. Consistent with recent reports on the apoptotic role of phospho-H2AX, knockdown of H2AX prior to PKCδ inhibition mitigated the induction of caspase-dependent apoptosis. To explore the potential of B106 further, melanoma cell lines harboring BRAF mutations that had evolved resistance to a BRAF inhibitor, PLX4032 (vemurafenib), were developed. B106 effectively induced cytotoxicity in these cells, suggesting the potential clinical application of targeting PKCδ in patients who have relapsed following treatment with PLX4032. Taken together, this work suggests that inhibition of PKCδ causes caspase-dependent apoptosis in melanomas with NRAS mutations and in PLX4032-resistant BRAF mutant melanomas. This apoptosis is mediated via activation of the JNK-H2AX pathway, which involves a novel role for phospho-H2AX in the execution of apoptosis

Frequency domain studies of impedance characteristics of biological cells using micropipet technique. I. Erythrocyte.

This study aims at precise measurement of the membrane capacity and its frequency dependence of small biological cells using the micropipet technique. The use of AC fields as an input signal enables the magnitude and phase angle of membrane impedance to be measured at various frequencies. The micropipet technique was applied to human erythrocyte, and passive membrane capacity and conductivity were determined between 4 Hz and 10 KHz. Membrane capacity thus determined changed from 1.05 to 0.73 microF/cm2 between 4 Hz and 10 KHz. In addition to the micropipet technique, we used suspension method between 50 KHz and 10 MHz for the purpose of supplementing the new method with the one which has been in use for many years. We obtained a membrane capacity of 0.65-0.8 microF/cm2 using this technique. These values agree with the capacitance obtained with the micropipet method. Although this paper discusses only human erythrocytes, the study has been performed with lymphocytes and various forms of cancer cells. This paper is the first of the series of reports on frequency domain studies of the impedance characteristics of various biological cells

Frequency domain analysis of membrane capacitance of cultured cells (HeLa and myeloma) using the micropipette technique.

The membrane capacitance and conductance of cultured cells (HeLa and mouse myeloma) are investigated using the micropipette method. Mean values of the membrane capacities were found to be 1.9 microF/cm2 for HeLa cells and 1.0 microF/cm2 for myeloma cells. These values are in agreement with those obtained using the suspension method. Whereas the suspension method is unable to provide the information on membrane conductance, the micropipette method is able to measure even an extremely small membrane conductance if leakage current is negligibly small. The membrane conductances were found, using this technique, to be approximately 90-100 microS/cm2 for both HeLa and myeloma cells. One of the purposes of this study is to establish the frequency profile of membrane capacitance. It was found, however, that membrane capacitances of these cells are independent of frequency between 1 Hz and 1 KHz within the resolution of this technique

Dielectric spectroscopy of Tobacco Mosaic Virus

The dielectric properties of the Tobacco Mosaic Virus (TMV) have been measured using time domain dielectric spectroscopy (TDDS) in the temperature range from 1 to 40 jC. A single dielectric dispersion is observed in the MHz range. The activation energy of the process is found to be in the range 1–2 kcal/mol. The experimental data could not be completely accounted for by current theoretical models, but evidence indicates that the dielectric loss arises from polarisation of charge on and around the virus