Involvement of p53 and Raf/MEK/ERK pathways in hematopoietic drug resistance.

A cytokine-dependent (FL5.12), drug-sensitive, p53 wild type (WT) and a doxorubicin-resistant derivative line (FL/Doxo) were used to determine the mechanisms that could result in drug resistance of early hematopoietic precursor cells. Drug resistance was associated with decreased p53 induction after doxorubicin treatment, which was due to a higher level of proteasomal degradation of p53. Dominant-negative (DN) p53 genes increased the resistance to chemotherapeutic drugs, MDM-2 and MEK inhibitors, further substantiating the role of p53 in therapeutic sensitivity. The involvement of signal transduction and apoptotic pathways was examined, as drug resistance did not appear to be due to increased drug efflux. Drug-resistant FL/Doxo cells had higher levels of activated Raf/MEK/ERK signaling and decreased induction of apoptosis when cultured in the presence of doxorubicin than drug-sensitive FL5.12 cells. Introduction of DN MEK1 increased drug sensitivity, whereas constitutively active (CA) MEK1 or conditionally active BRAF augmented resistance, documenting the importance of the Raf/MEK/ERK pathway in drug resistance. MEK inhibitors synergized with chemotherapeutic drugs to reduce the IC(50). Thus the p53 and Raf/MEK/ERK pathways play key roles in drug sensitivity. Targeting these pathways may be effective in certain drug-resistant leukemias that are WT at p53.

Oncogenic BRAF regulates beta-Trcp expression and NF-kappaB activity in human melanoma cells.

Mutational activation of BRAF is a frequent event in human malignant melanomas suggesting that BRAF-dependent signaling is conducive to melanoma cell growth and survival. Previously published work reported that melanoma cells exhibit constitutive anti-apoptotic nuclear factor kappaB (NF-kappaB) transcription factor activation triggered by proteolysis of its inhibitor IkappaB. IkappaB degradation is dependent upon its phosphorylation by the IkappaB kinase (IKK) complex and subsequent ubiquitination facilitated by beta-Trcp E3 ubiquitin ligase. Here, we report that melanocytes expressing a conditionally oncogenic form of BRAF(V600E) exhibit enhanced beta-Trcp expression, increased IKK activity and a concomitant increase in the rate of IkappaBalpha degradation. Conversely, inhibition of BRAF signaling using either a broad-spectrum Raf inhibitor (BAY 43-9006) or by selective knock-down of BRAF(V600E) expression by RNA interference in human melanoma cells leads to decreased IKK activity and beta-Trcp expression, stabilization of IkappaB, inhibition of NF-kappaB transcriptional activity and sensitization of these cells to apoptosis. Taken together, these data support a model in which mutational activation of BRAF in human melanomas contributes to constitutive induction of NF-kappaB activity and to increased survival of melanoma cells.

A strategy for primary high throughput cytotoxicity screening in pharmaceutical toxicology.

PURPOSE: Recent advances in combinatorial chemistry and high throughput screens for pharmacologic activity have created an increasing demand for in vitro high throughput screens for toxicological evaluation in the early phases of drug discovery. METHODS: To develop a strategy for such a screen, we have conducted a data mining study of the National Cancer Institute's Developmental Therapeutics Program (DTP) cytotoxicity database. RESULTS: Using hierarchical cluster analysis, we confirmed that the different tissues of origin and individual cell lines showed differential sensitivity to compounds in the DTP Standard Agents database. Surprisingly, however, approaching the data globally, linear regression analysis showed that the differences were relatively minor. Comparison with the literature on acute toxicity in mice showed that the predictive power of growth inhibition was marginally superior to that of cell death. CONCLUSIONS: This datamining study suggests that in designing a strategy for high throughput cytotoxicity screening: a single cell line, the choice of which may not be critical, can be used as a primary screen; a single end point may be an adequate measure and a cut off value for 50% growth inhibition between 10(-6) and 10(-8) M may be a reasonable starting point for accepting a cytotoxic compound for scale up and further study.