ABSTRACT
The t(8;21)(q22;q22) rearrangement represents the most common chromosomal translocation in acute myeloid leukemia (AML). It results in a transcript encoding for the fusion protein AML1-ETO (AE) with transcription factor activity. AE is considered to be an attractive target for treating t(8;21) leukemia. However, AE expression alone is insufficient to cause transformation, and thus the potential of such therapy remains unclear. Several genes are deregulated in AML cells, including KIT that encodes a tyrosine kinase receptor. Here, we show that AML cells transduced with short hairpin RNA vector targeting AE mRNAs have a dramatic decrease in growth rate that is caused by induction of apoptosis and deregulation of the cell cycle. A reduction in KIT mRNA levels was also observed in AE-silenced cells, but silencing KIT expression reduced cell growth but did not induce apoptosis. Transcription profiling of cells that escape cell death revealed activation of a number of signaling pathways involved in cell survival and proliferation. In particular, we find that the extracellular signal-regulated kinase 2 (ERK2; also known as mitogen-activated protein kinase 1 (MAPK1)) protein could mediate activation of 23 out of 29 (79%) of these upregulated pathways and thus may be regarded as the key player in establishing the t(8;21)-positive leukemic cells resistant to AE suppression.
Subject(s)
Apoptosis/genetics , Core Binding Factor Alpha 2 Subunit/genetics , Gene Expression Regulation, Leukemic , Leukemia, Myeloid, Acute/genetics , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Proteins c-kit/genetics , Signal Transduction/genetics , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation , Down-Regulation/genetics , HEK293 Cells , Humans , Leukemia, Myeloid, Acute/pathology , Models, Genetic , RNA, Small Interfering/genetics , RUNX1 Translocation Partner 1 ProteinABSTRACT
Here we describe a system based on recombinant lentiviral vectors for the safe screening of potential anti-HIV drugs. The system allows to evaluate the sensitivity of HIVl-1 reverse transcriptase and integrase (wild-type as well as mutant forms of these enzymes detected in drug-resistant virus isolates) towards different drugs and substances, but also to screen inhibitors of other stages of HIV-1 life cycle.
Subject(s)
Anti-HIV Agents/pharmacology , HIV Integrase Inhibitors/pharmacology , HIV-1/drug effects , High-Throughput Screening Assays , Virus Replication/drug effects , Drug Resistance, Viral , Flow Cytometry , Gene Expression , Genes, Reporter , Genetic Vectors , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , HIV Integrase/genetics , HIV Integrase/metabolism , HIV Reverse Transcriptase/antagonists & inhibitors , HIV Reverse Transcriptase/genetics , HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , HIV-1/genetics , Humans , Lentivirus/genetics , Transduction, Genetic , Virion/drug effects , Virion/growth & developmentABSTRACT
Acute myeloid leukemia is the most common acute leukemia affecting adults, and its incidence increases with age. Along with chromosomal translocations in leukemic cells mutations in the genes of receptor tyrosine kinases KIT and FLT3 were found with a high frequency. Here we show that transgenic progenitor of B-cells BAF3/FLT3-ITD are much more sensitive to the ribonuclease binase cytotoxic effects than the original BAF3 cells. The principal difference between BAF3/FLT3-ITD and the original BAF3 cells is the expression of FLT3-ITD oncogene, which leads to a change in the normal cell signaling pathways. Earlier, we described a similar effect for the cytotoxic action of binase on Kasumi-1 and FDC-P1-N822K cells, which express the activated KIT-N822K oncogene. Increased binase cytotoxicity toward the cells, expressing FLT3-ITD oncogene, suggests that, as in the case of FDC-P1 cells, transduced by KIT oncogene, the expression of an activated oncogene determines the sensitivity of cells to binase.
