alvocidib and geldanamycin

Similarities and differences in the cell cycle components, apoptosis and cytoskeleton-related molecules among mouse skin fibroblast cells (MSFs), mouse squamous cell lung carcinomas (SqCLCs) and mouse embryonic stem cells (mESCs) are important determinants of the behaviour and differentiation capacity of these cells. To reveal apoptotic pathways and to examine the distribution and the role of cell cycle-cell skeleton comparatively would necessitate tumour biology and stem cell biology to be assessed together in terms of oncogenesis and embryogenesis. The primary objectives of this study are to investigate the effects of flavopiridol, a cell cycle inhibitor, and geldanamycin, a heat shock protein inhibitor on mouse somatic, tumour and embryonic stem cells, by specifically focusing on alterations in cytoskeletal proteins, cell polarity and motility as well as cell cycle regulators. To meet these objectives, expression of several genes, cell cycle analysis and immunofluorescence staining of intracellular cytoskeletal molecules were performed in untreated and flavopiridol- or geldanamycin-treated cell lines. Cytotoxicity assays showed that SqCLCs are more sensitive to flavopiridol than MSFs and mESCs. Keratin-9 and keratin-2 expressions increased dramatically whereas cell cycle regulatory genes decreased significantly in the flavopiridol-treated MSFs. Flavopiridol-treated SqCLCs displayed a slight increase in several cell cytoskeleton regulatory genes as well as cell cycle regulatory genes. However, gene expression profiles of mESCs were not affected after flavopiridol treatment except the Cdc2a. Cytotoxic concentrations of geldanamycin were close to each other for all cell lines. Cdkn1a was the most increased gene in the geldanamycin-treated MSFs. However, expression levels of cell cytoskeleton-associated genes were increased dramatically in the geldanamycin-treated SqCLCs. Our results revealing differences in molecular mechanisms between embryogenesis and carcinogenesis may prove crucial in developing novel therapeutics that specifically target cancer cells.

Topics: Actins; Animals; Apoptosis; Benzoquinones; Cell Cycle; Cell Line, Tumor; Embryonic Stem Cells; Epithelial-Mesenchymal Transition; Fibroblasts; Flavonoids; Keratin-2; Lactams, Macrocyclic; Lung Neoplasms; Mice; Piperidines

Selective modulation of cell death is important for rational chemotherapy. By depleting Hsp90-client oncoproteins, geldanamycin (GA) and 17-allylamino-17-demethoxy-GA (17-AAG) (heat-shock protein-90-active drugs) render certain oncoprotein-addictive cancer cells sensitive to chemotherapy. Here we investigated effects of GA and 17-AAG in apoptosis-prone cells such as HL60 and U937. In these cells, doxorubicin (DOX) caused rapid apoptosis, whereas GA-induced heat-shock protein-70 (Hsp70) (a potent inhibitor of apoptosis) and G1 arrest without significant apoptosis. GA blocked caspase activation and apoptosis and delayed cell death caused by DOX. Inhibitors of translation and transcription and siRNA Hsp70 abrogated cytoprotective effects of GA. Also GA failed to protect HL60 cells from cytotoxicity of actinomycin D and flavopiridol (FL), inhibitors of transcription. We next compared cytoprotection by GA-induced Hsp70, caspase inhibitors (Z-VAD-fmk) and cell-cycle arrest. Whereas cell-cycle arrest protected HL60 cells from paclitaxel (PTX) but not from FL and DOX, Z-VAD-fmk prevented FL-induced apoptosis but was less effective against DOX and PTX. Thus, by inducing Hsp70, GA protected apoptosis-prone cells in unique and cell-type selective manner. Since GA does not protect apoptosis-reluctant cancer cells, we envision a therapeutic strategy to decrease side effects of chemotherapy without affecting its therapeutic efficacy.

Topics: Amino Acid Chloromethyl Ketones; Antibiotics, Antineoplastic; Apoptosis; Benzoquinones; Caspase 9; Caspase Inhibitors; Cell Cycle; Cell Line, Tumor; Cytoprotection; Dactinomycin; Doxorubicin; Enzyme Activation; Flavonoids; HSP70 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Paclitaxel; Piperidines; Protein Biosynthesis; RNA, Small Interfering; Transcriptional Activation

By activating anti-apoptotic factors (e.g., Hsp70, Raf-1, Bcl-xL), Bcr-Abl blocks apoptotic pathways at multiple levels, thus rendering leukemia cells resistant to chemotherapeutic agents such as doxorubicin (DOX). In Bcr-Abl-transfected HL60 (HL/Bcr-Abl) cells, procaspase-9 was increased and partially processed. The Bcr-Abl inhibitor imatinib (Gleevec, STI-571) released the apoptotic stream. Also, HL/Bcr-Abl cells were hyper-sensitive to geldanamycin (GA), which depletes Bcr-Abl and Raf-1. Raf-1 and Bcr-Abl-transfected FDC-P1 hematopoietic cells were selectively sensitive to GA and imatinib, respectively. Remarkably, cell clones with high levels of Bcr-Abl that could not be depleted by GA were relatively resistant to both GA and imatinib. GA and flavopiridol sensitized such resistant cells to imatinib. These data suggest bi-phasic sensitivity to mechanism-based therapeutic agents. Although Bcr-Abl renders cells hyper-sensitive, an excess of Bcr-Abl results in resistance (due to the remaining activity). We discuss therapeutic approaches to overcome bi-phasic resistance to mechanisms-based agents.

Topics: Antineoplastic Agents; Benzamides; Benzoquinones; Cell Survival; Clone Cells; Drug Interactions; Drug Resistance, Neoplasm; Flavonoids; Fusion Proteins, bcr-abl; HL-60 Cells; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Imatinib Mesylate; K562 Cells; Lactams, Macrocyclic; Models, Biological; Piperazines; Piperidines; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-raf; Pyrimidines; Quinones; Time Factors