Project description:The cell division cycle is tightly controlled in normal cells. Cancer cells are characterized by deregulation in cell division cycle, which is associated with increased DNA replication and elevated cellular proliferation. Recently, genetic studies indicate that cyclin dependent kinases CDK2, CDK4 and CDK6 are not essential for the mammalian cell cycle, instead, they are only required for the proliferation of specific cell types.Thus, targeting the activities of CDK2, CDK4 and CDK6 may be a promising approach for cancer treatment. Rocaglamides (Roc) (= Flavaglines), derived from the traditional Chinese medicinal plant Aglaia, are a group of naturally occurring herbal chemicals characterized by a cyclopenta[b]benzofurans skeleton. A number of Roc derivatives have been found to have potent inhibitory effects on tumor growth with IC50 concentrations at the nM rages. Roc-mediated inhibition of proliferation was first found to be associated with inhibition of protein synthesis in 1998. In this study, using the representative Roc-A compound we revealed a novel mechanistic function of Roc. We show that Roc-A induces rapid activation of ATM (ataxiatelangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related) kinases. Activated ATM and ATR in turn activate the downstream checkpoint kinases CHK1 and CHK2. The latter then phosphorylate Cdc25A that leads to a phosphorylation-mediated degradation of Cdc25A. Usually, ATR and ATM are activated in response to DNA damage. However, in this study we show that Roc-A does not directly induce DNA breaks. To elucidate the molecular mechanism by which Roc-A activates ATM and ATR we performed a time-resolved microarray analysis to compare Roc-A-inducible genes in malignant vs. normal T lymphocytes. Based on the dynamic gene responses we found that Roc-A stimulates in Jurkat, but not in normal T lymphocytes, a set of genes responsive to DNA damage and nucleotide excision repair. This finding provides a hint of the molecular bases for Roc-induced activation of ATM and ATR. This finding also revealed a new scope for cancer treatment using Roc-A. The isolation time points for Roc A treated D6 T-cells were recorded at t=[1,2,3,4,6,8,10,14,20,28,36] hours after stimulation. Post-stimulation time points for Jurkat cells are t=[0.5,1,2,3,4,5,6,7,8,10,12,14,16,20,24,28,32,36]. Unstimulated control time points were taken at t=[0,4,10,16,24,36] hours for Jurkat cells at t=[0,6,14,24,36] hours.

Project description:The cell division cycle is tightly controlled in normal cells. Cancer cells are characterized by deregulation in cell division cycle, which is associated with increased DNA replication and elevated cellular proliferation. Recently, genetic studies indicate that cyclin dependent kinases CDK2, CDK4 and CDK6 are not essential for the mammalian cell cycle, instead, they are only required for the proliferation of specific cell types.Thus, targeting the activities of CDK2, CDK4 and CDK6 may be a promising approach for cancer treatment. Rocaglamides (Roc) (= Flavaglines), derived from the traditional Chinese medicinal plant Aglaia, are a group of naturally occurring herbal chemicals characterized by a cyclopenta[b]benzofurans skeleton. A number of Roc derivatives have been found to have potent inhibitory effects on tumor growth with IC50 concentrations at the nM rages. Roc-mediated inhibition of proliferation was first found to be associated with inhibition of protein synthesis in 1998. In this study, using the representative Roc-A compound we revealed a novel mechanistic function of Roc. We show that Roc-A induces rapid activation of ATM (ataxiatelangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related) kinases. Activated ATM and ATR in turn activate the downstream checkpoint kinases CHK1 and CHK2. The latter then phosphorylate Cdc25A that leads to a phosphorylation-mediated degradation of Cdc25A. Usually, ATR and ATM are activated in response to DNA damage. However, in this study we show that Roc-A does not directly induce DNA breaks. To elucidate the molecular mechanism by which Roc-A activates ATM and ATR we performed a time-resolved microarray analysis to compare Roc-A-inducible genes in malignant vs. normal T lymphocytes. Based on the dynamic gene responses we found that Roc-A stimulates in Jurkat, but not in normal T lymphocytes, a set of genes responsive to DNA damage and nucleotide excision repair. This finding provides a hint of the molecular bases for Roc-induced activation of ATM and ATR. This finding also revealed a new scope for cancer treatment using Roc-A.

Project description:Pilotexp_IMAC_TiO2_eluate_ATM_ATR_dep_phos_15min_postIR: Proteins were isolated from 4 different conditions of Arabidopsis thaliana plants: wild type untreated, wild type irradiated, atm/atr mutant untreated, atm/atr mutant irradiated The proteins were tryptically digested and labelled each with one channel ofthe iTRAQ4plex reagent. The samples were mixed and subjected to phosphopeptide enrichment by IMAC followed by TiO2. The enriched phosphopeptides were separated by SCX and each fraction was analysed by nano-reversed phase HPLC coupled online to a LTQ Orbitrap Velos. Each peptide was fragmented both by CID (MSA) and HCD, using the iontrap-MS analyser for CID and the Orbitrap detector for HCD scans. IMAC_TiO2_eluate_ATM_ATR_dep_phos_15min_postIR: Proteins were isolated from 4 different conditions of Arabidopsis thaliana plants: wild type untreated, wild type irradiated, atm/atr mutant untreated, atm/atr mutant irradiated The proteins were tryptically digested and labelled each with one channel ofthe iTRAQ4plex reagent. The samples were mixed and subjected to phosphopeptide enrichment by IMAC followed by TiO2. The enriched phosphopeptides were separated by SCX and each fraction was analysed by nano-reversed phase HPLC coupled online to a LTQ Orbitrap Velos. Each peptide was fragmented both by CID (MSA) and HCD, using the iontrap-MS analyser for CID and the Orbitrap detector for HCD scans. IMAC_TiO2_FT_ATM_ATR_dep_phos_15min_postIR: Proteins were isolated from 4 different conditions of Arabidopsis thaliana plants: wild type untreated, wild type irradiated, atm/atr mutant untreated, atm/atr mutant irradiated The proteins were tryptically digested and labelled each with one channel ofthe iTRAQ4plex reagent. The samples were mixed and subjected to phosphopeptide enrichment by IMAC followed by TiO2. The unphosphorylated peptides found in the flowthrough of the TiO2 column were separated by SCX and each fraction was analysed by nano-reversed phase HPLC coupled online to a LTQ Orbitrap Velos. Each peptide was fragmented both by CID and HCD, using the iontrap-MS analyser for CID and the Orbitrap detector for HCD scans. The iTRAQ ratio obtained for the unmodified peptides from this flowthrough sample was used to calculate protein ratios for the 4 different conditions.

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Expression data from 5 day old Arabidopsis thaliana seedlings

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