| 1. |
- Kimbung, Siker, et al.
(författare)
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Co-targeting of the PI3K pathway improves the response of BRCA1 deficient breast cancer cells to PARP1 inhibition.
- 2012
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Ingår i: Cancer Letters. - : Elsevier BV. - 1872-7980 .- 0304-3835. ; 319:2, s. 232-241
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Tidskriftsartikel (refereegranskat)abstract
- Although pre-clinical and clinical studies on PARP1 inhibitors, alone and in combination with DNA-damaging agents, show promising results, further ways to improve and broaden the scope of application of this therapeutic approach are warranted. To this end, we have investigated the possibility of improving the response of BRCA1 mutant breast cancer cells to PARP1 inhibition by co-targeting the PI3K pathway. Human breast cancer cell lines with or without the expression of BRCA1 and/or PTEN were treated with PARP1 and PI3K inhibitors as single agents and in combination. PARP1 inhibition induced DNA damage conferring a G2/M arrest and decrease in viability, paralleled by the induction of apoptosis. PI3K inhibition alone caused a G1 arrest and decreased cell growth. Most importantly, sequential combination of PARP and PI3K inhibitors interacted synergistically to significantly decrease growth compared to PARP inhibition alone. Global transcriptional profiling revealed that this decrease in growth was associated with down-regulation of macromolecule biosynthesis and the induction of apoptosis. Taken together, these results suggest an improved treatment strategy for BRCA1-mutant and possibly also triple-negative breast cancers with similar molecular defects.
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| 2. |
- Kuang, Qie, et al.
(författare)
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A refined atomic model for microsomal glutathione transferase 1 from electron crystallography
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional (2D) crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The 2D crystals were of the p6 two-sided plane group symmetry. For the refinement, information to 3.5 Å resolution from 225 electron diffraction patterns recorded from specimens at tilt angles up to 66° was used. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two hydrocarbon chains, and one glutathione (GSH) molecule. Interactions between subunits form trimers centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer. The location of GSH is supported by mutagenesis data in vitro.
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| 3. |
- Kuang, Qie, et al.
(författare)
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Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation
- 2017
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two aliphatic chains and one glutathione (GSH) molecule. The functional unit is a homotrimer centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer supported by new in vitro mutagenesis data. Mutation of Arginine 130 to alanine resulted in complete loss of activity consistent with a role for Arginine 130 in stabilizing the strongly nucleophilic GSH thiolate required for catalysis. Based on the new model and an electron diffraction data set from crystals soaked with trinitrobenzene, that forms a dead-end Meisenheimer complex with GSH, a difference map was calculated. The map reveals side chain movements opening a cavity that defines the second substrate site.
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| 4. |
- Ottosson Wadlund, Astrid
(författare)
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Apoptosis signaling in leukemia and lymphoma: understanding mechanisms of chemoresistance
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Apoptosis (programmed cell death) is a basic physiological process, essential in the balance between life and death of cells of normal tissues in the body. Apoptosis can be considered as cellular “suicide” initiated by the cell itself when infected by a virus or transformed into a cancer cell. Cancer is a genetic disease and in cancer cells the molecules involved in initiation and execution of apoptosis are frequently lost or inactivated. Blockade of apoptosis is associated with resistance to conventional cancer drugs. The importance of intact apoptosis signaling pathways in leukemia and lymphoma was addressed in the current thesis. We found that apoptotic protease activating factor 1 (Apaf-1) is required for second mitochondria activator of caspases (Smac)-dependent potentiation of protein kinase inhibitor staurosporine- and proteasome inhibitor lactacystin-triggered apoptosis in chemoresistant Burkitt lymphoma cell lines Raji and DG-75. Furthermore, the importance of elevated levels of cellular inhibitor of apoptosis 2 (cIAP2), in these cells was examined in cellular extracts from Raji cells overexpressing cytosolic Apaf-1. Subsequently cytochrome c- dependent caspase activation in Raji cells immunodepleted for cIAP2 was assessed. We found immunodepletion of cIAP2 to potentiate caspase activation only in Raji cells stably transfected with cytosolic Apaf-1. To further study the importance of Apaf-1 in response to proteasome inhibitors we used a T cell acute lymphoblastic leukemia (T- ALL) cell line, Jurkat, stably transfected with shRNA against Apaf-1. The clinically relevant proteasome inhibitor bortezomib (Velcade®) failed to induce apoptosis in Jurkat cells without Apaf-1. The bortezomib-induced apoptosis was associated with induction of pro-apoptotic factor Noxa upstream of mitochondria. Moreover, we examined primary leukemic blasts from patients with T-ALL for Apaf-1 protein expression and responsiveness to bortezomib-induced apoptosis ex vivo. The Apaf-1 protein expression varied amongst the different patient samples and although the sample number was low we noted the lowest increase in bortezomib-induced apoptosis in the patient sample completely deficient for Apaf-1. In order to elucidate the importance of other mediators of apoptosis, anti-apoptotic HS-associated protein X-1 (HAX-1) was assessed at the protein and transcript level in malignant lymphomas. We thus determined the mRNA expression of HAX1 in two public transcriptomics databases. HAX-1 protein expression was assessed in a panel of 50 samples from patients with B lymphoma. We found that HAX-1 mRNA and protein was highly expressed in B lymphoma. Furthermore, we found a positive association with the proliferation marker Ki67 at the protein level in diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma samples, as well as an inverse correlation with Bcl-2 at the protein and transcript level in follicular lymphoma. Finally, the actions of the specific inhibitor of chymotrypsin-like serine proteases, TPCK and the NF-κB inhibitor Bay-11 7082 were elucidated in chemoresistant cell lines Raji and DG-75. Both compounds induced caspase-independent apoptosis as well as a decrease in constitutive NF-κB activity. Moreover, we found that TPCK and Bay-11 7082 reduced protein and mRNA expression of the NF-κB target HAX-1, which may contribute to the sensitization to apoptosis. In summary, these studies contribute to our understanding of the importance of intact apoptotic signaling pathways in sensitivity to apoptosis induced by anti-cancer substances. Studies of different pro- and anti-apoptotic molecules may lead to the identification of novel targets for therapy.
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| 5. |
- Ottosson-Wadlund, Astrid, et al.
(författare)
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Requirement of Apoptotic Protease-Activating Factor-1 for Bortezomib-Induced Apoptosis but Not for Fas-Mediated Apoptosis in Human Leukemic Cells
- 2013
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Ingår i: Molecular Pharmacology. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 1521-0111 .- 0026-895X. ; 83:1, s. 245-255
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Tidskriftsartikel (refereegranskat)abstract
- Bortezomib is a highly selective inhibitor of the 26S proteasome and has been approved for clinical use in the treatment of relapsing and refractory multiple myeloma and mantle cell lymphoma. Clinical trials are also underway to assess the role of bortezomib in several other human malignancies, including leukemia. However, the mechanism(s) by which bortezomib acts remain to be fully understood. Here, we studied the molecular requirements of bortezomib-induced apoptosis using the human T-cell leukemic Jurkat cells stably transfected with or without shRNA against apoptotic protease-activating factor-1 (Apaf-1). The Apaf-1-deficient Jurkat T cells were resistant to bortezomib-induced apoptosis, as assessed by caspase-3 activity, poly(ADP-ribose) polymerase cleavage, phosphatidylserine externalization, and hypodiploid DNA content. In contrast, Apaf-1-deficient cells were sensitive to Fas-induced apoptosis. Bortezomib induced an upregulation of the pro-apoptotic protein Noxa, loss of mitochondrial transmembrane potential, and release of cytochrome c in cells expressing or not expressing Apaf-1. Transient silencing of Apaf-1 expression in RPMI 8402 T-cell leukemic cells also diminished bortezomib-induced apoptosis. Fas-associated death domain (FADD)-deficient Jurkat cells were resistant to Fas-mediated apoptosis yet remained sensitive to bortezomib. Our results show that bortezomib induces apoptosis by regulating pathways that are mechanistically different from those activated upon death receptor ligation. Furthermore, in silico analyses of public transcriptomics data-bases indicated elevated Apaf-1 expression in several hematologic malignancies, including acute lymphoblastic and myeloid leukemia. We also noted variable Apaf-1 expression in a panel of samples from patients with acute lymphoblastic leukemia. Our results suggest that the expression of Apaf-1 may be predictive of the response to proteasome inhibition.
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| 6. |
- Spahiu, Linda, et al.
(författare)
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Global Kinetic Mechanism of Microsomal Glutathione Transferase 1 and Insights into Dynamic Enzyme Activation
- 2017
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Ingår i: Biochemistry. - : AMER CHEMICAL SOC. - 0006-2960 .- 1520-4995. ; 56:24, s. 3089-3098
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Tidskriftsartikel (refereegranskat)abstract
- Microsomal glutathione transferase 1 (MGST1) has a unique ability to be activated, <= 30-fold, by modification with sulfhydryl reagents. MGST1 exhibits one-third-of-the-sites reactivity toward glutathione and hence heterogeneous binding to different active sites in the homotrimer. Limited turnover stopped-flow kinetic measurements of the activated enzyme allowed us to more accurately determine the KD for the "third" low-affinity GSH binding site (1.4 +/- 0.3 mM). The rate of thiolate formation, k(2) (0.77 +/- 0.06 s(-1)), relevant to turnover, could also be determined. By deriving the steadystate rate equation for a random sequential mechanism for MGST1, we can predict K-M, k(cat), and k(cat)/K-M values from these and previously determined pre-steady-state rate constants (all determined at 5 C). To assess whether the pre-steady-state behavior can account for the steady-state kinetic behavior, we have determined experimental values for kinetic parameters at 5 degrees C. For reactive substrates and the activated enzyme, data for the microscopic steps account for the global mechanism of MGST1. For the unactivated enzyme and more reactive electrophilic substrates, pre steady -state and steady-state data can be reconciled only if a more active subpopulation of MGST1 is assumed. We suggest that unactivated MGST1 can be partially activated in its unmodified form. The existence of an activated subpopulation (approximately 10%) could be demonstrated in limited turnover experiments. We therefore suggest that MSGT1 displays a preexisting dynamic equilibrium between high- and low-activity forms.
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