A rapid and quantitative coat protein complex II vesicle formation assay using luciferase reporters. J Chris Fromme,Jinoh Kim Analytical biochemistry
421
2012
Show Abstract
The majority of protein export from the endoplasmic reticulum (ER) is facilitated by coat protein complex II (COPII). The COPII proteins deform the ER membrane into vesicles at the ER exit sites. During the vesicle formation step, the COPII proteins load cargo molecules into the vesicles. Formation of COPII vesicles has been reconstituted in vitro in yeast and in mammalian systems. These in vitro COPII vesicle formation assays involve incubation of microsomal membranes and purified COPII proteins with nucleotides. COPII vesicles are separated from the microsomes by differential centrifugation. Interestingly, the efficiency of the COPII vesicle formation with purified recombinant mammalian COPII proteins is lower than that with cytosol, suggesting that an additional cytosolic factor(s) is involved in this process. Indeed, other studies have also implicated additional factors. To facilitate biochemical identification of such regulators, a rapid and quantitative COPII vesicle formation assay is necessary because the current assay is lengthy. To expedite this assay, we generated luciferase reporter constructs. The reporter proteins were packaged into COPII vesicles and yielded quantifiable luminescent signals, resulting in a rapid and quantitative COPII vesicle formation assay. | 22244805
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Ligand-independent higher-order multimerization of CXCR4, a G-protein-coupled chemokine receptor involved in targeted metastasis. Makiko Hamatake, Toru Aoki, Yuko Futahashi, Emiko Urano, Naoki Yamamoto, Jun Komano, Makiko Hamatake, Toru Aoki, Yuko Futahashi, Emiko Urano, Naoki Yamamoto, Jun Komano Cancer science
100
95-102
2009
Show Abstract
CXCR4, a G-protein-coupled receptor of CXCL12/stromal cell-derived factor-1alpha, mediates a wide range of physiological and pathological processes, including the targeted metastasis of cancer cells. CXCR4 has been shown to homo-oligomerize in several experimental systems. However, it remains unclear with which domains CXCR4 interacts homotypically, and whether it dimerizes or forms a higher-order complex. To address these issues, we used bioluminescent resonance energy transfer and bimolecular fluorescence complementation analyses to measure the homotypic interactions of CXCR4 in living cells. Both assays indicated that CXCR4 interacts homotypically, which is consistent with previous studies. By studying CXCR4 mutants lacking various domains, we found that multiple transmembrane domains probably serve as potential molecular interaction surfaces for oligomerization. The relative contribution of the amino- or carboxy-termini to oligomerization was small. To differentiate between a dimer and a multimer consisting of more than two molecules, bioluminescent resonance energy transfer-bimolecular fluorescence complementation analysis was conducted. It revealed that CXCR4 engages in higher-order oligomerization in a ligand-independent fashion. This is the first report providing direct experimental evidence for the higher-order multimerization of CXCR4 in vivo. We hypothesize that CXCR4 distributes to the cell surface as a multimer, in order to effectively sense, with increased avidity, the chemotaxis-inducing ligand in the microenvironment. Studying the structure and function of the oligomeric state of CXCR4 may lead us to develop novel CXCR4 inhibitors that disassemble the molecular cluster of CXCR4. | 19018754
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Cells resistant to HTI-286 do not overexpress P-glycoprotein but have reduced drug accumulation and a point mutation in alpha-tubulin. Frank Loganzo, Malathi Hari, Tami Annable, Xingzhi Tan, Daniel B Morilla, Sylvia Musto, Arie Zask, Joshua Kaplan, Albert A Minnick, Michael K May, Semiramis Ayral-Kaloustian, Marianne S Poruchynsky, Tito Fojo, Lee M Greenberger Molecular cancer therapeutics
3
1319-27
2004
Show Abstract
HTI-286, a synthetic analogue of hemiasterlin, depolymerizes microtubules and is proposed to bind at the Vinca peptide site in tubulin. It has excellent in vivo antitumor activity in human xenograft models, including tumors that express P-glycoprotein, and is in phase II clinical evaluation. To identify potential mechanisms of resistance induced by HTI-286, KB-3-1 epidermoid carcinoma cells were exposed to increasing drug concentrations. When maintained in 4.0 nmol/L HTI-286, cells had 12-fold resistance to HTI-286. Cross-resistance was observed to other Vinca peptide-binding agents, including hemiasterlin A, dolastatin-10, and vinblastine (7- to 28-fold), and DNA-damaging drugs, including Adriamycin and mitoxantrone (16- to 57-fold), but minimal resistance was seen to taxanes, epothilones, or colchicine (1- to 4-fold). Resistance to HTI-286 was retained when KB-HTI-resistant cells were grown in athymic mice. Accumulation of [(3)H]HTI-286 was lower in cells selected in intermediate (2.5 nmol/L) and high (4.0 nmol/L) concentrations of HTI-286 compared with parental cells, whereas accumulation of [(14)C]paclitaxel was unchanged. Sodium azide treatment partially reversed low HTI-286 accumulation, suggesting involvement of an ATP-dependent drug pump. KB-HTI-resistant cells did not overexpress P-glycoprotein, breast cancer resistance protein (BCRP/ABCG2/MXR), MRP1, or MRP3. No mutations were found in the major beta-tubulin isoform. However, 4.0 nmol/L HTI-286-selected cells had a point mutation in alpha-tubulin that substitutes Ser for Ala(12) near the nonexchangeable GTP-binding site of alpha-tubulin. KB-HTI-resistant cells removed from drug became less resistant to HTI-286, no longer had low HTI-286 accumulation, and retained the Ala(12) mutation. These data suggest that HTI-286 resistance may be partially mediated by mutation of alpha-tubulin and by an ATP-binding cassette drug pump distinct from P-glycoprotein, ABCG2, MRP1, or MRP3. | 15486199
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Efficacy of MGI 114 (HMAF) against the MRP+ metastatic MV522 lung carcinoma xenograft. M J Kelner, T C McMorris, L A Estes, M Y Oval, R J Rojas, J R Lynn, K A Lanham, K M Samson Anti-cancer drugs
11
217-24
2000
Show Abstract
This study is part of an effort to evaluate efficacy of the novel agent MGI 114 (HMAF) against tumors resistant to conventional chemotherapeutic agents. MGI 114 is a novel semisynthetic anticancer agent currently in chemotherapeutic phase II trials to evaluate activity against various solid tumors. Previous studies indicate MGI 114 was active against human MDR1/gp170+ solid tumor xenografts. Recent evidence suggests overexpression of the MRP protein may also be clinically relevant to development of drug resistance in solid tumors. We evaluated the efficacy of MGI 114 against a human MRP+ lung carcinoma xenograft. Parent MV522 lung carcinoma cells were transfected with a MRP cDNA expression vector and resistant cells selected by exposure to vinblastine (30-fold resistance). Analysis of resistant clones indicated 20- to 40-fold increases in expression of both MRP mRNA and MRP protein. Administration of MGI 114 at the maximum tolerated dose (7 mg/kg, 5 x/week for 3 weeks) to MRP tumor-bearing mice demonstrated this novel agent was active against MRP+ tumors and significantly extended their lifespan (p0.001). In contrast, other cytotoxic agents had minimal activity against this MRP+ xenograft. These results indicate MGI 114 should retain activity in vivo against MRP+ tumor types. The development of this MRP+ xenograft model, in conjunction with the parent MV522 and MDR1/gp170+ xenograft models, will be useful for screening new classes of agents for activity against multidrug-resistant tumors. | 10831281
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Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Cole, S P, et al. Science, 258: 1650-4 (1992)
1992
Show Abstract
The doxorubicin-selected lung cancer cell line H69AR is resistant to many chemotherapeutic agents. However, like most tumor samples from individuals with this disease, it does not overexpress P-glycoprotein, a transmembrane transport protein that is dependent on adenosine triphosphate (ATP) and is associated with multidrug resistance. Complementary DNA (cDNA) clones corresponding to messenger RNAs (mRNAs) overexpressed in H69AR cells were isolated. One cDNA hybridized to an mRNA of 7.8 to 8.2 kilobases that was 100- to 200-fold more expressed in H69AR cells relative to drug-sensitive parental H69 cells. Overexpression was associated with amplification of the cognate gene located on chromosome 16 at band p13.1. Reversion to drug sensitivity was associated with loss of gene amplification and a marked decrease in mRNA expression. The mRNA encodes a member of the ATP-binding cassette transmembrane transporter superfamily. | 1360704
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