A series of mammalian expression vectors and characterisation of their expression of a reporter gene in stably and transiently transfected cells

Morgenstern, Jay P.; Land, Hartmut

doi:10.1093/nar/18.4.1068

Cited by 297 publications

(186 citation statements)

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“…To this end, a full-length c-akt and an HA-tagged Cterminally truncated (aa 1 to 429) c-akt construct (HA-c-akt⌬tail) in the simian virus 40 vector pJ3⍀ (20) were transfected either separately or together into COS-1 cells. The cells were subsequently labelled with [ 35 S]methionine and lysed in an NP40 lysis buffer.…”

Section: Resultsmentioning

confidence: 99%

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AH/PH Domain-Mediated Interaction between Akt Molecules and Its Potential Role in Akt Regulation

Datta

Franke

Chan

et al. 1995

Molecular and Cellular Biology

154

131

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The cytoplasmic serine-threonine protein kinase coded for by the c-akt proto-oncogene features a protein kinase C-like catalytic domain and a unique NH 2 -terminal domain (AH domain). The AH domain is a member of a domain superfamily whose prototype was observed in pleckstrin (pleckstrin homology, or PH, domain). In this communication, we present evidence that the AH/PH domain is a domain of protein-protein interaction which mediates the formation of Akt protein complexes. The interaction between c-akt AH/PH domains is highly specific, as determined by the failure of this domain to bind AKT2. The AH/PH domain-mediated interactions depend on the integrity of the entire domain. Akt molecules with deletions of the NH 2 -terminal portion (amino acids 11 to 60) and AH/PH constructs with deletions of the C-terminal portion of this domain (amino acids 107 to 147) fail to interact with c-akt. To determine the significance of these findings, we carried out in vitro kinase assays using Akt immunoprecipitates from serum-starved and serum-starved, plateletderived growth factor-stimulated NIH 3T3 cells. Addition of maltose-binding protein-AH/PH fusion recombinant protein, which is expected to bind Akt, to the immunoprecipitates from serum-starved cells induced the activation of the Akt kinase.

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Section: Resultsmentioning

confidence: 99%

“…Transfections were carried out in COS-1 cells. The vector utilized in the generation of the expression constructs (pJ3⍀) contains the simian virus 40 origin of replication and early promoter (20). c-akt constructs express the entire c-akt open reading frame (aa 1 to 480).…”

Section: Methodsmentioning

confidence: 99%

AH/PH Domain-Mediated Interaction between Akt Molecules and Its Potential Role in Akt Regulation

Datta

Franke

Chan

et al. 1995

Molecular and Cellular Biology

154

131

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“…S1(MRP) was obtained after transfection of non-small cell lung cancer SW-1573/S1 cells with an expression vector containing MRP cDNA and a neomycin-resistance marker gene (pRc/RSV-MRP), followed by selection with geneticin (G418) (6). Sl(MDR1) was previously named S1(1.1) (24) and was obtained after transfection of S1 cells with the expression vector pJ3fl (25) containing MDR1 cDNA, followed by selection with 10 nM vincristine. GLC4/ADR is a MRP-overexpressing subline of the non-small cell lung cancer cell line GLC4 and was obtained by selection with doxorubicin (20,21).…”

Section: Methodsmentioning

confidence: 99%

Role of glutathione in the export of compounds from cells by the multidrug-resistance-associated protein.

Zaman

Lankelma

Tellingen

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

436

298

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Multidrug-resistance-associated protein [6][7][8] and is able to decrease cellular drug levels against a concentration gradient (4, 6). However, recent work has also indicated interesting differences between MRP and Pgp. Increased cellular MRP levels are associated with increased reduced glutathione (GSH) S-conjugate carrier (GS-X pump) activity in isolated plasma membrane vesicles (9-11). This suggests that MRP is a GS-X pump (12) present in many, if not all, mammalian cells (9-13). These pumps transport substrates containing a large hydrophobic moiety and at least two negative charges (12, 13), as present in drug GSH Sconjugates. Moreover, recent studies indicate that GS-X pumps are also involved in the export of cisplatin (14-16) and arsenite (11). Indeed, some cell lines overexpressing MRP are moderately resistant to arsenite (4, 11). These results link MRP to older experiments in which resistance to anthracyclines was found to correlate with increased levels of cellular GSH, GSH synthesis, or GSH Stransferases (17)(18)(19). This link is supported by the strong decrease in drug resistance in two MDR lung carcinoma cell lines that overexpress MRP by DL-buthionine (S,R)-sulfoximine (BSO) (20)(21)(22), an inhibitor of y-glutamylcysteine synthetase, the enzyme that catalyzes the first step in GSH synthesis (23). The interpretation of these inhibitor experiments is not unambiguous, however. In both cell lines, other resistance mechanisms (e.g., alterations in topoisomerase II) contribute to resistance, and it is not clear whether the GSH depletion in these cells does not result in membrane damagee.g., by lipid peroxidation. Damage of the plasma membrane could increase drug influx and, hence, decrease resistance. To test whether GSH is specifically required for MDR caused by MRP but not by Pgp, we have analyzed the effects of BSO treatment on lung cancer cells transfected with an expression vector containing either MRP cDNA or MDR1 cDNA. MATERIALS AND METHODSCell Lines. S1(MRP) was obtained after transfection of non-small cell lung cancer SW-1573/S1 cells with an expression vector containing MRP cDNA and a neomycin-resistance marker gene (pRc/RSV-MRP), followed by selection with geneticin (G418) (6). Sl(MDR1) was previously named S1(1.1) (24) and was obtained after transfection of S1 cells with the expression vector pJ3fl (25) containing MDR1 cDNA, followed by selection with 10 nM vincristine. GLC4/ADR is a MRP-overexpressing subline of the non-small cell lung cancer cell line GLC4 and was obtained by selection with doxorubicin (20,21).Clonogenic Survival Assay. In six-well dishes, 400 cells per well were seeded and incubated in medium with or without 25 ,uM BSO for 24 hr prior to incubation with increasing concentrations of drug. After 1 hr, drug was removed, the wells were rinsed with phosphate-buffered saline, and drug-free medium without BSO was added. Seven days after the start of the experiment, the percentage of cells that were able to produce a colony of >50 cells was used as a measure of cell sur...

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“…The expression plasmid containing an activated H-ras cDNA (valine-12 mutation, RasV 12 ) in the pBabe retroviral vector (Morgenstern and Land, 1990) was provided by Dr. Scott Lowe (Cold Spring Harbor Laboratory, Cold Spring, NY). This Ras cDNA construct was sequenced to verify the presence of the codon 12 mutation using primers derived from the cDNA sequence of Ras and primers derived from the pBabe plasmid sequence 5Ј to the ATG of the Ras cDNA.…”

Section: Ras Transfectionmentioning

confidence: 99%

RasMutation Impairs Epithelial Barrier Function to a Wide Range of Nonelectrolytes

Mullin

Leatherman

Valenzano

et al. 2005

MBoC

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Although ras mutations have been shown to affect epithelial architecture and polarity, their role in altering tight junctions remains unclear. Transfection of a valine-12 mutated ras construct into LLC-PK 1 renal epithelia produces leakiness of tight junctions to certain types of solutes. Transepithelial permeability of D-mannitol increases sixfold but transepithelial electrical resistance increases >40%. This indicates decreased paracellular permeability to NaCl but increased permeability to nonelectrolytes. Permeability increases to D-mannitol (M r 182), polyethylene glycol (M r 4000), and 10,000-M r methylated dextran but not to 2,000,000-M r methylated dextran. This implies a "ceiling" on the size of solutes that can cross a ras-mutated epithelial barrier and therefore that the increased permeability is not due to loss of cells or junctions. Although the abundance of claudin-2 declined to undetectable levels in the ras-overexpressing cells compared with vector controls, levels of occludin and claudins 1, 4, and 7 increased. The abundance of claudins-3 and -5 remained unchanged. An increase in extracellular signal-regulated kinase-2 phosphorylation suggests that the downstream effects on the tight junction may be due to changes in the mitogen-activated protein kinase signaling pathway. These selective changes in permeability may influence tumorigenesis by the types of solutes now able to cross the epithelial barrier. INTRODUCTIONMutations in the three closely related ras genes, H-ras, K-ras, and N-ras, are among the most common mutations found in human cancer, reaching 50% in some types of tumors, such as colorectal carcinoma (Forrester et al., 1987;Andreyev et al., 1997Andreyev et al., , 1998Andreyev et al., , 2001). Vogelstein and colleagues have shown that mutations in K-ras occur early in the development of colon carcinomas (Vogelstein et al., 1988). This dominantly acting mutation in ras results in its constitutive activation and the subsequent triggering of its signaling pathway by influencing GTPase activity. It is clear that mutations in ras contribute both to the recurrence of the disease and to decreased survival (Andreyev et al., 1998). It is less clear, however, what the consequences of ras mutations are during the early stages of tumorigenesis. In this regard, one important issue is the effect of Ras activation on epithelial barrier function, perhaps the most basic of all epithelial functions, and a function shared by all epithelial tissues. Ras can exert fundamental effects on epithelial barrier function through the extracellular signal-regulated kinase-mitogen-activated protein (ERK-MAP) kinase pathway or through direct binding to the tight junction-associated protein, AF-6, which binds to ZO-1, which in turn links the tight junction to the actin cytoskeleton. Activated Ras is known to inhibit interaction between the tight junction-associated proteins, AF-6 and ZO-1, with resultant perturbation of intercellular junctions (Yamamoto et al., 1997). Modulation of tight junctions by G proteins in ge...

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A series of mammalian expression vectors and characterisation of their expression of a reporter gene in stably and transiently transfected cells

Cited by 297 publications

References 5 publications

AH/PH Domain-Mediated Interaction between Akt Molecules and Its Potential Role in Akt Regulation

AH/PH Domain-Mediated Interaction between Akt Molecules and Its Potential Role in Akt Regulation

Role of glutathione in the export of compounds from cells by the multidrug-resistance-associated protein.

RasMutation Impairs Epithelial Barrier Function to a Wide Range of Nonelectrolytes

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