Cap-independent protein translation is initially responsible for 4-(N-methylnitrosamino)-1-(3-pyridyl)-butanone (NNK)-induced apoptosis in normal human bronchial pithelial cells

Moon, Seo Hyun; Kim, Hyun Woo; Kim, Jun Sung; Park, Jin‐Hong; Kim, Hwa; Eu, Gook Jong; Cho, Hyun Sun; Kang, Ga Mi; Lee, Kee Ho; Cho, Myung‐Haing

doi:10.4142/jvs.2004.5.4.369

Cited by 4 publications

(3 citation statements)

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“…It is known that rapamycin inhibits 4E-BP1 phosphorylation, leading to increased association between 4E-BP1 and eIF-4E (cap-binding subunit of initiation complex), inhibiting 5′ cap-dependent translation; rapamycin has been used as a specific inhibitor of cap-dependent translation. 1,22,23 HT1080 cells expressing DIL were pretreated with rapamycin (25 nM, a nontoxic dose) and then treated with MTX (100 nM). Cells were collected following 0, 24, 48, 72, and 96 hours of MTX treatment, and levels of DHFR were determined by Western blotting.…”

Section: Resultsmentioning

confidence: 99%

“…Rapamycin treatment inhibits cap-dependent translation (by inhibiting 4E-BP1 phosphorylation, leading to increased association between 4E-BP1 and the cap-binding protein eIF-4E). [21][22][23] Antifolatemediated upregulation of DHFR protein upstream to IRES is inhibited by rapamycin pretreatment, indicating that the upregulation of DHFR protein upstream IRES is 5′ cap dependent, whereas translational upregulation of reporter protein downstream to IRES was not inhibited by rapamycin pretreatment, indicating that MTX-mediated induction of downstream reporter protein is 5′ cap independent in a bicistronic mRNA. Given that cap-dependent translation is more stringently regulated in the cell than IRES-mediated translation, the levels of upstream protein induce early but drop back to normal as soon as the drug is metabolized in the cell, whereas levels of downstream protein stay induced for longer periods.…”

Section: Discussionmentioning

confidence: 99%

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Translational Modulation of Proteins Expressed from Bicistronic Vectors

et al. 2009

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Bicistronic vectors are useful tools for exogenous expression of two gene products from a single promoter element; however, reduced expression of protein from the second cistron compared with the first cistron is a common limitation to this approach. To overcome this limitation, we explored use of dihydrofolate reductase (DHFR) complementary DNA encoded in bicistronic vectors to induce a second protein of interest by methotrexate (MTX) treatment. Previous studies have demonstrated that levels of DHFR protein and DHFR fusion protein can be induced translationally following MTX treatment of cells. We demonstrated that in response to MTX treatment, DHFR partner protein in a bicistronic construct is induced for longer periods of time when compared with endogenous DHFR and DHFR fusion protein, in vitro and in vivo. Using rapamycin pretreatment followed by MTX treatment, we also devised a strategy to modulate levels of two proteins expressed from a bicistronic construct in a cap-independent manner. To our knowledge, this is the first report demonstrating that levels of proteins in DHFR-based bicistronic constructs can be induced and modulated using MTX and rapamycin treatment.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Translational Modulation of Proteins Expressed from Bicistronic Vectors

et al. 2009

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“…It is now evident that many environmental chemicals exert their toxicity via apoptotic cell signalling [19]. For instance, it has been demonstrated that numerous food mutagens [20][21][22] and tobacco specific N-nitrosamine [23] induce apoptosis. Moreover, apoptosis induced by carcinogens seems to have an important role in cancer development.…”

Section: Introductionmentioning

confidence: 99%

Apoptosis Induced by N-Nitrosamines in Two Cancer Cell Lines

Morales¹,

Arranz²,

Haza³

2010

JEP

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In the present study, we investigated the induction of apoptosis by N-nitrosopyrrolidine (NPYR) and N-nitrosodimethy-lamine (NDMA) in two human cell lines: HL-60 (leukemia) and HepG2 (hepatoma). Apoptotic cells were identified by: 1) chromatin condensation, 2) flow cytometry analysis and 3) poly (ADP-ribose) polymerase cleavage. Both cell lines exhibited morphological changes consistent with apoptotic events following treatment with N-nitrosamines. Flow cytometry analysis showed that both N-nitrosamines induced apoptotic cell death in a concentration and time dependent- manner. NPYR was stronger than NDMA, since it induced a significant apoptotic cell death after 72 h starting from a concentration of 10 mM, whereas NDMA was effective at 27 mM. Furthermore, NPYR and NDMA caused the cleavage of PARP in HL-60 cells whereas no PARP cleavage was detected in HepG2 cells. However, NPYR- and NDMA-induced cell death in HepG2 cells was prevented by specific caspase inhibitors. Caspase-8 mediated main pathway and was responsible for 76% (NPYR) and 64% (NDMA) inhibition of apoptosis. The data demonstrate that NPYR and NDMA induce apoptosis in HL-60 and HepG2 cell lines via caspase-dependent pathway

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Induction of apoptosis and reactive oxygen species production by N‐nitrosopiperidine and N‐nitrosodibutylamine in human leukemia cells

García¹,

Morales²,

Arranz³

et al. 2007

J of Applied Toxicology

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N-nitrosopiperidine (NPIP) and N-nitrosodibutylamine (NDBA) belong to a group of N-nitrosamines that are widely distributed in foodstuffs and the occupational environment. In the present study, the human promyelocytic leukemia cell line HL-60, was used to characterize the apoptotic effects of N-nitrosamines, and to examine the production of reactive oxygen species (ROS). Apoptotic cells were identified by (i) chromatin condensation (ii) flow cytometry analysis and (iii) poly(ADP-ribose) polymerase (PARP) cleavage. NPIP and NDBA induced morphological changes consistent with apoptotic events in HL-60 cells. Flow cytometry analysis showed that both N-nitrosamines induced apoptotic cell death in a concentration and time dependent-manner. It was observed that NDBA was stronger than NPIP, since it induced a significant apoptotic cell death after 18 h starting from a concentration of 2 mm, whereas NPIP was effective at 10 mm. Furthermore, PARP was markedly cleaved with 0.5 mm of NDBA and 5 mm of NPIP after treatments for 3 and 18 h, respectively. Finally, the ROS level was found to be elevated after 0.5 h of treatment with both N-nitrosamines. Antioxidant N-acetylcysteine (NAC) completely inhibited the ROS production induced by NPIP and NDBA. However, this action seems not to be associated with the apoptosis because NAC did not block N-nitrosamines-induced apoptosis. The data demonstrate that NPIP and NDBA induce apoptosis and ROS production in HL-60 cells.

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Cap-independent protein translation is initially responsible for 4-(N-methylnitrosamino)-1-(3-pyridyl)-butanone (NNK)-induced apoptosis in normal human bronchial pithelial cells

Cited by 4 publications

References 0 publications

Translational Modulation of Proteins Expressed from Bicistronic Vectors

Translational Modulation of Proteins Expressed from Bicistronic Vectors

Apoptosis Induced by N-Nitrosamines in Two Cancer Cell Lines

Induction of apoptosis and reactive oxygen species production by N‐nitrosopiperidine and N‐nitrosodibutylamine in human leukemia cells

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