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NNK, a tobacco-specific carcinogen, inhibits the expression of lysyl oxidase, a tumor suppressor.

Cheng G, Li J, Zheng M, Zhao Y, Zhou J, Li W - Int J Environ Res Public Health (2014)

Bottom Line: Exposure of cells to NNK reduced levels of steady-states LOX mRNA and new transcript synthesis.At the promoter level, NNK enhanced methylation of CpG, but decreased acetylation of histone H3 at the core promoter region of the LOX gene.These results indicated that transcriptional and translational processes of LOX are major targets for NNK.

View Article: PubMed Central - PubMed

Affiliation: The Central Lab, Hebei United University Affinity Hospital, Tangshan 063000, China. gcheng911@163.com.

ABSTRACT
A tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is believed to contribute to the cancer burden in cigarette smokers. To evaluate NNK effects on the expression of lysyl oxidase (LOX), a tumor suppressor, we examined this enzyme at various levels in NNK-treated rat fetal lung fibroblasts (RFL6). Exposure of cells to NNK reduced levels of steady-states LOX mRNA and new transcript synthesis. NNK inhibited all LOX protein species in a dose-dependent manner. Although 300 µM NNK markedly decreased the level in the 46 kDa preproenzyme, under same conditions, there was no detectable amounts of the 50 kDa proenzyme and the 32 kDa mature enzyme suggesting NNK perturbing the LOX protein processing to its mature form. Moreover, NNK also suppressed LOX activities in conditioned media of treated cells. At the promoter level, NNK enhanced methylation of CpG, but decreased acetylation of histone H3 at the core promoter region of the LOX gene. These results indicated that transcriptional and translational processes of LOX are major targets for NNK. Thus, inactivation of tumor suppressor gene LOX may play a critical role in NNK carcinogenesis.

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Inactivation of the LOX core promoter in NNK treated cells. ChIP and PCR assays were performed to elucidate the active status of the LOX core promoter in treated cells by assessing acetylated histone H3 binding to the core promoter region. DNAs were isolated from control and NNK treated cells each with 2 × 106, sonicated and immunoprecipitated with an antibody against acetylated histone H3 or RNA-PolyII. Using immunoprecipitated DNA as a template, the PCR with primer pairs as shown under Methods amplified the acetylated histone H3-bound LOX core promoter region with 136 bp, and the RNA-Poly II bound fragment of the GAPDH promoter (an internal control) with 90 bp, respectively. PCR products were analyzed on 2.2% agarose gels and densities of DNA bands measured by the 1D Scan software.
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ijerph-12-00064-f006: Inactivation of the LOX core promoter in NNK treated cells. ChIP and PCR assays were performed to elucidate the active status of the LOX core promoter in treated cells by assessing acetylated histone H3 binding to the core promoter region. DNAs were isolated from control and NNK treated cells each with 2 × 106, sonicated and immunoprecipitated with an antibody against acetylated histone H3 or RNA-PolyII. Using immunoprecipitated DNA as a template, the PCR with primer pairs as shown under Methods amplified the acetylated histone H3-bound LOX core promoter region with 136 bp, and the RNA-Poly II bound fragment of the GAPDH promoter (an internal control) with 90 bp, respectively. PCR products were analyzed on 2.2% agarose gels and densities of DNA bands measured by the 1D Scan software.

Mentions: To elucidate the active status of the LOX core promoter in response to NNK, quantitation of acetylated histone H3 at the LOX Inr-DPE region was performed by the ChIP assay [19]. The anti-diacetylated histone H3 antibody was used to precipitate DNA fragments isolated from control and NNK-treated cells. Using primers as described, the PCR amplified a 136 bp fragment (−95/+41, relative to ATG) containing the LOX core promoter from −53 to −14 and an intact transcription start site cluster from −78 to −51 [19]. As shown in Figure 6, in comparison to the internal control, the GAPDH fragment bound with the RNA-PolyII, the histone H3 acetylated at the tested region of the LOX promoter in NNK-treated cells was reduced to 74, 41, 11 and 5% of the control for cells treated with 10, 30, 100 and 300 µM NNK, respectively. These results indicated NNK inactivation of the LOX core promoter as a key mechanism for down-regulation of this enzyme at the promoter level.


NNK, a tobacco-specific carcinogen, inhibits the expression of lysyl oxidase, a tumor suppressor.

Cheng G, Li J, Zheng M, Zhao Y, Zhou J, Li W - Int J Environ Res Public Health (2014)

Inactivation of the LOX core promoter in NNK treated cells. ChIP and PCR assays were performed to elucidate the active status of the LOX core promoter in treated cells by assessing acetylated histone H3 binding to the core promoter region. DNAs were isolated from control and NNK treated cells each with 2 × 106, sonicated and immunoprecipitated with an antibody against acetylated histone H3 or RNA-PolyII. Using immunoprecipitated DNA as a template, the PCR with primer pairs as shown under Methods amplified the acetylated histone H3-bound LOX core promoter region with 136 bp, and the RNA-Poly II bound fragment of the GAPDH promoter (an internal control) with 90 bp, respectively. PCR products were analyzed on 2.2% agarose gels and densities of DNA bands measured by the 1D Scan software.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306850&req=5

ijerph-12-00064-f006: Inactivation of the LOX core promoter in NNK treated cells. ChIP and PCR assays were performed to elucidate the active status of the LOX core promoter in treated cells by assessing acetylated histone H3 binding to the core promoter region. DNAs were isolated from control and NNK treated cells each with 2 × 106, sonicated and immunoprecipitated with an antibody against acetylated histone H3 or RNA-PolyII. Using immunoprecipitated DNA as a template, the PCR with primer pairs as shown under Methods amplified the acetylated histone H3-bound LOX core promoter region with 136 bp, and the RNA-Poly II bound fragment of the GAPDH promoter (an internal control) with 90 bp, respectively. PCR products were analyzed on 2.2% agarose gels and densities of DNA bands measured by the 1D Scan software.
Mentions: To elucidate the active status of the LOX core promoter in response to NNK, quantitation of acetylated histone H3 at the LOX Inr-DPE region was performed by the ChIP assay [19]. The anti-diacetylated histone H3 antibody was used to precipitate DNA fragments isolated from control and NNK-treated cells. Using primers as described, the PCR amplified a 136 bp fragment (−95/+41, relative to ATG) containing the LOX core promoter from −53 to −14 and an intact transcription start site cluster from −78 to −51 [19]. As shown in Figure 6, in comparison to the internal control, the GAPDH fragment bound with the RNA-PolyII, the histone H3 acetylated at the tested region of the LOX promoter in NNK-treated cells was reduced to 74, 41, 11 and 5% of the control for cells treated with 10, 30, 100 and 300 µM NNK, respectively. These results indicated NNK inactivation of the LOX core promoter as a key mechanism for down-regulation of this enzyme at the promoter level.

Bottom Line: Exposure of cells to NNK reduced levels of steady-states LOX mRNA and new transcript synthesis.At the promoter level, NNK enhanced methylation of CpG, but decreased acetylation of histone H3 at the core promoter region of the LOX gene.These results indicated that transcriptional and translational processes of LOX are major targets for NNK.

View Article: PubMed Central - PubMed

Affiliation: The Central Lab, Hebei United University Affinity Hospital, Tangshan 063000, China. gcheng911@163.com.

ABSTRACT
A tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is believed to contribute to the cancer burden in cigarette smokers. To evaluate NNK effects on the expression of lysyl oxidase (LOX), a tumor suppressor, we examined this enzyme at various levels in NNK-treated rat fetal lung fibroblasts (RFL6). Exposure of cells to NNK reduced levels of steady-states LOX mRNA and new transcript synthesis. NNK inhibited all LOX protein species in a dose-dependent manner. Although 300 µM NNK markedly decreased the level in the 46 kDa preproenzyme, under same conditions, there was no detectable amounts of the 50 kDa proenzyme and the 32 kDa mature enzyme suggesting NNK perturbing the LOX protein processing to its mature form. Moreover, NNK also suppressed LOX activities in conditioned media of treated cells. At the promoter level, NNK enhanced methylation of CpG, but decreased acetylation of histone H3 at the core promoter region of the LOX gene. These results indicated that transcriptional and translational processes of LOX are major targets for NNK. Thus, inactivation of tumor suppressor gene LOX may play a critical role in NNK carcinogenesis.

Show MeSH
Related in: MedlinePlus