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Tumor suppressor BLU inhibits proliferation of nasopharyngeal carcinoma cells by regulation of cell cycle, c-Jun N-terminal kinase and the cyclin D1 promoter.

Zhang X, Liu H, Li B, Huang P, Shao J, He Z - BMC Cancer (2012)

Bottom Line: BLU was re-expressed in nasopharyngeal carcinoma cells by transfection or viral infection.The data for each pair of groups were compared with Student t tests.BLU inhibits clonogenic growth of nasopharyngeal carcinoma cells, arrests cell cycle at G1 phase, downregulates JNK and cyclin D1 promoter activities, and inhibits phosphorylation of c-Jun.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathophysiology, Guangdong Medical College, 1 Xincheng Road, Song-Shan Lake SSL Science Technology and Industrial Park Dongguan, Guangdong, 523808, China. Zhangxn_2006@126.com

ABSTRACT

Background: Tumor suppressor genes function to regulate and block tumor cell proliferation. To explore the mechanisms underlying the tumor suppression of BLU/ZMYND10 gene on a frequently lost human chromosomal region, an adenoviral vector with BLU cDNA insert was constructed.

Methods: BLU was re-expressed in nasopharyngeal carcinoma cells by transfection or viral infection. Clonogenic growth was assayed; cell cycle was analyzed by flow cytometry-based DNA content detection; c-Jun N-terminal kinase (JNK) and cyclin D1 promoter activities were measured by reporter gene assay, and phosphorylation was measured by immunoblotting. The data for each pair of groups were compared with Student t tests.

Results: BLU inhibits clonogenic growth of nasopharyngeal carcinoma cells, arrests cell cycle at G1 phase, downregulates JNK and cyclin D1 promoter activities, and inhibits phosphorylation of c-Jun.

Conclusions: BLU inhibits growth of nasopharyngeal carcinoma cells by regulation of the JNK-cyclin D1 axis to exert tumor suppression.

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Related in: MedlinePlus

BLUexpression downregulates cyclin D1 promoter and JNK activity, and inhibits phosphorylation of c-Jun. (A) CNE-2 cells were transfected with pCD316 vector or pCD316-BLU, and cell lysates were immunoblotted and probed with goat anti-human BLU polyclonal antibody. The membranes were stripped and re-probed with anti-actin mAb clone C4. (B) Expression of BLU inhibits cyclin D1 promoter activity. pCD316 and pCD316p-BLU plasmids were co-transfected with cyclin D1 promoter reporter at concentration ratios of 1:1.5 and 1:4, respectively. The luciferase activity was measured for the two conditions, and the reporter activity was presented as the ratio of the two. The data are presented as the mean ± SD, and are derived from at least three independent tests. *Indicates t < 0.05 when compared with the measured values from the two groups. (C) pCD316-BLU and empty vector were co-transfected with JNK reporter at concentration ratios of 1:1.5 and 1:4, and the reporter activity was calculated. The data re presented as mean ± SD, and were derived from at least three independent tests. *Indicates P < 0.05 when comparing the calculated values for the relative light units between the two groups. (D) CNE-2 cells were infected with 0, 10, 50 and 100 PFU Ad BLU (lanes 1, 2, 3, 4), and ectopic expression was demonstrated by immunoblotting and probing with anti BLU goat polyclonal, anti actin mAb, anti phospho-c-Jun (P-c-Jun) and anti-c-Jun rabbit polyclonal antibodies. c-Jun phosphorylation was inhibited by infection with 100 PFU Ad BLU.
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Figure 3: BLUexpression downregulates cyclin D1 promoter and JNK activity, and inhibits phosphorylation of c-Jun. (A) CNE-2 cells were transfected with pCD316 vector or pCD316-BLU, and cell lysates were immunoblotted and probed with goat anti-human BLU polyclonal antibody. The membranes were stripped and re-probed with anti-actin mAb clone C4. (B) Expression of BLU inhibits cyclin D1 promoter activity. pCD316 and pCD316p-BLU plasmids were co-transfected with cyclin D1 promoter reporter at concentration ratios of 1:1.5 and 1:4, respectively. The luciferase activity was measured for the two conditions, and the reporter activity was presented as the ratio of the two. The data are presented as the mean ± SD, and are derived from at least three independent tests. *Indicates t < 0.05 when compared with the measured values from the two groups. (C) pCD316-BLU and empty vector were co-transfected with JNK reporter at concentration ratios of 1:1.5 and 1:4, and the reporter activity was calculated. The data re presented as mean ± SD, and were derived from at least three independent tests. *Indicates P < 0.05 when comparing the calculated values for the relative light units between the two groups. (D) CNE-2 cells were infected with 0, 10, 50 and 100 PFU Ad BLU (lanes 1, 2, 3, 4), and ectopic expression was demonstrated by immunoblotting and probing with anti BLU goat polyclonal, anti actin mAb, anti phospho-c-Jun (P-c-Jun) and anti-c-Jun rabbit polyclonal antibodies. c-Jun phosphorylation was inhibited by infection with 100 PFU Ad BLU.

Mentions: Cyclin D1 protein is involved in the process of permitting cells to enter S phase. Consistent with its role in regulating G1/S phase progression, BLU expression dramatically reduced cyclin D1 level (Figure 3A). This is in contrast to previous findings in RASSF1A-ectopically expressing cells, in that regulation of the cyclin D1 promoter does not appear to be inhibited upon RASSF1A expression [11]. However, expression of BLU in CNE-2 cells significantly inhibited the activity of the cyclin D1 promoter, as manifested by the different levels of the luciferase gene co-expressed with pCD316-BLU (Figure 3B). Transcription of cyclin D1 has been suggested to be regulated by the c-Jun transcription factor, because of the presence of a c-Jun activation site on its promoter [11]. Modulation of JNK activity by BLU was tested by co-transfection of the AP1 reporter, in which luciferase expression is driven by activated JNK. We found that BLU dramatically blocks the reporter (Figure 3A) and leads to the inhibition of c-Jun phosphorylation at a dose of 100 PFU per cell (Figure 3C and D).


Tumor suppressor BLU inhibits proliferation of nasopharyngeal carcinoma cells by regulation of cell cycle, c-Jun N-terminal kinase and the cyclin D1 promoter.

Zhang X, Liu H, Li B, Huang P, Shao J, He Z - BMC Cancer (2012)

BLUexpression downregulates cyclin D1 promoter and JNK activity, and inhibits phosphorylation of c-Jun. (A) CNE-2 cells were transfected with pCD316 vector or pCD316-BLU, and cell lysates were immunoblotted and probed with goat anti-human BLU polyclonal antibody. The membranes were stripped and re-probed with anti-actin mAb clone C4. (B) Expression of BLU inhibits cyclin D1 promoter activity. pCD316 and pCD316p-BLU plasmids were co-transfected with cyclin D1 promoter reporter at concentration ratios of 1:1.5 and 1:4, respectively. The luciferase activity was measured for the two conditions, and the reporter activity was presented as the ratio of the two. The data are presented as the mean ± SD, and are derived from at least three independent tests. *Indicates t < 0.05 when compared with the measured values from the two groups. (C) pCD316-BLU and empty vector were co-transfected with JNK reporter at concentration ratios of 1:1.5 and 1:4, and the reporter activity was calculated. The data re presented as mean ± SD, and were derived from at least three independent tests. *Indicates P < 0.05 when comparing the calculated values for the relative light units between the two groups. (D) CNE-2 cells were infected with 0, 10, 50 and 100 PFU Ad BLU (lanes 1, 2, 3, 4), and ectopic expression was demonstrated by immunoblotting and probing with anti BLU goat polyclonal, anti actin mAb, anti phospho-c-Jun (P-c-Jun) and anti-c-Jun rabbit polyclonal antibodies. c-Jun phosphorylation was inhibited by infection with 100 PFU Ad BLU.
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Related In: Results  -  Collection

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Figure 3: BLUexpression downregulates cyclin D1 promoter and JNK activity, and inhibits phosphorylation of c-Jun. (A) CNE-2 cells were transfected with pCD316 vector or pCD316-BLU, and cell lysates were immunoblotted and probed with goat anti-human BLU polyclonal antibody. The membranes were stripped and re-probed with anti-actin mAb clone C4. (B) Expression of BLU inhibits cyclin D1 promoter activity. pCD316 and pCD316p-BLU plasmids were co-transfected with cyclin D1 promoter reporter at concentration ratios of 1:1.5 and 1:4, respectively. The luciferase activity was measured for the two conditions, and the reporter activity was presented as the ratio of the two. The data are presented as the mean ± SD, and are derived from at least three independent tests. *Indicates t < 0.05 when compared with the measured values from the two groups. (C) pCD316-BLU and empty vector were co-transfected with JNK reporter at concentration ratios of 1:1.5 and 1:4, and the reporter activity was calculated. The data re presented as mean ± SD, and were derived from at least three independent tests. *Indicates P < 0.05 when comparing the calculated values for the relative light units between the two groups. (D) CNE-2 cells were infected with 0, 10, 50 and 100 PFU Ad BLU (lanes 1, 2, 3, 4), and ectopic expression was demonstrated by immunoblotting and probing with anti BLU goat polyclonal, anti actin mAb, anti phospho-c-Jun (P-c-Jun) and anti-c-Jun rabbit polyclonal antibodies. c-Jun phosphorylation was inhibited by infection with 100 PFU Ad BLU.
Mentions: Cyclin D1 protein is involved in the process of permitting cells to enter S phase. Consistent with its role in regulating G1/S phase progression, BLU expression dramatically reduced cyclin D1 level (Figure 3A). This is in contrast to previous findings in RASSF1A-ectopically expressing cells, in that regulation of the cyclin D1 promoter does not appear to be inhibited upon RASSF1A expression [11]. However, expression of BLU in CNE-2 cells significantly inhibited the activity of the cyclin D1 promoter, as manifested by the different levels of the luciferase gene co-expressed with pCD316-BLU (Figure 3B). Transcription of cyclin D1 has been suggested to be regulated by the c-Jun transcription factor, because of the presence of a c-Jun activation site on its promoter [11]. Modulation of JNK activity by BLU was tested by co-transfection of the AP1 reporter, in which luciferase expression is driven by activated JNK. We found that BLU dramatically blocks the reporter (Figure 3A) and leads to the inhibition of c-Jun phosphorylation at a dose of 100 PFU per cell (Figure 3C and D).

Bottom Line: BLU was re-expressed in nasopharyngeal carcinoma cells by transfection or viral infection.The data for each pair of groups were compared with Student t tests.BLU inhibits clonogenic growth of nasopharyngeal carcinoma cells, arrests cell cycle at G1 phase, downregulates JNK and cyclin D1 promoter activities, and inhibits phosphorylation of c-Jun.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathophysiology, Guangdong Medical College, 1 Xincheng Road, Song-Shan Lake SSL Science Technology and Industrial Park Dongguan, Guangdong, 523808, China. Zhangxn_2006@126.com

ABSTRACT

Background: Tumor suppressor genes function to regulate and block tumor cell proliferation. To explore the mechanisms underlying the tumor suppression of BLU/ZMYND10 gene on a frequently lost human chromosomal region, an adenoviral vector with BLU cDNA insert was constructed.

Methods: BLU was re-expressed in nasopharyngeal carcinoma cells by transfection or viral infection. Clonogenic growth was assayed; cell cycle was analyzed by flow cytometry-based DNA content detection; c-Jun N-terminal kinase (JNK) and cyclin D1 promoter activities were measured by reporter gene assay, and phosphorylation was measured by immunoblotting. The data for each pair of groups were compared with Student t tests.

Results: BLU inhibits clonogenic growth of nasopharyngeal carcinoma cells, arrests cell cycle at G1 phase, downregulates JNK and cyclin D1 promoter activities, and inhibits phosphorylation of c-Jun.

Conclusions: BLU inhibits growth of nasopharyngeal carcinoma cells by regulation of the JNK-cyclin D1 axis to exert tumor suppression.

Show MeSH
Related in: MedlinePlus