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cAMP responsive element binding protein-1 is a transcription factor of lysosomal-associated protein transmembrane-4 Beta in human breast cancer cells.

Zhang M, Xu JJ, Zhou RL, Zhang QY - PLoS ONE (2013)

Bottom Line: Lysosomal-associated protein transmembrane-4 beta (LAPTM4B) is a potential proto-oncogene, whose overexpression is involved in cancer occurrence and progression.However, its transcriptional regulation mechanism is still unclear.The +10+292 promoter region was possessed the highest transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical laboratory, Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute, Beijing, China.

ABSTRACT
Lysosomal-associated protein transmembrane-4 beta (LAPTM4B) is a potential proto-oncogene, whose overexpression is involved in cancer occurrence and progression. Its transcript is up-regulated in various types of solid tumors including breast cancer. However, its transcriptional regulation mechanism is still unclear. To investigate the mechanism of transcriptional regulation of LAPTM4B in human breast cancer cells, a series of luciferase reporter constructs and construct with mutated binding site for cAMP responsive element binding protein-1 (CREB1) were generated by PCR amplification and transiently transfected into breast cancer cells to determine the transcriptional activities of different promoter regions. The +10+292 promoter region was possessed the highest transcriptional activity. The ability of CREB1 to bind the LAPMT4B promoter was confirmed by electrophoretic mobility shift assay, super-shift and RNA interference experiments. Our study identified the core promoter region responsible for constitutive expression of LAPTM4B and clarified that CREB1 played an important role in LAPTM4B transcriptional regulation in human breast cancer cells.

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Related in: MedlinePlus

Analysis of the MCF7 nuclear extract proteins ability to bind dsDNA oligo targeting the LAPTM4B promoter.(A) EMSA. Nuclear extract proteins were incubated with biotin-labeled dsDNA oligo with excess of unlabeled competitors or CREB1 conserved binding site mutated competitors. (B) Super-shift. Nuclear extract proteins were incubated with biotin-labeled probe with different concentrations of CREB1 antibody. The antibody-CREB1-probe complex forms the supershit signal.
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pone-0057520-g004: Analysis of the MCF7 nuclear extract proteins ability to bind dsDNA oligo targeting the LAPTM4B promoter.(A) EMSA. Nuclear extract proteins were incubated with biotin-labeled dsDNA oligo with excess of unlabeled competitors or CREB1 conserved binding site mutated competitors. (B) Super-shift. Nuclear extract proteins were incubated with biotin-labeled probe with different concentrations of CREB1 antibody. The antibody-CREB1-probe complex forms the supershit signal.

Mentions: To determine whether CREB1 binds to the LAPTM4B core promoter, EMSA and super-shift assays were performed with MCF7 nuclear extract and biotin-labeled wild-type, unlabeled wild-type and mutant probes. After incubation with nuclear proteins, the 25 bp wild-type oligonucleotide containing the CREB1 binding motif formed protein-DNA complexes, but not with the mutant probe, which was competed out by the addition of unlabeled oligonucleotide probe. As showed in Fig. 4A, lane 1 was the migration of free-probe in the absence of nuclear extract and therefore no shift observed. Lane 2 was the CREB1-containing double-stranded DAN probe and showed a signal shift due to transcription factor binding (arrow). Lane 3 was bio-probe with 2-fold nuclear extract. The shift signal was stronger than lane 2. Lanes 4–6 showed that the shift signal can be inhibited from 50-fold, 100-fold, 200-fold excess unlabeled probe. Lanes 7–9 were CREB1 conserved binding site mutated competitors. The shift signal could not be inhibited from 50-fold, 100-fold, 200-fold excess mutated probe. Fig. 4B was the result from super-shift experiment. Lane 1 was the free probe. Lanes 2–3 were bio-probe with nuclear extract and 2-fold protein (arrow). Lanes 4–6 showed that the dsDNA-protein mixes with 1-fold, 2-fold and 4-fold specific anti-CREB1 antibodies had slower electrophoretic mobility (dashed arrow).


cAMP responsive element binding protein-1 is a transcription factor of lysosomal-associated protein transmembrane-4 Beta in human breast cancer cells.

Zhang M, Xu JJ, Zhou RL, Zhang QY - PLoS ONE (2013)

Analysis of the MCF7 nuclear extract proteins ability to bind dsDNA oligo targeting the LAPTM4B promoter.(A) EMSA. Nuclear extract proteins were incubated with biotin-labeled dsDNA oligo with excess of unlabeled competitors or CREB1 conserved binding site mutated competitors. (B) Super-shift. Nuclear extract proteins were incubated with biotin-labeled probe with different concentrations of CREB1 antibody. The antibody-CREB1-probe complex forms the supershit signal.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585361&req=5

pone-0057520-g004: Analysis of the MCF7 nuclear extract proteins ability to bind dsDNA oligo targeting the LAPTM4B promoter.(A) EMSA. Nuclear extract proteins were incubated with biotin-labeled dsDNA oligo with excess of unlabeled competitors or CREB1 conserved binding site mutated competitors. (B) Super-shift. Nuclear extract proteins were incubated with biotin-labeled probe with different concentrations of CREB1 antibody. The antibody-CREB1-probe complex forms the supershit signal.
Mentions: To determine whether CREB1 binds to the LAPTM4B core promoter, EMSA and super-shift assays were performed with MCF7 nuclear extract and biotin-labeled wild-type, unlabeled wild-type and mutant probes. After incubation with nuclear proteins, the 25 bp wild-type oligonucleotide containing the CREB1 binding motif formed protein-DNA complexes, but not with the mutant probe, which was competed out by the addition of unlabeled oligonucleotide probe. As showed in Fig. 4A, lane 1 was the migration of free-probe in the absence of nuclear extract and therefore no shift observed. Lane 2 was the CREB1-containing double-stranded DAN probe and showed a signal shift due to transcription factor binding (arrow). Lane 3 was bio-probe with 2-fold nuclear extract. The shift signal was stronger than lane 2. Lanes 4–6 showed that the shift signal can be inhibited from 50-fold, 100-fold, 200-fold excess unlabeled probe. Lanes 7–9 were CREB1 conserved binding site mutated competitors. The shift signal could not be inhibited from 50-fold, 100-fold, 200-fold excess mutated probe. Fig. 4B was the result from super-shift experiment. Lane 1 was the free probe. Lanes 2–3 were bio-probe with nuclear extract and 2-fold protein (arrow). Lanes 4–6 showed that the dsDNA-protein mixes with 1-fold, 2-fold and 4-fold specific anti-CREB1 antibodies had slower electrophoretic mobility (dashed arrow).

Bottom Line: Lysosomal-associated protein transmembrane-4 beta (LAPTM4B) is a potential proto-oncogene, whose overexpression is involved in cancer occurrence and progression.However, its transcriptional regulation mechanism is still unclear.The +10+292 promoter region was possessed the highest transcriptional activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical laboratory, Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute, Beijing, China.

ABSTRACT
Lysosomal-associated protein transmembrane-4 beta (LAPTM4B) is a potential proto-oncogene, whose overexpression is involved in cancer occurrence and progression. Its transcript is up-regulated in various types of solid tumors including breast cancer. However, its transcriptional regulation mechanism is still unclear. To investigate the mechanism of transcriptional regulation of LAPTM4B in human breast cancer cells, a series of luciferase reporter constructs and construct with mutated binding site for cAMP responsive element binding protein-1 (CREB1) were generated by PCR amplification and transiently transfected into breast cancer cells to determine the transcriptional activities of different promoter regions. The +10+292 promoter region was possessed the highest transcriptional activity. The ability of CREB1 to bind the LAPMT4B promoter was confirmed by electrophoretic mobility shift assay, super-shift and RNA interference experiments. Our study identified the core promoter region responsible for constitutive expression of LAPTM4B and clarified that CREB1 played an important role in LAPTM4B transcriptional regulation in human breast cancer cells.

Show MeSH
Related in: MedlinePlus