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The immunoglobulin heavy chain VH6-1 promoter regulates Ig transcription in non-B cells.

Wu L, Liu Y, Zhu X, Zhang L, Chen J, Zhang H, Hao P, Zhang S, Huang J, Zheng J, Zhang Y, Zhang Y, Qiu X - Cancer Cell Int. (2014)

Bottom Line: Our data showed that the sequence 1200 bp upstream of VH6-1 exhibited promoter activity in both B and non-B cells.No new regulatory elements were identified within the region 1200 bp to 300 bp upstream of VH6-1.The regulatory mechanisms among different cell types controlling the production of IgM heavy chains are worth discussing.

View Article: PubMed Central - PubMed

Affiliation: Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Central Laboratory, Peking University Cancer Hospital & Institute, Beijing, 100142 China.

ABSTRACT

Background: Non-B cell immunoglobulins (Igs) are widely expressed in epithelial cancer cells. The past 20 years of research have demonstrated that non-B cell Igs are associated with cancer cell proliferation, the cellular cytoskeleton and cancer stem cells. In this study we explored the transcriptional mechanism of IgM production in non-B cells.

Methods: The promoter region of a V-segment of the heavy mu chain gene (VH6-1) was cloned from a colon cancer cell line HT-29. Next, the promoter activities in non-B cells and B-cells were detected using the dual-luciferase reporter assay. Then the transcription factor binding to the promoter regions was evaluated by electrophoretic mobility shift assays (EMSAs) and gel supershift experiments.

Results: Our data showed that the sequence 1200 bp upstream of VH6-1 exhibited promoter activity in both B and non-B cells. No new regulatory elements were identified within the region 1200 bp to 300 bp upstream of VH6-1. In addition, Oct-1 was found to bind to the octamer element of the Ig gene promoter in cancer cells, in contrast to B cells, which utilize the transcriptional factor Oct-2.

Conclusion: The regulatory mechanisms among different cell types controlling the production of IgM heavy chains are worth discussing.

No MeSH data available.


Related in: MedlinePlus

The 5′-flanking sequence of VH6-1 exhibits promoter activity in non-B cancer cells. (A) Schematic diagram of 5′-flanking 1.2-kb pGL3 construct. (B) The 1.2-kb fragments amplified from upstream of VH6-1 in HT-29 cells by PCR. (C) The 1.2-kb pGL3 construct was transfected into HeLa, HeLa MR, HeLa S3, HT-29, MDA-MB-231, HEK 293,L02, HK2, Raji or Jurkat cells. Luciferase activity was measured using a dual-luciferase reporter system. The results are representative of three independent experiments after normalization to renilla luciferase activity (internal controls). Each bar represents mean ± SD.
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Fig1: The 5′-flanking sequence of VH6-1 exhibits promoter activity in non-B cancer cells. (A) Schematic diagram of 5′-flanking 1.2-kb pGL3 construct. (B) The 1.2-kb fragments amplified from upstream of VH6-1 in HT-29 cells by PCR. (C) The 1.2-kb pGL3 construct was transfected into HeLa, HeLa MR, HeLa S3, HT-29, MDA-MB-231, HEK 293,L02, HK2, Raji or Jurkat cells. Luciferase activity was measured using a dual-luciferase reporter system. The results are representative of three independent experiments after normalization to renilla luciferase activity (internal controls). Each bar represents mean ± SD.

Mentions: The Ig VH core promoter is primarily composed of a conserved TATA box and the octamer element that is located within 100 bp of the transcriptional start site. We first amplified the upstream 1.2-kb DNA fragment of VH6-1 from HT-29 (a colon cancer cell line) by PCR and cloned this fragment into a pGL3 luciferase reporter vector named pGL3-1.2 kb (Figure 1A&B). To examine the promoter activity of pGL3-1.2 kb, dual-luciferase reporter assays were performed in ten cell lines, including HT-29, three cervical cancer cell lines HeLa, HeLa MR and HeLa S3,a breast cancer cell line MDA-MB-231, three immortalized diploid cell lines HEK293, L02 and HK2, Raji cells as a positive control and Jurkat cells as a negative control. The 5′ flanking sequence of VH6-1 demonstrated higher promoter activity in nine cell lines than the pGL3 control and even exhibited higher activity than the Simian Virus 40 (SV40) promoter, which demonstrated high activity in mammalian cell lines (Figure 1C). As expected, the 1.2-kb construct exhibited no activity in Jurkat cells.Figure 1


The immunoglobulin heavy chain VH6-1 promoter regulates Ig transcription in non-B cells.

Wu L, Liu Y, Zhu X, Zhang L, Chen J, Zhang H, Hao P, Zhang S, Huang J, Zheng J, Zhang Y, Zhang Y, Qiu X - Cancer Cell Int. (2014)

The 5′-flanking sequence of VH6-1 exhibits promoter activity in non-B cancer cells. (A) Schematic diagram of 5′-flanking 1.2-kb pGL3 construct. (B) The 1.2-kb fragments amplified from upstream of VH6-1 in HT-29 cells by PCR. (C) The 1.2-kb pGL3 construct was transfected into HeLa, HeLa MR, HeLa S3, HT-29, MDA-MB-231, HEK 293,L02, HK2, Raji or Jurkat cells. Luciferase activity was measured using a dual-luciferase reporter system. The results are representative of three independent experiments after normalization to renilla luciferase activity (internal controls). Each bar represents mean ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4260249&req=5

Fig1: The 5′-flanking sequence of VH6-1 exhibits promoter activity in non-B cancer cells. (A) Schematic diagram of 5′-flanking 1.2-kb pGL3 construct. (B) The 1.2-kb fragments amplified from upstream of VH6-1 in HT-29 cells by PCR. (C) The 1.2-kb pGL3 construct was transfected into HeLa, HeLa MR, HeLa S3, HT-29, MDA-MB-231, HEK 293,L02, HK2, Raji or Jurkat cells. Luciferase activity was measured using a dual-luciferase reporter system. The results are representative of three independent experiments after normalization to renilla luciferase activity (internal controls). Each bar represents mean ± SD.
Mentions: The Ig VH core promoter is primarily composed of a conserved TATA box and the octamer element that is located within 100 bp of the transcriptional start site. We first amplified the upstream 1.2-kb DNA fragment of VH6-1 from HT-29 (a colon cancer cell line) by PCR and cloned this fragment into a pGL3 luciferase reporter vector named pGL3-1.2 kb (Figure 1A&B). To examine the promoter activity of pGL3-1.2 kb, dual-luciferase reporter assays were performed in ten cell lines, including HT-29, three cervical cancer cell lines HeLa, HeLa MR and HeLa S3,a breast cancer cell line MDA-MB-231, three immortalized diploid cell lines HEK293, L02 and HK2, Raji cells as a positive control and Jurkat cells as a negative control. The 5′ flanking sequence of VH6-1 demonstrated higher promoter activity in nine cell lines than the pGL3 control and even exhibited higher activity than the Simian Virus 40 (SV40) promoter, which demonstrated high activity in mammalian cell lines (Figure 1C). As expected, the 1.2-kb construct exhibited no activity in Jurkat cells.Figure 1

Bottom Line: Our data showed that the sequence 1200 bp upstream of VH6-1 exhibited promoter activity in both B and non-B cells.No new regulatory elements were identified within the region 1200 bp to 300 bp upstream of VH6-1.The regulatory mechanisms among different cell types controlling the production of IgM heavy chains are worth discussing.

View Article: PubMed Central - PubMed

Affiliation: Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Central Laboratory, Peking University Cancer Hospital & Institute, Beijing, 100142 China.

ABSTRACT

Background: Non-B cell immunoglobulins (Igs) are widely expressed in epithelial cancer cells. The past 20 years of research have demonstrated that non-B cell Igs are associated with cancer cell proliferation, the cellular cytoskeleton and cancer stem cells. In this study we explored the transcriptional mechanism of IgM production in non-B cells.

Methods: The promoter region of a V-segment of the heavy mu chain gene (VH6-1) was cloned from a colon cancer cell line HT-29. Next, the promoter activities in non-B cells and B-cells were detected using the dual-luciferase reporter assay. Then the transcription factor binding to the promoter regions was evaluated by electrophoretic mobility shift assays (EMSAs) and gel supershift experiments.

Results: Our data showed that the sequence 1200 bp upstream of VH6-1 exhibited promoter activity in both B and non-B cells. No new regulatory elements were identified within the region 1200 bp to 300 bp upstream of VH6-1. In addition, Oct-1 was found to bind to the octamer element of the Ig gene promoter in cancer cells, in contrast to B cells, which utilize the transcriptional factor Oct-2.

Conclusion: The regulatory mechanisms among different cell types controlling the production of IgM heavy chains are worth discussing.

No MeSH data available.


Related in: MedlinePlus