Quantitative Evaluation of Alternative Promoter Usage and 3′ Splice Variants for Parathyroid Hormone-related Protein by Real-Time Reverse Transcription-PCR

Richard, Virgile; Luchin, Alexander I.; Brena, Romulo M.; Plass, Christoph; Rosol, Thomas J.

doi:10.1373/49.8.1398

Cited by 31 publications

(26 citation statements)

References 28 publications

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“…Quantitative evaluation of HTLV-1 tax/rex and PTHrP mRNA expression and alternative promoter usage was performed as previously described. 22 RT reactions were carried out with 5 mg of total RNA according to the manufacturer's protocol using oligo(dT) [12][13][14][15][16][17][18] and 50 U of Superscript II reverse transcriptase (Invitrogen) and were diluted to 50 ml after RT. cDNA standards were prepared by PCR amplification of cDNA from human lung carcinoma cells BEN (PTHrP and b2-microglobulin) or MT-2 cells (tax/rex standards) and calibrators ranging from 250 to 2.5 Â 10 8 copies/ml were prepared by serial dilution.…”

Section: Real-time Rt-pcrmentioning

confidence: 99%

“…P1 and P3 contain a typical TATA box, [16][17][18] while P2 is a GC-rich promoter region. 19 Previous evaluation of PTHrP alternative promoter usage by qualitative 20,21 and quantitative reverse transcription (RT)-PCR 22 revealed that P3-initiated transcripts were detectable at high levels in most tumors and cell lines examined, including the (HTLV-1)-positive cell line MT-2, whereas P1 or P2-derived transcripts were not detected or expressed only in a subset of tumors.…”

Section: Introductionmentioning

confidence: 99%

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Transcriptional regulation of parathyroid hormone-related protein promoter P3 by ETS-1 in adult T-cell leukemia/lymphoma

et al. 2005

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Parathyroid hormone-related protein (PTHrP) plays a primary role in the development of humoral hypercalcemia of malignancy seen in the majority of adult T-cell leukemia/lymphoma (ATLL) patients with human T-cell lymphotropic virus type-1 (HTLV-1) infection. HTLV-1 Tax has been shown to complex with ETS-1 and SP1 to transactivate the PTHrP P3 promoter. Previously, we established a SCID/bg mouse model of human ATL with RV-ATL cells and showed that PTHrP expression was independent of Tax. In this study, we report an inverse correlation of PTHrP with tax/rex mRNA in multiple HTLV-1-positive cell lines and RV-ATL cells. Stimulation of Jurkat T cells with PMA/ionomycin upregulated the PTHrP P3 promoter by a previously characterized Ets binding site and also induced protein/DNA complex formation identical to that observed in RV-ATL cells. Further, we provide evidence that cotransfection with Ets-1 and constitutively active Mek-1 in HTLV-1-negative transformed T cells with stimulation by PMA/ionomycin not only resulted in a robust induction of PTHrP P3 but also formed a complex with ETS-1/P3 EBS similar to that in ATLL cells. Our data demonstrate that transcriptional regulation of PTHrP in ATLL cells can be controlled by T-cell receptor signaling and the ETS and MAPK ERK pathway in a Tax-independent manner.

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Section: Real-time Rt-pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Transcriptional regulation of parathyroid hormone-related protein promoter P3 by ETS-1 in adult T-cell leukemia/lymphoma

et al. 2005

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“…38 We identified two putative NF-kB-binding sequences in the human PTHrP P2 promoter that are located 1558 (kB2) and 152 (kB1) nucleotides upstream from the transcription initiation start site. Vasavada et al 39 have shown that the SmaI/Sau3A fragment of the P2 promoter had significant activity in renal carcinoma cells.…”

Section: Discussionmentioning

confidence: 99%

“…Several studies have shown that P2/P3 promoter usage is prevalent in many cancers such as breast cancer and bone cancer in addition to HTLV-1-positive and ATLL cells. [8][9][10][11] Furthermore, Brandt et al 12 reported a preferential usage of P2 over P1 and P3 promoters in different cancer cell lines examined. Although this stimulation and activity of the PTHrP P2 promoter was identified 10 years ago, little is known regarding the transcriptional mechanisms involving cis-acting regulatory sequences within this promoter.…”

Section: Introductionmentioning

confidence: 99%

Transcriptional regulation of parathyroid hormone-related protein promoter P2 by NF-κB in adult T-cell leukemia/lymphoma

et al. 2007

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Parathyroid hormone-related protein (PTHrP) plays a primary role in the development of humoral hypercalcemia of malignancy (HHM) that occurs in the majority of patients with adult T-cell leukemia/lymphoma (ATLL) due to human T-cell lymphotropic virus type-1 (HTLV-1) infection. We previously showed that ATLL cells constitutively express high levels of PTHrP via activation of promoters P2 and P3, resulting in HHM. In this study, we characterized a nuclear factor-jB (NF-jB) binding site in the P2 promoter of human PTHrP. Using electrophoretic mobility shift assays, we detected a specific complex in Taxexpressing human T cells composed of p50/c-Rel, and two distinct complexes in ATLL cells consisting of p50/p50 homodimers and a second unidentified protein(s). Chromatin immunoprecipitation assays confirmed in vivo binding of p50 and c-Rel on the PTHrP P2 promoter. Using transient co-transfection with NF-jB expression plasmids and PTHrP P2 luciferase reporter-plasmid, we showed that NF-jB p50/p50 alone and p50/ c-Rel or p50/Bcl-3 cooperatively upregulated the PTHrP P2 promoter. Furthermore, inhibition of NF-jB activity by Bay 11-7082 reduced PTHrP P2 promoter-initiated transcripts in HTLV-1-infected T cells. In summary, the data demonstrated that transcriptional regulation of PTHrP in ATLL cells can be controlled by NF-jB activation and also suggest a Taxindependent mechanism of activation of PTHrP in ATLL.

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“…P1 and P3 are canonical TATA promoters (Mangin et al, 1990;Campos et al, 1992;Suva et al, 1989) and initiate transcription at exons 1 and 4 respectively, while P2 is a GC-rich promoter that initiates transcription upstream of exon IC (Vasavada et al, 1993). Evaluation of PTHrP alternative promoter usage by qualitative (Southby et al, 1995 and and quantitative reverse transcription RT-PCR (Bouizar et al, 1999;Richard et al, 2003) revealed high concentrations of P3-initiated transcripts in most tumors, including breast cancers (Bouizar et al, 1999) and many tumor cell lines (Cataisson et al, 2003). The amino acid sequences encoded by only two of the 9 exons in the human gene are present in all transcripts.…”

Section: Gene Structurementioning

confidence: 99%

Parathyroid Hormone Related Protein: A Marker of Breast Tumor Progression and Outcome

Bouizar¹

2011

Breast Cancer - Recent Advances in Biology, Imaging and Therapeutics

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Quantitative Evaluation of Alternative Promoter Usage and 3′ Splice Variants for Parathyroid Hormone-related Protein by Real-Time Reverse Transcription-PCR

Cited by 31 publications

References 28 publications

Transcriptional regulation of parathyroid hormone-related protein promoter P3 by ETS-1 in adult T-cell leukemia/lymphoma

Transcriptional regulation of parathyroid hormone-related protein promoter P3 by ETS-1 in adult T-cell leukemia/lymphoma

Transcriptional regulation of parathyroid hormone-related protein promoter P2 by NF-κB in adult T-cell leukemia/lymphoma

Parathyroid Hormone Related Protein: A Marker of Breast Tumor Progression and Outcome

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