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. 2008 Aug 12:9:74.
doi: 10.1186/1471-2199-9-74.

Differential expression of the TFIIIB subunits Brf1 and Brf2 in cancer cells

Stephanie Cabarcas  1 , Joby Jacob, Ingrid Veras, Laura Schramm
Affiliations

Differential expression of the TFIIIB subunits Brf1 and Brf2 in cancer cells

Stephanie Cabarcas et al. BMC Mol Biol. .

Abstract

Background: RNA polymerase (pol) III transcription is specifically elevated in a variety of cancers and is a target of regulation by a variety of tumor suppressors and oncogenes. Accurate initiation by RNA pol III is dependent on TFIIIB. In higher eukaryotes, two forms of TFIIIB have been characterized. TFIIIB required for proper initiation from gene internal RNA pol III promoters is comprised of TBP, Bdp1, and Brf1. Proper initiation from gene external RNA pol III promoters requires TBP, Bdp1, and Brf2. We hypothesized that deregulation of RNA polymerase III transcription in cancer may be a consequence of altered TFIIIB expression

Results: Here, we report: (1) the TFIIIB subunits Brf1 and Brf2 are differentially expressed in a variety of cancer cell lines: (2) the Brf1 and Brf2 promoters differ in activity in cancer cell lines, and (3) VAI transcription is universally elevated, as compared to U6, in breast, prostate and cervical cancer cells.

Conclusion: Deregulation of TFIIIB-mediated transcription may be an important step in tumor development. We demonstrate that Brf1 and Brf2 mRNA are differentially expressed in a variety of cancer cells and that the Brf2 promoter is more active than the Brf1 promoter in all cell lines tested. We also demonstrate, that Brf1-dependent VAI transcription was significantly higher than the Brf2-dependent U6 snRNA transcription in all cancer cell lines tested. The data presented suggest that Brf2 protein expression levels correlate with U6 promoter activity in the breast, cervical and prostate cell lines tested. Interestingly, the Brf1 protein levels did not vary considerably in HeLa, MCF-7 and DU-145 cells, yet Brf1 mRNA expression varied considerably in breast, prostate and cervical cancer cell lines tested. Thus, Brf1 promoter activity and Brf1 protein expression levels did not correlate well with Brf1-dependent transcription levels. Taken together, we reason that deregulation of Brf1 and Brf2 expression could be a key mechanism responsible for the observed deregulation of RNA pol III transcription in cancer cells.

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Figures

Figure 1
Figure 1
The TFIIIB subunits Brf1 and Brf2 are differentially expressed in cancer cell lines. Total RNA was isolated from asynchronous growing Caski, HeLa, RKO, SIHA, Du 145, ZR75-1, MCF-7, MDA-MB-453, MDA-MB-231, and MDA-MB-468 cells. After first strand cDNA synthesis, diluted cDNA was used in PCR using the primers depicted in Table 1. The mRNA expression of (A) β-actin, (B) Brf1, and (C) Brf2 are shown. The expected sizes of the PCR products of the different TFIIIB subunits are depicted.
Figure 2
Figure 2
The Brf1 and Brf2 promoters differ in activity in breast, cervical and prostate cancer cell lines. (A) Schematic representation of the Brf1 promoter identified. The empty pGL3 vector (300 ng), as well as increasing concentrations of Brf1-pGL3 (100 ng, 200 ng, 300 ng) and Brf2-pGL3 (100 ng, 200 ng, 300 ng) were transiently transfected into asynchronous (A) HeLa, (B) DU145, and (C) MCF-7 cells. All luciferase assay results are expressed as relative light units (RLU): the average of the Photinus pyralis firefly activity observed divided by the average of the activity recorded from the Renilla luciferase vector. Experiments were done in triplicate, repeated three times, and representative experiments are depicted.
Figure 3
Figure 3
VAI transcription is higher than U6 snRNA transcription in HeLa, DU145, and MCF-7 cells. The empty pGL3 vector (300 ng), as well as increasing concentrations of pGL3-U6 (100 ng, 200 ng, 300 ng) and pGL3-VAI (100 ng, 200 ng, 300 ng) were transiently transfected into asynchronous (A) HeLa, (B) DU145, and (C) MCF-7 cells. All luciferase assay results are expressed as relative light units (RLU): the average of the Photinus pyralis firefly activity observed divided by the average of the activity recorded from the Renilla luciferase vector. Experiments were done in triplicate, repeated three times, and representative experiments are depicted. (D) Western blot analysis of Brf1 and Brf2 protein levels in HeLa, MCF-7 and DU145 cells. 25 μg of nuclear extract was immunoblotted with anti-Brf1 (CS1043) and anti-Brf2 (CS1228) antibodies. As loading control, membrane was also immunoblotted with an anti-actin antibody, bottom panel. Arrows depict migration of actin, Brf1 and Brf2.
Figure 4
Figure 4
Brf1 and Brf2 have a high sequence similarity to the codon determinant region of c-myc. (A) Schematic representation of Brf1, Brf2 and c-myc. Structural features are indicated. Location of nucleotide sequences aligned (red text) in lower panel are indicated by red brackets. (B) Sequence alignment of the codon determinant region of c-myc, Brf1 and Brf2. Identical amino acids in all three sequences are depicted in yellow. Sequences identical in two of the three sequences are highlighted in blue. Consensus sequence is indicated.
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