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Aberrant over-expression of a forkhead family member, FOXO1A, in a brain tumor cell line.

Dallas PB, Egli S, Terry PA, Kees UR - BMC Cancer (2007)

Bottom Line: In addition, two novel FOXO1A transcripts were isolated that differ from the canonical transcript by alternative splicing within the 3'-UTR.The FOXO1A open reading frame is wild type in the PER-453 cell line and the abnormally high FOXO1A mRNA expression is not due to mutations affecting the 5'-UTR or proximal promoter.Over expression of FOXO1A may be the result of PER-453 specific epimutations or imbalances in regulatory factors acting at the promoter and/or 3'-UTR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Children's Leukemia and Cancer Research, Telethon Institute for Child Health Research and Centre for Child Health Research, University of Western Australia, Perth, Australia. peterd@ichr.uwa.edu.au

ABSTRACT

Background: The mammalian FOXO (forkhead box, O subclass) proteins are a family of pleiotropic transcription factors involved in the regulation of a broad range of cellular processes critical for survival. Despite the essential and diverse roles of the FOXO family members in human cells and their involvement in tumor pathogenesis, the regulation of FOXO expression remains poorly understood. We have addressed the mechanisms underlying the high level of expression of the FOXO1A gene in a cell line, PER-453, derived from a primitive neuroectodermal tumor of the central nervous system (CNS-PNET).

Methods: The status of the FOXO1A locus in the PER-453 CNS-PNET cell line was investigated by Southern blotting and DNA sequence analysis of the proximal promoter, 5'-UTR, open reading frame and 3'-UTR. FOXO1A expression was assessed by conventional and quantitative RT-PCR, Northern and Western blotting.

Results: Quantitative real-time RT-PCR (qRT-PCR) data indicated that after normalization to ACTB mRNA levels, canonical FOXO1A mRNA expression in the PER-453 cell line was 124-fold higher than the average level of five other CNS-PNET cell lines tested, 24-fold higher than the level in whole fetal brain, and 3.5-fold higher than the level in fetal brain germinal matrix cells. No mutations within the FOXO1A open reading frame or gross rearrangements of the FOXO1A locus were detected. However, a single nucleotide change within the proximal promoter and several nucleotide changes within the 3'-UTR were identified. In addition, two novel FOXO1A transcripts were isolated that differ from the canonical transcript by alternative splicing within the 3'-UTR.

Conclusion: The CNS-PNET cell line, PER-453, expresses FOXO1A at very high levels relative to most normal and cancer cells from a broad range of tissues. The FOXO1A open reading frame is wild type in the PER-453 cell line and the abnormally high FOXO1A mRNA expression is not due to mutations affecting the 5'-UTR or proximal promoter. Over expression of FOXO1A may be the result of PER-453 specific epimutations or imbalances in regulatory factors acting at the promoter and/or 3'-UTR.

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DNA sequence of the alternatively spliced region of the FOXO1A 3'-UTR. The alternatively spliced regions of the FOXO1A-2 and FOXO1A-3 variants are in plain text. DNA sequence flanking the spliced region is in bold. The alternatively spliced region of the FOXO1A-2 transcript is underlined and is 34 bp shorter than the region spliced from the FOXO1A-3 transcript. The FOXO1A stop codon is boxed. Primers used to amplify the alternatively spliced region are in italics and underlined with an arrow.
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Figure 4: DNA sequence of the alternatively spliced region of the FOXO1A 3'-UTR. The alternatively spliced regions of the FOXO1A-2 and FOXO1A-3 variants are in plain text. DNA sequence flanking the spliced region is in bold. The alternatively spliced region of the FOXO1A-2 transcript is underlined and is 34 bp shorter than the region spliced from the FOXO1A-3 transcript. The FOXO1A stop codon is boxed. Primers used to amplify the alternatively spliced region are in italics and underlined with an arrow.

Mentions: The FOXO1A mRNA has a particularly long 3'-UTR of 3356 nt representing nearly 60% of the length of the transcript (Fig. 2c). The extent of the FOXO1A 3'-UTR suggests that this region may play an important role in the regulation of FOXO1A transcript stability or translation. We did not identify any gross rearrangements in the FOXO1A 3'-UTR from PER-453 by DNA sequence analysis. However, we identified five single nucleotide differences including two previously identified SNPs (Table 3 and Fig. 3a). In addition to the canonical cDNA sequence, we identified two FOXO1A splice variants designated FOXO1A-2 and FOXO1A-3, both of which are differentially spliced in a manner consistent with the GT/AG rule from the same splice donor site as the canonical transcript, 14 bp downstream from the stop codon within the 3'-UTR (Fig. 3b and Fig 4). The sequences of these cDNAs were identical to the canonical FOXO1A cDNA except for the splicing out of 492 bp (extending from nt 2368 – 2859, FOXO1A-2) and 526 bp (extending from nt 2368–2893, FOXO1A-3)(Fig. 4). We did not obtain evidence for any other FOXO1A alternative splicing events from any of our RT-PCR analyses, which also included amplification of the entire FOXO1A cDNA as two overlapping RT-PCR products. These data indicated that the FOXO1A-2 and FOXO1A-3 cDNAs were derived from transcripts that were identical to the canonical transcript except for the alternative splicing within the 3'-UTR. The lower signal intensity and size of the ~5.4 kb molecular weight band relative to the canonical ~5.9 kb transcript visible on the Northern blot (Fig. 1a) was consistent both with the expected sizes of the smaller alternative transcripts and the levels of the RT-PCR products generated from the two splice variants relative to the canonical FOXO1A RT-PCR product when observed on ethidium bromide stained agarose gels (Fig. 3b).


Aberrant over-expression of a forkhead family member, FOXO1A, in a brain tumor cell line.

Dallas PB, Egli S, Terry PA, Kees UR - BMC Cancer (2007)

DNA sequence of the alternatively spliced region of the FOXO1A 3'-UTR. The alternatively spliced regions of the FOXO1A-2 and FOXO1A-3 variants are in plain text. DNA sequence flanking the spliced region is in bold. The alternatively spliced region of the FOXO1A-2 transcript is underlined and is 34 bp shorter than the region spliced from the FOXO1A-3 transcript. The FOXO1A stop codon is boxed. Primers used to amplify the alternatively spliced region are in italics and underlined with an arrow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1878494&req=5

Figure 4: DNA sequence of the alternatively spliced region of the FOXO1A 3'-UTR. The alternatively spliced regions of the FOXO1A-2 and FOXO1A-3 variants are in plain text. DNA sequence flanking the spliced region is in bold. The alternatively spliced region of the FOXO1A-2 transcript is underlined and is 34 bp shorter than the region spliced from the FOXO1A-3 transcript. The FOXO1A stop codon is boxed. Primers used to amplify the alternatively spliced region are in italics and underlined with an arrow.
Mentions: The FOXO1A mRNA has a particularly long 3'-UTR of 3356 nt representing nearly 60% of the length of the transcript (Fig. 2c). The extent of the FOXO1A 3'-UTR suggests that this region may play an important role in the regulation of FOXO1A transcript stability or translation. We did not identify any gross rearrangements in the FOXO1A 3'-UTR from PER-453 by DNA sequence analysis. However, we identified five single nucleotide differences including two previously identified SNPs (Table 3 and Fig. 3a). In addition to the canonical cDNA sequence, we identified two FOXO1A splice variants designated FOXO1A-2 and FOXO1A-3, both of which are differentially spliced in a manner consistent with the GT/AG rule from the same splice donor site as the canonical transcript, 14 bp downstream from the stop codon within the 3'-UTR (Fig. 3b and Fig 4). The sequences of these cDNAs were identical to the canonical FOXO1A cDNA except for the splicing out of 492 bp (extending from nt 2368 – 2859, FOXO1A-2) and 526 bp (extending from nt 2368–2893, FOXO1A-3)(Fig. 4). We did not obtain evidence for any other FOXO1A alternative splicing events from any of our RT-PCR analyses, which also included amplification of the entire FOXO1A cDNA as two overlapping RT-PCR products. These data indicated that the FOXO1A-2 and FOXO1A-3 cDNAs were derived from transcripts that were identical to the canonical transcript except for the alternative splicing within the 3'-UTR. The lower signal intensity and size of the ~5.4 kb molecular weight band relative to the canonical ~5.9 kb transcript visible on the Northern blot (Fig. 1a) was consistent both with the expected sizes of the smaller alternative transcripts and the levels of the RT-PCR products generated from the two splice variants relative to the canonical FOXO1A RT-PCR product when observed on ethidium bromide stained agarose gels (Fig. 3b).

Bottom Line: In addition, two novel FOXO1A transcripts were isolated that differ from the canonical transcript by alternative splicing within the 3'-UTR.The FOXO1A open reading frame is wild type in the PER-453 cell line and the abnormally high FOXO1A mRNA expression is not due to mutations affecting the 5'-UTR or proximal promoter.Over expression of FOXO1A may be the result of PER-453 specific epimutations or imbalances in regulatory factors acting at the promoter and/or 3'-UTR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Children's Leukemia and Cancer Research, Telethon Institute for Child Health Research and Centre for Child Health Research, University of Western Australia, Perth, Australia. peterd@ichr.uwa.edu.au

ABSTRACT

Background: The mammalian FOXO (forkhead box, O subclass) proteins are a family of pleiotropic transcription factors involved in the regulation of a broad range of cellular processes critical for survival. Despite the essential and diverse roles of the FOXO family members in human cells and their involvement in tumor pathogenesis, the regulation of FOXO expression remains poorly understood. We have addressed the mechanisms underlying the high level of expression of the FOXO1A gene in a cell line, PER-453, derived from a primitive neuroectodermal tumor of the central nervous system (CNS-PNET).

Methods: The status of the FOXO1A locus in the PER-453 CNS-PNET cell line was investigated by Southern blotting and DNA sequence analysis of the proximal promoter, 5'-UTR, open reading frame and 3'-UTR. FOXO1A expression was assessed by conventional and quantitative RT-PCR, Northern and Western blotting.

Results: Quantitative real-time RT-PCR (qRT-PCR) data indicated that after normalization to ACTB mRNA levels, canonical FOXO1A mRNA expression in the PER-453 cell line was 124-fold higher than the average level of five other CNS-PNET cell lines tested, 24-fold higher than the level in whole fetal brain, and 3.5-fold higher than the level in fetal brain germinal matrix cells. No mutations within the FOXO1A open reading frame or gross rearrangements of the FOXO1A locus were detected. However, a single nucleotide change within the proximal promoter and several nucleotide changes within the 3'-UTR were identified. In addition, two novel FOXO1A transcripts were isolated that differ from the canonical transcript by alternative splicing within the 3'-UTR.

Conclusion: The CNS-PNET cell line, PER-453, expresses FOXO1A at very high levels relative to most normal and cancer cells from a broad range of tissues. The FOXO1A open reading frame is wild type in the PER-453 cell line and the abnormally high FOXO1A mRNA expression is not due to mutations affecting the 5'-UTR or proximal promoter. Over expression of FOXO1A may be the result of PER-453 specific epimutations or imbalances in regulatory factors acting at the promoter and/or 3'-UTR.

Show MeSH
Related in: MedlinePlus