Limits...
Identification of a human TFPI-2 splice variant that is upregulated in human tumor tissues.

Kempaiah P, Chand HS, Kisiel W - Mol. Cancer (2007)

Bottom Line: Previous studies have shown that the expression of tissue factor pathway inhibitor-2 (TFPI-2), a matrix-associated Kunitz-type serine proteinase inhibitor, is markedly down-regulated in several tumor cells through hypermethylation of the TFPI-2 gene promoter.In the present study, RT-PCR analysis of total RNA from both human normal and tumor cells revealed a novel 289 nucleotide splice variant of the TFPI-2 transcript designated as aberrantly-spliced TFPI-2 (asTFPI-2).In spite of the absence of a 5'-UTR or poly (A)+ tail, the asTFPI-2 variant exhibited a half-life of ~16 h in tumor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA. pkempaiah@salud.unm.edu

ABSTRACT

Background: Previous studies have shown that the expression of tissue factor pathway inhibitor-2 (TFPI-2), a matrix-associated Kunitz-type serine proteinase inhibitor, is markedly down-regulated in several tumor cells through hypermethylation of the TFPI-2 gene promoter. In the present study, RT-PCR analysis of total RNA from both human normal and tumor cells revealed a novel 289 nucleotide splice variant of the TFPI-2 transcript designated as aberrantly-spliced TFPI-2 (asTFPI-2).

Results: Nucleotide sequence analyses indicated that asTFPI-2 consists of complete exons II and V, fused with several nucleotides derived from exons III and IV, as well as six nucleotides derived from intron C. 5'- and 3'-RACE analyses of total RNA amplified exclusively the wild-type TFPI-2 transcript, indicating that asTFPI-2 lacks either a 5'-untranslated region (UTR) or a 3'-poly (A)+ tail. Quantitative real-time RT-PCR analyses revealed that several human tumor cells contain 4 to 50-fold more copies of asTFPI-2 in comparison to normal cells. In spite of the absence of a 5'-UTR or poly (A)+ tail, the asTFPI-2 variant exhibited a half-life of ~16 h in tumor cells.

Conclusion: Our studies reveal the existence of a novel, aberrantly-spliced TFPI-2 transcript predominantly expressed in tumor cells and provides suggestive evidence for an additional mechanism for tumor cells to down-regulate TFPI-2 protein expression enhancing their ability to degrade the extracellular matrix.

Show MeSH

Related in: MedlinePlus

A schematic representation of the full-length TFPI-2 gene and a novel TFPI-2 splice variant. (A) The full-length TFPI-2 gene consists of 5 exons (designated by roman numerals) and 4 introns (designated by letters). (B) The novel splice variant reported here is generated by splicing of complete exon II, 15 nucleotides of exon III (AAAGTTCCCAAAGTT), 6 nucleotides of (GAATCC) intron C, 7 nucleotides of exon IV (GCAAAAG) and complete exon V. (B) The complete (289 bp) asTFPI-2 cDNA sequence is shown in 5' to 3' orientation. (C) The nucleotide sequences at the splice site junctions that result in the generation of asTFPI-2. The arabic numbers in the TFPI-2 gene diagram (A) correspond to the splice site locations. Splice site numbers 1–3 and 8–9 have normal consensus splice sites, whereas sites 4–7 and 10 are non-consensus splice sites. The intronic sequences are presented in lower case letters, while the exonic sequence is presented in upper case letters.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1828166&req=5

Figure 1: A schematic representation of the full-length TFPI-2 gene and a novel TFPI-2 splice variant. (A) The full-length TFPI-2 gene consists of 5 exons (designated by roman numerals) and 4 introns (designated by letters). (B) The novel splice variant reported here is generated by splicing of complete exon II, 15 nucleotides of exon III (AAAGTTCCCAAAGTT), 6 nucleotides of (GAATCC) intron C, 7 nucleotides of exon IV (GCAAAAG) and complete exon V. (B) The complete (289 bp) asTFPI-2 cDNA sequence is shown in 5' to 3' orientation. (C) The nucleotide sequences at the splice site junctions that result in the generation of asTFPI-2. The arabic numbers in the TFPI-2 gene diagram (A) correspond to the splice site locations. Splice site numbers 1–3 and 8–9 have normal consensus splice sites, whereas sites 4–7 and 10 are non-consensus splice sites. The intronic sequences are presented in lower case letters, while the exonic sequence is presented in upper case letters.

Mentions: In preliminary studies designed to assess the levels of TFPI-2 transcripts in various normal and tumor tissues, co-amplification of a lower molecular weight cDNA was observed following RT-PCR of total RNA. The low Mr cDNA was faintly visible in normal tissues (lung, colon and liver), but was markedly upregulated in the corresponding tumor tissues. Nucleotide sequence analyses of the low Mr cDNA amplified from the total RNA of lung tumor tissue revealed a novel, 289 nucleotide, aberrantly-spliced form of the TFPI-2 transcript designated as asTFPI-2 (Fig. 1B). Subsequent studies revealed that the nucleotide sequence of the low Mr cDNA from HepG2 cells was identical to that observed in lung tumor tissue (data not shown). Both 5' and 3' RACE analyses of total RNA derived from several tissues and cell lines tested resulted exclusively in the amplification of the normal TFPI-2 transcript. In these RACE analyses, several attempts were made to identify any 5' and 3'-untranslated regions (UTRs) by varying reaction conditions and using different sets of primers, but each attempt only amplified the 5' and 3' ends of normal TFPI-2 (data not shown). Moreover, the 5' and 3' boundaries of the asTFPI-2 were also assessed by primer walking studies using a series of primer combinations spanning the entire regions of exon I, intron A and the 3' UTR (Fig. 1). Thus, these results indicate that the aberrantly-spliced asTFPI-2 reported here lacks any unique 5' and 3'-UTRs and consists of complete exons II and V, fused with 14 nucleotides derived from exon III, 7 nucleotides derived from exon IV, and 6 nucleotides of intron C (Fig. 1A).


Identification of a human TFPI-2 splice variant that is upregulated in human tumor tissues.

Kempaiah P, Chand HS, Kisiel W - Mol. Cancer (2007)

A schematic representation of the full-length TFPI-2 gene and a novel TFPI-2 splice variant. (A) The full-length TFPI-2 gene consists of 5 exons (designated by roman numerals) and 4 introns (designated by letters). (B) The novel splice variant reported here is generated by splicing of complete exon II, 15 nucleotides of exon III (AAAGTTCCCAAAGTT), 6 nucleotides of (GAATCC) intron C, 7 nucleotides of exon IV (GCAAAAG) and complete exon V. (B) The complete (289 bp) asTFPI-2 cDNA sequence is shown in 5' to 3' orientation. (C) The nucleotide sequences at the splice site junctions that result in the generation of asTFPI-2. The arabic numbers in the TFPI-2 gene diagram (A) correspond to the splice site locations. Splice site numbers 1–3 and 8–9 have normal consensus splice sites, whereas sites 4–7 and 10 are non-consensus splice sites. The intronic sequences are presented in lower case letters, while the exonic sequence is presented in upper case letters.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1828166&req=5

Figure 1: A schematic representation of the full-length TFPI-2 gene and a novel TFPI-2 splice variant. (A) The full-length TFPI-2 gene consists of 5 exons (designated by roman numerals) and 4 introns (designated by letters). (B) The novel splice variant reported here is generated by splicing of complete exon II, 15 nucleotides of exon III (AAAGTTCCCAAAGTT), 6 nucleotides of (GAATCC) intron C, 7 nucleotides of exon IV (GCAAAAG) and complete exon V. (B) The complete (289 bp) asTFPI-2 cDNA sequence is shown in 5' to 3' orientation. (C) The nucleotide sequences at the splice site junctions that result in the generation of asTFPI-2. The arabic numbers in the TFPI-2 gene diagram (A) correspond to the splice site locations. Splice site numbers 1–3 and 8–9 have normal consensus splice sites, whereas sites 4–7 and 10 are non-consensus splice sites. The intronic sequences are presented in lower case letters, while the exonic sequence is presented in upper case letters.
Mentions: In preliminary studies designed to assess the levels of TFPI-2 transcripts in various normal and tumor tissues, co-amplification of a lower molecular weight cDNA was observed following RT-PCR of total RNA. The low Mr cDNA was faintly visible in normal tissues (lung, colon and liver), but was markedly upregulated in the corresponding tumor tissues. Nucleotide sequence analyses of the low Mr cDNA amplified from the total RNA of lung tumor tissue revealed a novel, 289 nucleotide, aberrantly-spliced form of the TFPI-2 transcript designated as asTFPI-2 (Fig. 1B). Subsequent studies revealed that the nucleotide sequence of the low Mr cDNA from HepG2 cells was identical to that observed in lung tumor tissue (data not shown). Both 5' and 3' RACE analyses of total RNA derived from several tissues and cell lines tested resulted exclusively in the amplification of the normal TFPI-2 transcript. In these RACE analyses, several attempts were made to identify any 5' and 3'-untranslated regions (UTRs) by varying reaction conditions and using different sets of primers, but each attempt only amplified the 5' and 3' ends of normal TFPI-2 (data not shown). Moreover, the 5' and 3' boundaries of the asTFPI-2 were also assessed by primer walking studies using a series of primer combinations spanning the entire regions of exon I, intron A and the 3' UTR (Fig. 1). Thus, these results indicate that the aberrantly-spliced asTFPI-2 reported here lacks any unique 5' and 3'-UTRs and consists of complete exons II and V, fused with 14 nucleotides derived from exon III, 7 nucleotides derived from exon IV, and 6 nucleotides of intron C (Fig. 1A).

Bottom Line: Previous studies have shown that the expression of tissue factor pathway inhibitor-2 (TFPI-2), a matrix-associated Kunitz-type serine proteinase inhibitor, is markedly down-regulated in several tumor cells through hypermethylation of the TFPI-2 gene promoter.In the present study, RT-PCR analysis of total RNA from both human normal and tumor cells revealed a novel 289 nucleotide splice variant of the TFPI-2 transcript designated as aberrantly-spliced TFPI-2 (asTFPI-2).In spite of the absence of a 5'-UTR or poly (A)+ tail, the asTFPI-2 variant exhibited a half-life of ~16 h in tumor cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA. pkempaiah@salud.unm.edu

ABSTRACT

Background: Previous studies have shown that the expression of tissue factor pathway inhibitor-2 (TFPI-2), a matrix-associated Kunitz-type serine proteinase inhibitor, is markedly down-regulated in several tumor cells through hypermethylation of the TFPI-2 gene promoter. In the present study, RT-PCR analysis of total RNA from both human normal and tumor cells revealed a novel 289 nucleotide splice variant of the TFPI-2 transcript designated as aberrantly-spliced TFPI-2 (asTFPI-2).

Results: Nucleotide sequence analyses indicated that asTFPI-2 consists of complete exons II and V, fused with several nucleotides derived from exons III and IV, as well as six nucleotides derived from intron C. 5'- and 3'-RACE analyses of total RNA amplified exclusively the wild-type TFPI-2 transcript, indicating that asTFPI-2 lacks either a 5'-untranslated region (UTR) or a 3'-poly (A)+ tail. Quantitative real-time RT-PCR analyses revealed that several human tumor cells contain 4 to 50-fold more copies of asTFPI-2 in comparison to normal cells. In spite of the absence of a 5'-UTR or poly (A)+ tail, the asTFPI-2 variant exhibited a half-life of ~16 h in tumor cells.

Conclusion: Our studies reveal the existence of a novel, aberrantly-spliced TFPI-2 transcript predominantly expressed in tumor cells and provides suggestive evidence for an additional mechanism for tumor cells to down-regulate TFPI-2 protein expression enhancing their ability to degrade the extracellular matrix.

Show MeSH
Related in: MedlinePlus