Characterization of human mesothelin transcripts in ovarian and pancreatic cancer

Abstract

Background: Mesothelin is an attractive target for cancer immunotherapy due to its restricted expression in normal tissues and high level expression in several tumor types including ovarian and pancreatic adenocarcinomas. Three mesothelin transcript variants have been reported, but their relative expression in normal tissues and tumors has been poorly characterized. The goal of the present study was to clarify which mesothelin transcript variants are commonly expressed in human tumors.

Methods: Human genomic and EST nucleotide sequences in the public databases were used to evaluate sequences reported for the three mesothelin transcript variants in silico. Subsequently, RNA samples from normal ovary, ovarian and pancreatic carcinoma cell lines, and primary ovarian tumors were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing to directly identify expressed transcripts.

Results: In silico comparisons of genomic DNA sequences with available EST sequences supported expression of mesothelin transcript variants 1 and 3, but there were no sequence matches for transcript variant 2. Newly-derived nucleotide sequences of RT-PCR products from tissues and cell lines corresponded to mesothelin transcript variant 1. Mesothelin transcript variant 2 was not detected. Transcript variant 3 was observed as a small percentage of total mesothelin amplification products from all studied cell lines and tissues. Fractionation of nuclear and cytoplasmic RNA indicated that variant 3 was present primarily in the nuclear fraction. Thus, mesothelin transcript variant 3 may represent incompletely processed hnRNA.

Conclusion: Mesothelin transcript variant 1 represents the predominant mature mRNA species expressed by both normal and tumor cells. This conclusion should be important for future development of cancer immunotherapies, diagnostic tests, and gene microarray studies targeting mesothelin.

Figure 1

Amino acid sequences of mesothelin transcript variants 1, 2 and 3. Amino acids in single-letter code are displayed for maximal sequence alignment of translated mesothelin variant 1 (tr1, GenBank accession NM_005823), variant 2 (tr2, NM_013404) and variant 3 (tr3, AF180951). (.) indicates amino acid identity, and (-) represents gaps for optimal sequence alignment. Note that tr3 is a partial cDNA lacking N-terminal sequence.

Figure 2

Human genomic DNA sequence alignment with mesothelin transcript variants. Selected portions of human genome sequences corresponding to the mesothelin gene (AL031258) were aligned with corresponding sequences of reported mesothelin transcript variants. (-) represents sequence gaps for optimal alignment. A. The 5'-ends of mesothelin transcript variants 1 and 2 (tr1, NM_005823; tr2, NM_013404) aligned with the genomic DNA sequence. Lower case letters represent presumed untranslated nucleotide sequences. B. Nucleotide sequence at the exon 13 5'-boundary using the same reference database sequences as in (A). Lower case letters in the genomic sequence indicate presumed introns for transcript variant 1. Splice acceptor sites potentially utilized by transcript variants 1 and 2 are underlined, and the arrowhead indicates a third splice acceptor detected by sequence analysis in this study. C. Genomic sequence for the intron 16 region are aligned with sequences for transcript variants 1 (tr1, NM_005823) and 3 (tr3, AF180951). Lower case letters represent nucleotides presumed to be untranslated for tr1. Expected amino acid sequences are shown above each transcript, highlighting the intron 16 insert and reading frame shift predicted for tr3.

Figure 3

RT-PCR analysis of mesothelin transcripts. Primers designed to amplify full length mesothelin from transcript variants 1 and 2 (Table 1, line 1) were used in RT-PCR analyses of total RNA from cell lines and tissues, and analyzed by agarose gel electrophoresis. M, molecular size markers; Lane 1, HeLa; Lane 2, OVCAR-3; Lane 3, Caov-4; Lane 4, SKOV-3; Lane 5, UCI 101; Lane 6, AsPC-1; Lane7, PANC-1; Lanes 8–10, three individual normal ovary tissues; Lanes 11–16, six individual primary ovarian tumors; Lane 17, no template PCR control.

Figure 4

RT-PCR analysis of potential mesothelin exon 13 splicing differences between transcript variants 1 and 2. A. Diagram of the PCR strategy, showing predicted size of amplified products from transcript variants 1 and 2. PCR primers (Table 1, line 2) are indicated by arrows. B. Agarose gel analysis of representative RT-PCR results. Lane M, molecular size marker; Lane 1, HeLa; Lane 2, OVCAR-3; Lane 3, Caov-4; Lane 4, SKOV-3; Lane 5, UCI 101; Lane 6, AsPC-1; Lane7, PANC-1; Lanes 8–9, two individual normal human ovaries; Lanes 10–14, five individual primary ovarian tumors; Lane 15, no template PCR control.

Figure 5

RT-PCR analysis of potential mesothelin exon 16–17 splicing differences between transcript variants 1 and 3. A. Diagram of the PCR strategy, showing predicted size of amplified products from transcript variants 1 and 3. PCR primers (Table 1, line 3) are indicated by arrows. B. Agarose gel analysis of representative RT-PCR results. Lane M, molecular size markers; Lane 1, no template PCR control; Lane 2, PANC-1; Lane 3, AsPc-1; Lane 4, HeLa; Lane 5, OVCAR-3; Lane 5, normal ovary tissue sample; Lane 6–8, three individual primary ovarian tumors. The product with a size between 262 and 344 bp represents heteroduplex formation between transcript variants 1 and 3 (tr1, tr3). C. RT-PCR and agarose gel analysis of full length mesothelin (transcript 1 versus 3) in cytoplasmic (C) versus nuclear (N) RNA fractions as a template, using primers in Table 1, line 4. Lane M, molecular size marker; Lane 1, OVCAR-3 cytoplasmic fraction; Lane 2, OVCAR-3 nuclear fraction; Lane 3, AsPC-1 cytoplasmic fraction; Lane 4, AsPC-1 nuclear fraction; Lane 5, HeLa cytoplasmic fraction; Lane 6, HeLa nuclear fraction; (--), no template PCR control.