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[Cancer Research 62, 947-952, February 1, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

A Misspliced Form of the Cholecystokinin-B/Gastrin Receptor in Pancreatic Carcinoma

Role of Reduced Cellular U2AF35 and a Suboptimal 3'-Splicing Site Leading to Retention of the Fourth Intron1

Wei-Qun Ding, Susan M. Kuntz and Laurence J. Miller2

Center for Basic Research in Digestive Diseases and the Department of Biochemistry/Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abnormal splicing of primary RNA transcripts of normal genes is a recognized mechanism for the production of some abnormal proteins found in cancer cells. A misspliced form of the cholecystokinin-B/gastrin (CCK-B) receptor recently was reported to be present in colon carcinoma, where it was postulated to play a role in stimulating tumor growth (M. R. Hellmich et al., J. Biol. Chem., 275: 32122–32128, 2000). Here, we report the presence of the same abnormal protein in pancreatic carcinoma and explore the molecular basis for this missplicing event. Reverse transcription-PCR and sequencing were used to demonstrate the presence of a misspliced form of the CCK-B receptor having its fourth intron retained in three pancreatic cancer cell lines and in tumor tissue, but not in surrounding healthy pancreas, from two patients with pancreatic carcinoma. A mini-gene construct representing the region of this gene from its third through its fifth exon and containing the two intervening introns was produced and transiently expressed in the MIA PaCa-2 human pancreatic cancer cell line. Specific reverse transcription-PCR reactions with both vector-derived and receptor-specific primers demonstrated the presence of both correctly fully spliced and selectively misspliced forms of this receptor. Mutagenesis of the mini-gene demonstrated that a suboptimal sequence at the 3'-end of intron 4 contributed to this missplicing. This focused attention on the U2 small nuclear ribonucleoprotein particle auxiliary splicing factors (U2AFs) known to interact specifically with this domain. Indeed, quantitative real-time PCR demonstrated a reduced level of expression of one of these factors, U2AF35, in pancreatic cancer cells compared with healthy pancreas. Furthermore, the relative amount of missplicing of the CCK-B receptor mini-gene in the pancreatic cancer cell line was reversed by transfection of the cells with U2AF35 cDNA. This work describes the presence of an additional abnormal protein in pancreatic cancer and describes a new molecular mechanism for its production, providing additional potential therapeutic targets.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A large number of molecular changes have been described in cancer cells relative to their healthy cells of origin (1 , 2) . These include changes in levels in normal proteins and in mutations that change the actions or regulation of other proteins. Genetic instability, inadequate DNA mismatch repair, and increased oncogene expression have been well described in various cancers. Hormones and receptors are key molecules for the regulation of cellular functions such as cell growth, a process that can play a role in the development and progression of neoplasia.

The current work was focused on the CCK-B3 receptor, which has been reported to be expressed in human pancreas and in pancreatic carcinomas (3, 4, 5) , and has been described to have trophic effects on various normal and neoplastic tissues (6) . This includes the description of trophic actions of gastrin on pancreatic carcinoma cell lines (4 , 7) . Our examination of the expression of this receptor on pancreatic carcinomas and pancreatic cancer cell lines resulted in the identification of a misspliced form of this receptor in which intron 4 is retained. This is identical to the molecule identified recently in colon carcinomas (8) . In that report, this abnormal spliceoform of the CCK-B receptor was shown to have constitutive activity and to exert trophic effects on cells expressing it (8) .

Indeed, abnormal processing of primary RNA transcripts has been reported to occur in various pathological conditions (9 , 10) . Prominent among these are alternative splicing events that have been reported to occur in neoplasms (11, 12, 13, 14) .

We have extended the current descriptive observation with mechanistic studies to better understand the molecular basis for the abnormal expression of this variant spliceoform of the CCK-B receptor. This was accomplished with an in vitro model system to investigate the processing of RNA transcripts coming from a mini-gene construct spanning the region of the CCK-B receptor gene that was misspliced. This resulted in further focus on the 3'-end of the fourth intron and the U2AF known to act on that region (15) , and on the potential roles of its protein components in the pathogenesis of this event.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Pancreatic Tissue and Cell Lines.
Clinical biospecimens (pancreatic carcinomas and surrounding healthy pancreas in the excised specimens) were collected and used with the approval of the Mayo Clinic Institutional Review Board.

Human pancreatic carcinoma cell lines, MIA PaCa-2, Panc-1, and Capan-2 (American Type Culture Collection, Manassas, VA), were used for this study. MIA PaCa-2 and Panc-1 cells were cultured in DMEM supplemented with 88 mM sodium bicarbonate, 10% FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin. Capan-2 cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 100 units/ml penicillin, and 100 µg/ml streptomycin. All cells were kept at 37°C, in a humidified environment containing 5% CO2.

Determination of Forms of CCK-B Receptor and Other Relevant Molecules Expressed in Tissue and Cell Lines.
RT-PCR was used to define forms of CCK-B receptor mRNA expressed. Total RNA was isolated from tissues or cell lines using the SV Total RNA Isolation System (Promega, Madison, WI), following the recommended protocol. First-strand cDNA was synthesized using SuperScript II (Invitrogen, Carlsbad, CA) with 5 µg of RNA as template. The reactions contained 5 µM random hexamer oligonucleotide primers, 10 mM dithioerythritol, 0.5 mM dNTP mix, 1x first-strand buffer, and 20 units of SuperScript II in a total volume of 20 µl. RNA was denatured at 65°C for 5 min, chilled on ice, and incubated at room temperature for 10 min before adding reverse transcriptase. The reaction was allowed to proceed for 40 min at 42°C and was stopped by heating to 70°C for 15 min. Aliquots of 2 µl of the resulting cDNA solution were amplified by PCR in a 50-µl reaction volume containing 0.5 mM dNTP mix and 0.2 µM sense and antisense oligonucleotide primers (Table 1)Citation . The cDNA was denatured at 94°C for 5 min before 0.5 µl of Taq DNA polymerase (5 units) were added. Incubation and thermal cycling conditions were 94°C for 1 min, 56°C for 2 min, and 72°C for 3 min, repeating this for 35 cycles. For nested PCR, 2 µl of the initial PCR reaction mixture were subjected to a second round of PCR, following similar thermal cycling for 30 repetitions. The products were separated on 1% agarose gels containing ethidium bromide and were visualized under UV light.


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  		Table 1 Primers used for PCR amplification

For reference sequences, see GenBank accession numbers L10822 (CCK-B receptor), M96982 (U2AF35), and NM_007279 (U2AF65).

 
mRNA Quantitation.
Real-time PCR was performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, IN) to quantify relevant mRNA expression. Oligonucleotide primers used for U2AF35 and U2AF65 amplifications are shown in Table 1Citation . RNA (18S) was detected as an internal control. PCR reactions were carried out in 20-µl volumes containing 4 mM MgCl2, 0.2 µM dNTP mix, 0.5 µM sense and antisense primers, 0.05% BSA, 0.3x Syber Green dye, and 0.5 unit Platinum Gold Taq DNA polymerase. Amplification was allowed to proceed for 40 cycles of sequential melting (10 s, 95°C) and annealing/extension (20 s, 52°C) steps. The mRNA expression levels were measured by determining specific fluorescence peak areas for each reaction. This was normalized to 18S RNA amplification, with results expressed as relative fluorescence levels.

CCK-B Receptor Mini-Gene Construction and Mutation.
The TRIzol reagent (Invitrogen, Carlsbad, CA) was used to isolate DNA from MIA PaCa-2 cells to be used as a template for production of mini-gene constructs. DNA (1 µg) was subjected to PCR amplification with Taq DNA polymerase using oligonucleotide primers spanning the last three exons and two introns of the CCK-B receptor gene (sense primer: 2406–2424 of GenBank accession no. L10822, 5'-TGT CTG TGA GTG TGT CCA C-3'; antisense primer: 3867–3850, 5'-CCT CTA CTC CTC AGC CAG-3'). DNA was separated on a 1% agarose gel and a fragment corresponding to the size of the expected product was cut from the gel. This DNA fragment was purified using the Qiagen II DNA purification kit (Qiagen, Valencia, CA) and subcloned into the PCR II vector (Invitrogen). The identity of this mini-gene was verified by DNA sequencing. It was then excised and subcloned into the pBK-CMV eukaryotic expression vector (Stratagene, La Jolla, CA) at the BamHI/XhoI sites. This expression construct was modified using the QuikChange Kit (Stratagene), such that the mini-gene was located just downstream of the T3 promoter of the pBK-CMV vector. Primers used for this modification include the following: sense, 5'-CAC TAA AGG GAT GTC TGT GAG TGT GTC CAC GCT AAG CC-3'; and antisense, 5'-GGC TTA GCG TGG ACA CAC TCA CAG ACA TCC CTT TAG TG-3'. Mutation of the splicing sites of the mini-gene was performed using the QuikChange Kit (Stratagene).

Lipofectin (Invitrogen) was used to transfect MIA PaCa-2 cells for transient expression studies. Briefly, 0.5 x 106 cells were plated in 100-mm dishes 24 h before transfection. Lipofectin (25 µl) was mixed with 200 µl of Opti-MEM and was incubated at room temperature for 40 min before adding 5 µg of a cDNA-vector construct. Cells were washed once with the Opti-MEM and were covered with 3 ml of Opti-MEM containing the DNA-Lipofectin complex. After 5 h of incubation at 37°C, the medium was replaced with DMEM containing supplements.

U2AF cDNA Cloning and Expression Studies.
Oligonucleotide primers spanning the full length of U2AF35 and U2AF65 were used with PWO DNA polymerase (Roche Molecular Biochemicals) to amplify cDNA from MIA PaCa-2 cells (Table 1)Citation . The amplified 723-bp DNA fragment representing U2AF35 was separated on a 1% agarose gel, purified, and ligated into the pBK-CMV expression vector at BamHI/XbaI sites. The identity of the products was verified by direct DNA sequencing. Because the sequence of the U2AF35 that was isolated was distinct from the wild-type sequence that has been reported in GenBank, this construct was reengineered by mutagenesis using the QuikChange Kit (Stratagene) to achieve the wild-type sequence. This U2AF35 fragment was also cloned into the pEYFP-C1 vector (Clontech) at SacI/ApaI sites to produce a fluorescent fusion protein for morphological studies. After DNA sequencing, the 1.42-kb DNA fragment representing U2AF65 was also cloned into the pBK-CMV vector at BamHI/XbaI, and into the pEYFP-C1 vector at EcoRI/KpnI sites.

Confocal Microscopic Examination of the YFP-U2AF35 Constructs.
COS-1 and MIA PaCa-2 cells were transfected with the variant and wild-type YFP-U2AF35 constructs, and grown in DMEM with supplements. Twenty-four hours after transfection, cells were plated onto 24-well plates containing 12-mm coverslips (50,000 cells/well). Forty-eight hours after transfection, cells were washed with PBS and were fixed for 30 min at room temperature using 2% paraformaldehyde in PBS. After fixation, cells were washed three times with PBS, mounted on slides in Vectashield (Vector Laboratories, Burlingame, CA), and examined using a Zeiss confocal microscope.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Alternative Splicing of CCK-B Receptor Pre-mRNA in Pancreatic Cancer Cells.
The presence of CCK-B receptor mRNA was examined using PCR and nested PCR techniques. RNA isolated from pancreatic cancer specimens and tumor-free pancreatic tissue from two patients and three pancreatic cancer cell lines were subjected to two rounds of PCR, representing amplification of the full length of the receptor cDNA and subsequent nested reactions. Tumor-free pancreatic tissue only yielded a normal full-length PCR product and a single amplified DNA fragment in the region of interest, whereas two distinct fragments in this region were detected in the pancreatic carcinoma specimens from these patients (Fig. 1)Citation . These two DNA fragments were cloned into the PCR II vector (Invitrogen) and sequenced. This confirmed that the short fragment (314 bp) represented the correctly processed CCK-B receptor mRNA, whereas the longer one (521 bp) represented a misspliced variant of this receptor having the fourth intron (207 bp) retained. The same CCK-B receptor splice variant recently was reported to be present in some human colon carcinomas (8) .



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  		Fig. 1. Expression of CCK-B receptor mRNA in pancreatic cancer cells. Total RNA from normal (Nl panc) and neoplastic pancreatic tissue (PC) from two patients with pancreatic carcinoma and three pancreatic cancer cell lines (PANC-1, Capan-2, and MIA PaCa-2) were analyzed by RT-PCR. Tumor-free tissue from the same patients was used as a control. The results shown are representative of three experiments, showing the nested PCR reactions that demonstrate the variant products identified in the pancreatic cancer cells. Also shown is a schematic diagram of the structure of the CCK-B receptor gene and the primers used for the full-length and subsequent nested PCR reactions.

 
Critical Role of the Region of the 3'-Splice Site of the Fourth Intron in the Alternative Splicing of the CCK-B Receptor Pre-mRNA in Pancreatic Cancer Cells.
To study possible cellular mechanisms for the retention of the fourth intron in pancreatic cancer cells, a CCK-B receptor mini-gene spanning the last three exons and two introns of the gene was prepared and expressed in MIA PaCa-2 cells. These cells were able to transcribe and process, or partially process, the pre-mRNA coming from this template. A specific assay was established and validated that involved RT-PCR, using a vector-derived T3 oligonucleotide primer and a mini-gene internal primer (Fig. 2)Citation . DNA sequencing confirmed the identity of these two products as representing the normally processed form having both introns excised, and the misspliced variant in which the third intron was excised and the fourth intron was retained, similar to the form found in the cancer specimens.



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  		Fig. 2. Detection of the CCK-B receptor mini-gene expression in MIA PaCa-2 cells. MIA PaCa-2 cells were transfected with the CCK-B receptor mini-gene (4 µg of DNA per 100-mm dish) and cultured in DMEM with supplements. Forty-eight hours after transfection, total RNA was isolated from the cells and subjected to first strand cDNA synthesis and PCR using a vector-derived T3 primer and an internal mini-gene primer (strategy shown in the schematic diagram on the right). Shown is a typical pattern of amplification in control MIA PaCa-2 cells without transfection and those cells transfected with the CCK-B receptor mini-gene.

 
The role of the regions of the 5'- and 3'-splice sites of the fourth intron in this alternative splicing event was then examined using a series of mini-gene constructs. This was performed by reengineering of the 5'- and the 3'-ends of the fourth intron of the CCK-B receptor gene, to achieve the optimal consensus splicing sites (16 , 17 ; Table 2Citation ). Interestingly, optimization of the 5'-end had no effect on the splicing of the mini-gene, whereas modification of the 3'-end dramatically improved the splicing process. This was reflected in an increase in the amounts of intron-free, normally processed mRNA, and the concurrent reduction in the amounts of the spliceoform selectively retaining the fourth intron (Fig. 3)Citation . This indicates that the 3'-splice site of the fourth intron is suboptimal and plays an important role in the retention of the fourth intron of the CCK-B receptor gene in cancer cells.


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  		Table 2 Sequences of wild-type and reengineered splice sites in the fourth intron of the CCK-B receptor gene

Shown are the sequences for the 5' (donor)- and 3' (acceptor)-splice sites in the fourth intron and modifications made in these sites (underlined nucleotides). Consensus sequences were derived for mammalian splice sites (16 , 17) . The reengineered forms were designed to make the relevant sequences more like consensus ones (17) .

 


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  		Fig. 3. Effects of mutation of the 5'- and 3'-splice sites of the fourth intron on the splicing of the CCK-B receptor mini-gene in MIA PaCa-2 cells. MIA PaCa-2 cells were transfected with the CCK-B receptor mini-gene and its reengineered constructs, and assayed as described in Fig. 2Citation . RNA (18S) was amplified from the same cDNA, indicating similar amounts of cDNA in each sample. The right panel shows the densitometry of the ratio of the intron-free, normally processed fragment over the fragment retaining intron 4. Data are given as means ± SE of data from three independent experiments. *, P < 0.05 relative to processing of wild-type construct.

 
The Involvement of U2AF35 in the Retention of the Fourth Intron of the CCK-B Receptor in Pancreatic Cancer Cells.
We then focused our attention on the splicing factors known to act specifically on 3'-splice sites of introns. PCR reactions demonstrated the presence of full-length forms of both U2AF35 and U2AF65 in a series of pancreatic cancer cell lines, pancreatic carcinomas, and healthy pancreas, with no forms of variant length identified. Both of these auxiliary factors present in the MIA PaCa-2 cells were isolated and fully sequenced. The U2AF65 sequence was identical to the wild-type sequence in GenBank (18) , whereas the sequence of the U2AF35 that was isolated was distinct from the wild-type sequence (19) . The deduced amino acid sequence of U2AF35 that was isolated from this pancreatic cancer cell line was identical to that of wild-type U2AF35, except that one glycine was missing from a 12-glycine repeat and the arginine at position 151 of the peptide was replaced with a lysine (Fig. 4)Citation . Two independently amplified and isolated clones confirmed this variant sequence. This could represent a variant isoform or allele or even a mutation.



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  		Fig. 4. Deduced amino acid sequence of U2AF35 cloned from MIA PaCa-2 cells. Shown is the translation of the cloned cDNA of the isolated U2AF35 (Variant), and its comparison with the wild-type (WT) U2AF35 sequence from GenBank (differences highlighted).

 
As a first order indication of the possible functional effect of this variant sequence of the U2AF35, a fluorescent fusion protein construct was prepared, linking YFP to the cDNA. As controls, analogous fluorescent fusion constructs were prepared with the wild-type U2AF35 cDNA and with U2AF65. These were expressed transiently in the MIA PaCa-2 cells and in COS cells and studied morphologically. Of note, there was no difference in cellular localization of either U2AF35 construct in either cell (Fig. 5)Citation . Both the variant and wild-type forms localized to the nucleus of the model cell and of the pancreatic cancer cell line in which the missplicing event had been shown to occur. The wild-type U2AF65 was also localized to the nucleus (data not shown).



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  		Fig. 5. Expression of fluorescent U2AF35 in COS-1 and MIA PaCa-2 cells. Cells were transfected with expression constructs including YFP fused with cDNAs encoding for wild-type U2AF35 and variant U2AF35, as well as YFP as a soluble protein (4 µg of DNA per 100-mm dish). At 48 h after transfection, the cells were fixed and examined with a Zeiss 510 confocal microscope. The nuclear localization of both variants of U2AF35 in both types of cells was clearly evident, whereas soluble YFP is present diffusely throughout the cells. Shown are representative images of two experiments.

 
We also studied the levels of expression of the U2AF35 and the U2AF65 in the pancreatic cancer cell lines and in the pancreatic carcinomas using quantitative real-time PCR (Fig. 6)Citation . The expression level of U2AF35 in cancer cells was approximately 50% of that in tumor-free pancreatic tissue, whereas the expression level of U2AF65 was the same in all preparations. This raised the possibility that low expression of U2AF35 plus a suboptimal 3'-splice site leads to the retention of the fourth intron of the CCK-B receptor gene in the cancer environment. To test this hypothesis, cDNAs representing both U2AF65 and U2AF35 were inserted into expression vectors and were coexpressed with the CCK-B receptor mini-gene in MIA PaCa-2 cells. Overexpression of U2AF65 in these cells had no effect on the splicing of the mini-gene; but in cells that overexpressed the wild-type U2AF35 or the variant sequence U2AF35, which was isolated from the cancer cell line, the splicing of the CCK-B receptor mini-gene was significantly improved (Fig. 7)Citation . These data support the view that U2AF35 is a key cellular factor that is critically involved in the retention of the fourth intron of the CCK-B receptor gene in mRNA in cancer cells.



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  		Fig. 6. Quantitation of U2AF35 and U2AF65 mRNA in pancreatic cancer cells. RNA isolated from pancreatic cancer cells and adjacent healthy pancreas was subjected to real-time PCR reactions using specific primers (Table 1)Citation . The mRNA expression is presented as relative fluorescence levels that have been normalized relative to levels of 18S RNA. Data are given as means ± SE of data from three independent experiments. *, P < 0.05 relative to levels in normal pancreas.

 


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  		Fig. 7. Effects of overexpression of U2AF35 and U2AF65 on the splicing of the CCK-B receptor mini-gene in MIA PaCa-2 cells. MIA PaCa-2 cells were cotransfected with the CCK-B receptor mini-gene and the U2AF35 or U2AF65 cDNA constructs. A total of 3 µg of each DNA construct were used for cotransfection. In control cells, the CCK-B receptor mini-gene was cotransfected with empty vector. At 48 h after transfection, total RNA was isolated from the cells and subjected to first strand cDNA synthesis and PCR using the vector-derived T3 primer and the internal mini-gene primer. The lower panel shows the densitometry of the ratio of the intron-free, normally processed fragment over the fragment retaining intron 4. Data are given as means ± SE of data from three independent experiments. *, P < 0.05 relative to control ratio.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present work, we have identified the expression of a misspliced variant of the CCK-B receptor mRNA in which the fourth intron is retained in pancreatic carcinomas and pancreatic cancer cell lines but is not present in mRNA from healthy pancreas adjacent to the tumors. This is the same abnormal spliceoform of this receptor that was recently described to be present in colon carcinoma (8) , where it was constitutively active and had trophic effects (8) . This variant may mediate the trophic effect of gastrin in pancreatic cancer, which was described previously (4 , 7 , 20) and may play an important role in the development and progression of this highly lethal cancer.

There is a significant difference in the pattern of CCK-B receptor expression in the pancreatic carcinomas now described and in the colon carcinomas reported previously (8) . This difference relates to the exclusive presence of the misspliced form of this mRNA in the colon carcinomas and the simultaneous presence of normal and abnormal splicing in the pancreatic carcinomas. This may reflect the much more prominent expression of the normally processed CCK-B receptor gene in healthy pancreas than in colon (21) . Although gastrin has been reported to have a trophic influence on the normal colon (22 , 23) , it has been difficult or impossible to demonstrate a normal receptor that might mediate that response (21) . Although there is the assumption that the molecular basis of the missplicing of the pre-mRNA for the CCK-B receptor is similar in both of these neoplasms, the defect may be even more marked in the colon carcinomas than that identified in the current work. This has not yet been studied.

A key contribution of the present report relates to the molecular basis for the missplicing event observed. This was investigated using an in vitro mini-gene expression, transcription, and splicing system. The domain spanning the abnormally spliced region of the CCK-B receptor gene was expressed in the MIA PaCa-2 pancreatic cancer cell line. An assay was developed that was able to establish the pattern of processing of the pre-mRNA transcript generated from this mini-gene that was not affected by the endogenous expression of the CCK-B receptor in these cells. This was achieved by using RT-PCR with oligonucleotide primers that included a vector primer not found in the normal human genome, as well as a gene-specific primer. It is noteworthy that this assay system was able to demonstrate the same missplicing event in the pancreatic cancer cell line that was observed in the pancreatic carcinomas.

This assay was also able to gain insights into the molecular mechanism of the missplicing of this pre-mRNA in these cells. It was demonstrated that the reengineering of the 3'-splice site of intron 4, but not the 5'-splice site of this intron, to the consensus sequence was able to markedly improve the splicing of the primary transcript from the mini-gene in these cells. The misspliced mRNA was markedly reduced, whereas the normally spliced mRNA was increased. This was true with the cellular environment unchanged.

Similarly, the nonoptimal 3'-splice site in intron 4 of the CCK-B receptor gene was not enough to explain the missplicing event observed in pancreatic and colonic carcinomas. Clearly, that sequence is able to be recognized and allow the efficient excision of this intron in healthy cells. This was even true in the healthy pancreatic tissue surrounding the pancreatic carcinomas studied in this work. This points toward a primary event of abnormal splicing machinery in the cancer.

Indeed, there is strong precedent for aberrant pre-mRNA splicing in various pathological states (10 , 15 , 24) . Prominent among these are malignant neoplasms (11 , 12 , 14) . Changes in splicing factor activity during tumor development have also been demonstrated (13) . It is of considerable interest that one of the major settings for regulation of alternative splicing is during development (24) . The cellular processes of carcinogenesis often reflect events early in development. This may suggest that the program of changes in pre-mRNA splicing that is observed in cancer may relate to a developmental program that is normal for a different developmental stage.

There are more than 50 proteins that have been identified as playing a role in the processes relevant to RNA splicing (15) . The specific roles and sites of action of many of these proteins are now known. Because the 3'-splice site was shown to play an important role in the retention of the fourth intron of the CCK-B receptor gene, we focused our attention on the splicing factors that are believed to act on that portion of an intron during pre-mRNA splicing of mammalian genes. Recently, a model has been proposed that illustrates the U2AF interacting with the region of the intron near the 3'-splice site (25) . U2AF is a heterodimeric splicing factor composed of 65-kDa (U2AF65; 18 ) and 35-kDa (U2AF35; 19 ) subunits. The large subunit, U2AF65, recognizes the polypyrimidine tract, a sequence located close to the 3'-splice site dinucleotide AG that serves as an important splicing signal for pre-mRNA processing (26) . The small subunit, U2AF35, has been shown to specifically recognize the 3'-end dinucleotide AG, and is essential for pre-mRNA splicing (27, 28, 29) .

In the present study, real-time PCR revealed that the expression of U2AF65 was similar in pancreatic cancer cells and tumor-free tissue, whereas the expression of U2AF35 was significantly lower in cancer cells than in normal tissue. Not only was the U2AF35 expression level reduced in the pancreatic carcinomas and pancreatic cancer cell lines, but there was also a variant form of this protein expressed in the MIA PaCa-2 cells. The U2AF35 that was isolated from these cells differed from the wild-type protein sequence that was isolated previously and entered in GenBank by having two amino acid changes. These included the deletion of one glycine in a 12-glycine-repeat domain and the isocharge replacement of an arginine (151) with a lysine. It was important, therefore, to determine whether this represented functionally important changes in sequence. We achieved this by comparing the transfection of this variant sequence with the transfection of the reengineered wild-type sequence. Of note, there was no difference between the two, and they both were expressed and were localized to the nucleus of the cell after transfection. To our knowledge, the polymorphism of the U2AF35 demonstrated in this study has not been reported previously.

The involvement of the low expression of U2AF35 in the retention of the fourth intron of the CCK-B receptor gene was confirmed by the observation that transfection of the MIA PaCa-2 cells to express additional U2AF35, but not U2AF65, improved the splicing of the CCK-B receptor mini-gene. Little is known about the regulation of expression of U2AF35 in cells. The reduced expression level of this splicing factor in pancreatic cancer cells may reflect reduced transcription of this gene or increased degradation of the mRNA. These possibilities need to be investigated directly.


   ACKNOWLEDGMENTS
 
We acknowledge the helpful discussions with Dr. Eric Wieben and the assistance in manuscript and graphic preparation of E. M. Hadac.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the National Institutes of Health (Grant DK32878) and the Fiterman Foundation. Back

2 To whom requests for reprints should be addressed, at Center for Basic Research in Digestive Diseases, Guggenheim 17, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905. Phone: (507) 284-0680; Fax: (507) 284-0762; E-mail: miller{at}mayo.edu Back

3 The abbreviations used are: CCK-B, cholecystokinin-B/gastrin; RT-PCR, reverse transcription-polymerase chain reaction; dNTP, deoxynucleotide triphosphate; U2AF, U2 small nuclear ribonucleoprotein particle auxiliary splicing factor; YFP, yellow fluorescent protein. Back

Received 10/ 1/01. Accepted 12/ 3/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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