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[Cancer Research 60, 56-59, January 1, 2000]
© 2000 American Association for Cancer Research

Advances in Brief

A Novel Form of Prostate-specific Antigen Transcript Produced by Alternative Splicing1

Tsutomu Tanaka, Takahiro Isono, Tatsuhiro Yoshiki2, Takeshi Yuasa and Yusaku Okada

Department of Urology [T. T., T. Yo., T. Yu., Y. O.] and Central Research Laboratory [T. I.], Shiga University of Medical Science, Shiga 520-2192, Japan


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Molecular characterization of prostate-specific antigen (PSA) has not been well elucidated, despite a great deal of clinical study. We examined the heterogeneity of PSA using reverse transcription-PCR and direct sequencing. A novel, alternatively spliced variant of the PSA transcript was found in prostate cancer (PC), as well as in benign prostatic tissue. This alternative splicing leads to the deletion of 44 amino acid residues (amino acids 45–88) from mature PSA, resulting in the loss of asparagine 45, which is a binding site for a carbohydrate chain. By these nested reverse transcription-PCR systems, this novel, alternatively spliced PSA gene was recognized in 13 of 18 (72.2%) cases with noncancerous prostate tissue, 4 of 5 (80.0%) PC cases, and 3 of 12 (25.0%) blood samples from PC patients (noncancerous prostate tissue group versus blood sample group, P = 0.011). At present, the biological significance of this alternative splicing remains to be established.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
PC3 is currently the most common malignancy in elderly males in Western countries (1) . The recent popularization of administering serum PSA tests as part of a regular health checkup or clinic visit has resulted in an increase in the number of newly diagnosed PC patients. However, PSA is produced not only by malignant cells but also by noncancerous prostate epithelial cells. Therefore, there is a substantial overlap in serum PSA levels between men with BPH and those with PC (2, 3, 4) . Recently, different molecular forms of serum PSA have been characterized. The differences in the serum ratio between free (noncomplexed) and total PSA, including PSA complexes with {alpha}1-antichymotrypsin, have been introduced to assist in the differential diagnosis between BPH and PC (5, 6, 7) . Although the basis for the heterogeneity of molecular PSA forms remains unexplained at present, such heterogeneity is widely used. We questioned whether or not a specific difference existed between cancerous and noncancerous PSA. If, in fact, they were different, then that difference might be useful for distinguishing PC from BPH. Therefore, we developed a meticulous plan to obtain the full-length PSA gene and to investigate the sequences in detail from both PC cells and noncancerous cells, respectively. Such analyses also promised to contribute to the understanding of PSA heterogeneity, which has remained a mystery since its discovery. To obtain reliable samples that did not contain PSA derived from BPH (noncancerous) cells, we preferred to use peripheral blood from patients with advanced PC rather than PC tissue itself as the source for the RT-PCR analysis. In this study, we found a novel alternative splicing phenomenon of the PSA gene. Here we report the details and frequency of this spliced gene in cancerous and noncancerous prostatic epithelial cells.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Samples.
We analyzed normal prostatic tissue from 4 patients with advanced bladder cancer who underwent radical cystoprostatectomy and hypertrophic prostatic tissues from 14 patients with BPH who underwent transurethral resection of the prostate or retropubic prostatectomy. Five PC tissues including a metastatic lymph node were obtained by radical prostatectomy or autopsy. Specimens that were 5 mm in diameter were cut in half. One half of each sample was frozen immediately and stored at -80°C until RNA extraction; the other half was fixed with neutralized buffered formalin for routine histopathological examination. We also analyzed peripheral blood samples from 12 patients with PC who had clinically metastatic disease. Cells were isolated from blood samples (10 ml each) by a density separation method using Leucoprep (Becton & Dickinson, Franklin Lakes, NJ), washed once in PBS, and stored at -80°C (8) .

Analysis of the PSA Gene by RT-PCR and Direct Sequencing.
Total cellular RNA of tissues and blood samples was isolated using the TRIzol extraction kit (Life Technologies, Inc., Rockville, MD) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 5 µg of total RNA using 20 units of RAV-2 reverse transcriptase (Takara, Otsu, Japan) and random nonamers (Takara). Primer sequences were chosen within PSA gene regions that maximized the mismatches with other genes of the same family, such as the human kallikrein gene (9 , 10) , and these sequences are shown in Fig. 2ACitation . Portions (1 µl) of the cDNA were amplified by PCR using the S1 and A1 primers. The reaction mixture (50 µl) consisted of 20 mM Tris-HCl (pH 7.5), 8 mM MgCl2, 7.5 mM DTT, 12.5 µg of BSA, 0.2 µM deoxynucleotide triphosphates, 15 µg of activated calf thymus DNA, 1.25 units of Thermophilus aquaticus (KOD Dash) polymerase (Toyobo, Osaka, Japan), and 200 ng of each primer. Amplification was performed with 30 cycles of denaturation (98°C, 10 s), annealing (60°C, 2 s), and extension (74°C, 30 s). After amplification, 5 µl of the RT-PCR products were subjected to electrophoretic analysis on a 2% agarose gel with ethidium bromide. DNA sequencing of the PCR products was performed by the dideoxy chain termination method (11) using the ABI PRISM 310 Genetic Analyzer (Applied Biosystems; Perkin-Elmer, Foster City, CA; Ref. 8 ).



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  		Fig. 2. A, the nucleotide sequences of PSA and alternatively spliced PSA. Double underlining indicates the region that is absent in the alternatively spliced variant. PSA exons are boxed, and the oligonucleotide primer sequences used are underlined and designated. Arrows show the orientation of the primers. The dotted line shows the orientation of the alternative PSA-specific primer. B, parts of the deduced amino acid sequences of PSA and alternative PSA. *, asparagine 45, which is the only N-glycosylation binding site for the carbohydrate chain in PSA. Forty-four amino acid residues were deleted by alternative splicing, and lysine subsequently appeared.

 
Analysis of the Expression of an Alternative PSA Gene by Nested RT-PCR.
To verify the reliable expression of an alternative PSA gene, we synthesized the novel alternatively spliced PSA gene-specific primer (SS1, 5'-CTGCCCACTGCATCAGGAAGC-3'), which extends 2 bases past the splice junction, according to the nucleotide sequence of human alternative PSA (Figs. 2ACitation and 3A)Citation . A normal PSA transcript cannot be amplified by the SS1 primer because this primer crosses over the spliced junction site (Fig. 2A)Citation . Subsequently, only alternatively spliced PSA transcript is amplified. Nested RT-PCR for 18 nonmalignant prostatic tissues and 5 PC tissues was performed with the first 30 cycles of denaturation (98°C, 10 s), annealing (65°C, 2 s), and extension (74°C, 30 s) using S1 and A1 primers, and the next 20 cycles of the same temperature profile using SS1 and A3 primers. Nested RT-PCR for blood samples from 12 PC patients was performed with the first 40 cycles followed by an additional 40 cycles using the same temperature profile and the primer pairs described above. To certify the existence of the PSA transcript, nested RT-PCR was performed using S1 and A1 primers followed by S4 and A2 primers, which can amplify the common part (nucleotides 619–917) of normal and alternatively spliced PSA transcripts.



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  		Fig. 3. A, graphical representation of PSA and alternative PSA transcripts and primer sites. Top, PSA transcript with complete coding region. Bottom, alternative PSA transcript from this study. and , identical regions in PSA and alternatively spliced PSA transcript, respectively. {square}, the absent region in alternatively spliced PSA transcript. The alternative PSA gene-specific primer (SS1 primer) extends 2 bases past the splice junction. B, comparison of the expression of normal and alternative PSA transcripts by nested RT-PCR analysis. Lane 1, BPH1 was used as a negative sample in which alternative PSA transcript was not detected (normal transcript is 333 bp). Lane 2, BPH2 was used as a positive sample that contained an alternative PSA transcript (alternative PSA transcript is 204 bp). Lane 3, in the case of BPH1, an alternatively spliced product was negative. Lane 4, BPH2 showed a positive band using the same primer pairs. This PCR product is 366 bp. Lane M, a 100-bp ladder (Toyobo). C, nested RT-PCR analysis for the detection of the alternatively spliced PSA transcript. Top panel, S1 and A1 primers were used, followed by SS1 and A3 primers for spliced PSA. Bottom panel, S1 and A1 primers were used, followed by S4 and A2 primers to amplify the common part of both mature and spliced PSA genes. Lane M, a 100-bp ladder (Life Technologies, Inc.).

 
Western Blot Analysis.
To investigate the presence of PSA protein derived from alternatively spliced PSA transcript in prostate tissues, Western blot analysis was performed as described previously (12) . BPH tissues were used as the source for this experiment. Anti-PSA polyclonal antibody was kindly supplied by Wako Pure Chemical Industries, Ltd. (Osaka, Japan). In all gels, 2 µg of protein dissolved in sample buffer were loaded per lane. The proteins were separated by 16% SDS-PAGE under reducing conditions and then transferred onto a polyvinylidene difluoride membrane (TEFCO Corp., Tokyo, Japan). The polyvinylidene difluoride membrane was placed in Super Block (Pierce, Rockford, IL) to block nonspecific binding sites. The first antibody (diluted 1:500) was used to incubate the membrane overnight. Normal rabbit serum was used as a negative control instead of anti-PSA antibody. The immunoproducts were visualized using the Histofine streptavidin-biotin kit (Nichirei, Tokyo, Japan).


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Nucleotide Sequence of the Human PSA Gene.
We amplified a portion of the human PSA gene from prostatic tissues by RT-PCR using the S1 and A1 primers. In addition to the major product, which corresponded in size to the mature PSA, a minor product was shown more faintly by gel electrophoresis (Fig. 1)Citation . We determined the nucleotide sequence of each of the PCR products. The nucleotide sequence of the major product was identical to that reported previously for the human PSA gene (10) . However, the nucleotide sequence of one minor product was identical to that of the human PSA gene except for a 129-nucleotide deletion in the exon 3 (nucleotides 248–376; Fig. 2ACitation ). This result revealed that this minor product was a novel, alternatively spliced variant. By this alternative splicing, 44 codons (codons 45–88) were deleted from mature PSA composed of 237 amino acid residues (Fig. 2B)Citation , resulting in the loss of asparagine 45, which is a binding site of carbohydrate chains (13) . This novel PSA product was calculated to have a molecular weight of 21,071.19.



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  		Fig. 1. We amplified a portion of the human PSA gene from prostatic tissue by RT-PCR using the S1 and A1 primers. In addition to the major product, which corresponds in size to mature PSA, a faint minor product was seen at a lower position than the major product. Lane M, a 100-bp ladder (Toyobo).

 
Expression of the Alternative PSA Gene by Nested RT-PCR.
We analyzed the expression of the alternative PSA gene in noncancerous prostatic tissues, in PC tissues, and in the peripheral blood of patients with advanced PC by nested RT-PCR using an alternative PSA-specific primer (SS1). We confirmed that the primer pair SS1/A3 amplified 366-bp products from the alternatively spliced variant, but not from the normal transcript (Fig. 3B)Citation . The alternative PSA gene was recognized by these nested RT-PCR systems in 13 of 18 (72.2%) noncancerous prostate tissues, 4 of 5 (80.0%) PC tissues, and 3 of 12 (25.0%) blood samples from PC patients. Of the five PC tissues, one lymph node metastasis tissue did not contain the alternatively spliced PSA transcript. The other four primary PC tissues expressed both mature PSA transcripts and spliced ones. There was a statistically significant difference in alternative PSA gene expression between the first group and the last group in these three groups (P = 0.011 by {chi}2 test; Fig. 3CCitation ).

Existence of an Alternatively Spliced PSA Gene Product.
Western blotting analyses revealed multiple PSA protein bands. As predicted from the data regarding the deduced amino acid residues of variant PSA, a protein band corresponding to a molecular weight of approximately 21,000 was shown, in addition to a main band of Mr 33,000 (Fig. 4)Citation .



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  		Fig. 4. Western blot analysis for PSA in BPH tissue. Anti-PSA polyclonal antibody was used for Lanes 1 and 2, and normal rabbit serum was used for Lane 3. Lane 1, BPH tissue was used as the sample. A variant PSA band was observed at a molecular weight of approximately 21,000. Lane 2, human colon cancer cell line HCT116 was used as a negative control sample. Lane 3, normal rabbit serum showed no band against BPH tissue. Lane M, molecular weight standards.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
PSA is well known as the most powerful tool to diagnose and monitor patients with PC. However, its weak point has become apparent from numerous previous reports (2, 3, 4) . Best characterized as a differential antigen, PSA is not a cancer-specific protein. To overcome this potential problem, various expedients have been invented such as PSA density, PSA velocity, free-total PSA ratios, and so on (5, 6, 7 , 14 , 15) . In these modifications of the measurement of PSA, the differentiation of free-total PSA ratios would be caused by the variation of the molecular form.

We believed that the details of the PSA molecule should be investigated more intensively. Thus far, only a small number of studies have addressed this particular facet of PSA research. Among these studies, the products of Christensson et al. (5) are worthy of special mention. Christensson et al. (5) revealed the existence of binding proteins for PSA, which significantly affected the field of PC research. Their efforts bore fruit as the measurement of free-total PSA ratios. Unfortunately, the precise mechanisms and binding conditions by which some molecules of PSA display binding with {alpha}1-antichymotrypsin, {alpha}2-macroglobulin, or other are still unknown. Baffa et al. (10) evaluated the full sequence analyses of PSA genes derived from benign and malignant prostate tissues. They concluded that the PSA gene expressed in malignant prostate tissue is the wild-type gene. Their data stimulated us and led to a follow-up study.

In our study, we decided to use samples of peripheral blood from patients with advanced PC for amplification of the PSA gene from cancer cells to minimize possible contamination from benign prostate cells. Although there are significant issues with using circulating cells for patients with PC as a surrogate for PC cells obtained directly from cancer tissues, it can also be recognized that the complete elimination of benign prostatic secretory cells including noncancerous PSA gene is not easy when using PCR technology. The primers for RT-PCR were instituted at 5' and 3' untranslated regions to obtain the entire open reading frame. We found an extra product, consisting of a mature PSA product, at a lower position (Fig. 1)Citation . Using a direct sequencing method, this product was sequenced. The result revealed that there was a novel alternative splicing variant of the PSA transcript (Fig. 2A)Citation . The initial sequence of the spliced region was composed of GT, and the terminal portion was composed of AG, both of which are typical sequences at splice junction sites (16) . This splicing phenomenon was confirmed by RT-PCR analysis using a special primer that crosses over the splice junction site (Fig. 3B)Citation .

The spliced cDNA sequence is very interesting in that asparagine 45 is deleted, and this amino acid residue is the only N-glycosylation binding site for a carbohydrate chain in PSA (13) . This alternative splicing resulted in the loss of a total carbohydrate chain from the PSA molecule. This alternative PSA is a novel variant of mRNA, although there are a few studies concerning an alternative transcript of the PSA gene (17, 18, 19) . The sequences of the PCR products corresponding to the mature forms of PSA from several cases were identical to those reported previously for PSA (10) . The alternative PSA gene was observed by nested RT-PCR analysis in 13 noncancerous prostate tissues (72.2%) and 3 blood samples from PC patients (25.0%; P = 0.011 by {chi}2 test; Fig. 3CCitation ). The high positive rate (80%) for the alternative PSA transcript in our small series of PC tissues may have been caused by the aforementioned contamination of noncancerous cells, because it is not unusual for cancerous and noncancerous cell to coexist in PC tissues. In such a situation, it is almost impossible to distinguish whether a spliced PSA gene is derived from noncancerous or cancerous cells. However, a metastatic lymph node tissue sample, which never contains the noncancerous PSA gene, did not show a spliced variant band on RT-PCR analysis. This representative example clearly demonstrates that PC cells did not use this splicing system. Although it is noted here that the sample size of our study is small, this alternative splicing system was used more frequently in benign prostate cells than in PC cells. To verify the data, a large scale RT-PCR analysis using BPH tissues and PC tissues without noncancerous prostatic epithelial cells will need to be performed in the future. Western blotting revealed the product of an alternatively spliced PSA gene (Fig. 4)Citation . The existence of this variant PSA protein might be one of the causes of the different free-total PSA ratios for BPH tissue and PC tissue.

Further investigation should be done, and the question of whether this alternative PSA gene can be translated into a protein as efficiently as the more predominant form of PSA should also be investigated. In future studies, the clinical significance and utility of the splicing heterogeneity of PSA may be more fully discerned.


   ACKNOWLEDGMENTS
 
We thank Masafumi Suzaki (Central Research Laboratory, Shiga University of Medical Science, Shiga, Japan) for technical assistance.


   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 in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. Back

2 To whom requests for reprints should be addressed, at Department of Urology, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan. Phone: 81-77-548-2273; Fax: 81-77-548-2400; E-mail: yoshiki{at}belle.shiga-med.ac.jp Back

3 The abbreviations used are: PC, prostate cancer; PSA, prostate-specific antigen; BPH, benign prostate hypertrophy; RT-PCR, reverse transcription-PCR. Back

Received 8/11/99. Accepted 11/10/98.


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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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