Gandini et al.(1) claim that the prostate-specific expression of DD3PCA3 is restricted to exon 4 of the DD3PCA3 gene. The authors show that reverse transcription-PCR amplification of the DD3PCA3 transcript using primers specific for exons 1 and 3 also amplified a DD3PCA3-specific product in several nonprostate tissues and cell lines. On the basis of the critical analysis of our published data (2, 3), Gandini et al.(1) conclude that the use of exons 1–4-specific PCR primers and the use of a probe located in exon 4 of the DD3PCA3 gene (for Northern blot analysis) would explain our observed prostate (cancer)-specific expression of DD3PCA3. The presence of exon 3 in nonprostatic tissues has been suggested to be attributable to alternative splicing mechanisms, omitting exon 4 of the DD3PCA3 gene.

After our first publication of the DD3PCA3 gene (2), we now use exactly the same exon 1 to exon 3 primers as being described in the letter by Gandini et al.(1). In the past 4 years, we have amplified DD3PCA3 in many samples using these primers and have never observed nonprostatic expression of DD3PCA3. Although it is not clear from the letter how many cycles of PCR amplification Gandini et al.(1) performed, we never used >35 rounds of amplification. We cannot exclude that using more rounds of amplification low levels of expression will be detected. These levels of expression would be far below those observed in prostate cancer, normal prostate, and even prostate cancer cell lines. In addition, we also have performed Northern blot analysis using DD3PCA3 exon 3-specific probes, resulting in exactly the same prostate cancer-specific expression profiles as shown before (2). These data strongly argue against the alternative splicing of exon 3 in nonprostatic cells.

The former conclusion is further substantiated by our finding that an alternative exon 3 to 4 DD3PCA3 variant exists in nonprostatic cells (Fig. 1). The level of expression of this product is lower than in normal prostatic tissue and far below the expression in prostate cancer tissue. Strikingly, the DD3PCA3 variant in nonprostatic tissues is not spliced unlike in prostate-derived samples, as determined by DNA sequence analysis of the PCR products. In normal prostatic tissue the nonspliced transcript is also expressed, although at low levels. In prostate tumor tissue, the nonspliced variant is not expressed or not detectable because of the high overexpression of the spliced DD3PCA3 product that most probably will be preferentially amplified in the PCR reactions. In RNA samples not subjected to reverse transcription, no amplification product was found (data not shown), indicating that the nonspliced DD3PCA3 PCR products were not attributable to DNA contamination.

Several explanations for the presence of nonspliced DD3PCA3 transcripts can be postulated (Fig. 2). Firstly, in prostatic tissues, the DD3PCA3 transcript may be tissue specifically spliced, a phenomenon that has been described for several other genes (4). Secondly, an alternative ubiquitous promoter may exist in the DD3PCA3 gene, resulting in a second transcript that is not prostate specific. This option seems less likely, because that transcript seems to be nonspliced despite the strong splice consensus sequences flanking the DD3PCA3 exons (2). Thirdly, a ubiquitous antisense promoter may be present at the 3′ end of the DD3PCA3 gene, leading to antisense DD3PCA3 transcription in many human tissues. It has recently been reported that antisense transcription occurs widespread in the human genome (5), and therefore, it is not unlikely that an antisense DD3PCA3 transcript exists. Such a putative DD3PCA3 antisense transcript may be involved in the regulation of DD3PCA3 transcription in prostate cells or vice versa in prostate cells the DD3PCA3 transcript may affect the, thus far unidentified, antisense transcribed gene. Currently, we are investigating whether alternative splicing or alternative transcription initiation mechanisms are responsible for the expression of the nonprostatic DD3PCA3-like transcript.

In conclusion, we agree that transcription of the DD3PCA3 gene or a DD3PCA3-like gene is evident in tissues other than the prostate. However, these transcripts are either not spliced or are complementary (i.e., antisense) to the DD3PCA3 gene. We have never observed spliced DD3PCA3 variants (i.e., exon 1–3 product) in nonprostatic tissues. For the application of DD3PCA3 as a marker for prostate cancer this has one major implication: primers for the amplification of the DD3PCA3 transcripts in patient samples should cross the large (16 kb) first intron. This region of the DD3PCA3 gene may be present in the alternative nonspliced or antisense transcripts but is lacking from the prostate-specific spliced form of DD3PCA3. Therefore, using exon 1 to exon 3 or 4 primer pairs, only the prostate-specific spliced form of DD3PCA3 can be amplified (the large intron prevents amplification of this region in the nonspliced transcripts). We have developed two independent assays for the detection of DD3PCA3 mRNA in patient material, using an exon 1 forward and an exon 4 reverse primer and exon 4-specific detection probes (3, 6). The DD3PCA3 detection assays have now been applied on >200 patient samples and have been shown to be very specific and sensitive with a strong negative predictive value (6). Furthermore, the analysis of >100 control samples has never revealed any expression of DD3PCA3 outside the prostate.

Fig. 1.

Expression of DD3PCA3 in several human tissues using 32 cycles of DD3PCA3-specific reverse transcription-PCR with the following primers: forward 5′CAGGAAGCACAAAAGGAAGC-3′ (exon 3, position 443–462) and reverse 5′-TCCTGCCCATCCTTTAAGG-3′ (exon 4, position 593–575). The following tissues have been analyzed: normal prostate (Lane 1), prostate cancer (Lane 2), heart (Lane 3), lung (Lane 4), artery (Lane 5), kidney (Lane 6), liver (Lane 7), breast cancer (Lane 8), normal breast (Lane 9), cervix (Lane 10), endometrium (Lane 11), and testis (Lane 12). The arrowhead indicates the spliced DD3PCA3 transcript (151 bp) in prostate samples only and the arrow the nonspliced transcript (378 bp) in the other tissues. Note that the signal in Lane 2 is saturated. A β-microglobulin PCR was performed as a control (bottom panel).

Fig. 1.

Expression of DD3PCA3 in several human tissues using 32 cycles of DD3PCA3-specific reverse transcription-PCR with the following primers: forward 5′CAGGAAGCACAAAAGGAAGC-3′ (exon 3, position 443–462) and reverse 5′-TCCTGCCCATCCTTTAAGG-3′ (exon 4, position 593–575). The following tissues have been analyzed: normal prostate (Lane 1), prostate cancer (Lane 2), heart (Lane 3), lung (Lane 4), artery (Lane 5), kidney (Lane 6), liver (Lane 7), breast cancer (Lane 8), normal breast (Lane 9), cervix (Lane 10), endometrium (Lane 11), and testis (Lane 12). The arrowhead indicates the spliced DD3PCA3 transcript (151 bp) in prostate samples only and the arrow the nonspliced transcript (378 bp) in the other tissues. Note that the signal in Lane 2 is saturated. A β-microglobulin PCR was performed as a control (bottom panel).

Close modal
Fig. 2.

Schematic representation of the DD3PCA3 transcription unit. Boxes indicate the four DD3PCA3 exons and the solid arrowhead of the prostate-specific DD3PCA3 promoter. The arrows indicate the different (putative) DD3PCA3 (-like) transcripts; transcription start sites of the putative ubiquitous transcripts are not determined and are therefore arbitrarily chosen.

Fig. 2.

Schematic representation of the DD3PCA3 transcription unit. Boxes indicate the four DD3PCA3 exons and the solid arrowhead of the prostate-specific DD3PCA3 promoter. The arrows indicate the different (putative) DD3PCA3 (-like) transcripts; transcription start sites of the putative ubiquitous transcripts are not determined and are therefore arbitrarily chosen.

Close modal

References

1
Gandini O., Luci L., Stigliano A., Lucera R., Di Silverio F., Toscano V., Cardillo M. R. Is DD3 a new prostate-specific gene?.
Anticancer Res.
,
23(1A)
:
305
-308,  
2003
.
2
Bussemakers M. J., van Bokhoven A., Verhaegh G. W., Smit F. P., Karthaus H. F., Schalken J. A., Debruyne F. M., Ru N., Isaacs W. B. DD3: a new prostate-specific gene, highly overexpressed in prostate cancer.
Cancer Res.
,
59
:
5975
-5979,  
1999
.
3
de Kok J. B., Verhaegh G. W., Roelofs R. W., Hessels D., Kiemeney L. A., Aalders T. W., Swinkels D. W., Schalken J. A. DD3PCA3, a very sensitive and specific marker for to detect prostate tumors.
Cancer Res.
,
62
:
2695
-2698,  
2002
.
4
Black D. L. Mechanisms of alternative pre-messenger RNA splicing.
Annu. Rev. Biochem.
,
72
:
291
-336,  
2003
.
5
Yelin R., Dahary D., Sorek R., Levanon E. Y., Goldstein O., Shoshan A., Diber A., Biton S., Tamir Y., Khosravi R., Nemzer S., Pinner E., Walach S., Bernstein J., Savitsky K., Rotman G. Widespread occurrence of antisense transcription in the human genome.
Nat. Biotechnol.
,
21
:
379
-386,  
2003
.
6
Hessels D., Klein Gunnewiek J., Oort I., Karthaus H. F. M., van Leenders G. J. L., van Balken B., Kiemeney L. A., Witjes J. A., Schalken J. A. DD3PCA3-based molecular urine analysis for the diagnosis of prostate cancer.
Eur. Urol.
,
44
:
8
-16,  
2003
.