Novel expressed sequences identified in a model of androgen independent prostate cancer

Abstract

Background: Prostate cancer is the most frequently diagnosed cancer in American men, and few effective treatment options are available to patients who develop hormone-refractory prostate cancer. The molecular changes that occur to allow prostate cells to proliferate in the absence of androgens are not fully understood.

Results: Subtractive hybridization experiments performed with samples from an in vivo model of hormonal progression identified 25 expressed sequences representing novel human transcripts. Intriguingly, these 25 sequences have small open-reading frames and are not highly conserved through evolution, suggesting many of these novel expressed sequences may be derived from untranslated regions of novel transcripts or from non-coding transcripts. Examination of a large metalibrary of human Serial Analysis of Gene Expression (SAGE) tags demonstrated that only three of these novel sequences had been previously detected. RT-PCR experiments confirmed that the 6 sequences tested were expressed in specific human tissues, as well as in clinical samples of prostate cancer. Further RT-PCR experiments for five of these fragments indicated they originated from large untranslated regions of unannotated transcripts.

Conclusion: This study underlines the value of using complementary techniques in the annotation of the human genome. The tissue-specific expression of 4 of the 6 clones tested indicates the expression of these novel transcripts is tightly regulated, and future work will determine the possible role(s) these novel transcripts may play in the progression of prostate cancer.

Figure 1

Distribution of sequence matches in the suppression subtractive hybridization clone set. The 237 non-redundant clones identified through sequence analysis of the subtractive hybridization libraries were mapped to the human genome (v. 35) and classified as representing an annotated Ensembl cDNA, a previously identified expressed sequence tag (EST), or an unannotated region of the genome. Clones not mapping uniquely to the genome were classified as ambiguous matches.

Figure 2

The novel subtracted clones were expressed in a variety of human tissues. (A) RT-PCR was performed with primer pairs specific for each of six novel clones using cDNA from the LNCaP hollow fibre model. RT indicates the presence or absence of Reverse Transcriptase. (B) RT-PCR was performed using cDNAs generated from normal human tissues with each of the six novel clones. All clones were expressed in at least one of the normal tissues. (C) RT-PCR was performed using cDNAs generated from three samples of prostate cancer (T1 – T3) and their respective matched normal samples (N1 – N3). All six of the clones were expressed in at least one of these samples.

Figure 3

Two of the novel clones were part of transcripts containing a neighbouring SAGE tag. RT-PCR amplified transcript regions between clones 1E05 (A) and 2dB01 (B) and SAGE tags identified nearby each clone. RT indicates the presence or absence of Reverse Transcriptase.