BRIEF REPORT

Identification of Genes Differentially Expressed in Benign Prostatic Hyperplasia

Correspondence to: Anthony G. DiLella, Dept. of Pharmacology, Merck Research Laboratories, P.O. Box 4, West Point, PA 19486. E–mail: tony_dilella@merck.com

	Summary

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Differences between benign prostatic hyperplasia (BPH) and normal prostate tissue at the level of mRNA expression provide an opportunity to identify candidate genes for this disease. A cDNA subtraction procedure was used to isolate differentially expressed genes in BPH. The subtraction was done by solution hybridization of BPH cDNA against excess normal prostate cDNA. We identified known, EST, and novel genes by sequence and database analysis of the subtracted cDNAs. Several of these cDNAs were used as probes in Northern blotting analysis to confirm over-expression of their corresponding mRNAs in BPH tissues. One highly upregulated sequence of interest shared identity with a known mRNA encoding human NELL2, a protein containing epidermal growth factor-like domains. NELL2 was not previously reported to be expressed in prostate and may code for a novel prostatic growth factor. In situ hybridization analysis of hyperplastic prostate specimens demonstrated that NELL2 mRNA expression is predominantly localized in basal cells of the epithelium. Disease-related changes in the levels of NELL2 may contribute to alterations in epithelial–stromal homeostasis in BPH. (J Histochem Cytochem 49:669–670, 2001)

Key Words: benign prostatic hyperplasia, mRNA, cDNA subtraction, Northern blotting, in situ hybridization

	Introduction

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Benign prostatic hyperplasia (BPH) is an age-related disease, and most men over 50 years of age have urinary tract symptoms arising from this disorder. The cellular and molecular processes involved in the development and pathogenesis of BPH are not well understood. The objective of this study was to identify differentially expressed genes involved in the uncontrolled prostate growth that characterizes BPH.Prostate tissue was obtained after written consent from each patient and was approved by the local ethics committee. mRNA isolated from BPH and normal prostate mRNA was subjected to a subtractive and kinetic enrichment procedure (Hubank and Schatz 1994 ). For in situ hybridization (ISH), digoxigenin-labeled probes were hybridized to transurethral resectioned prostate (TURP) specimens and detected using the tyramide signal amplification method and DAB staining (NEN Life Sciences; Boston, MA).

Before subtraction, cDNA populations from normal and BPH tissue were indistinguishable (Fig 1A, Lanes 1 and 2, respectively). By an iterative process of subtractive and kinetic enrichment, cDNA fragments that were upregulated in BPH were selected (Fig 1A, Lane 3). Sequence and database analysis of these fragments identified at least 15 known, EST, and novel genes. Five clones were hybridized to Northern blots to verify expression patterns. Fig 1B shows the detection of highly upregulated mRNA in BPH (clones 1 and 3) in addition to mRNA specific for BPH (clones 2, 4, and 5) compared to normal prostate.

View larger version (48K): [in this window] [in a new window]   Figure 1. (A) Gel analysis of cDNA subtraction. (B) Hybridization analysis of normal (N) and BPH prostate using subtracted probes.

Sequences representative of clones 1 and 3 were further analyzed and revealed identity to lactoferrin and NELL2, respectively. Lactoferrin is an iron-binding glycoprotein expressed in inflamed prostate (Reese et al. 1992 ) and may play a role in providing protection against tumor development (Damiens et al. 1998 ). Lactoferrin is expressed in the secretory component of the epithelium in TURP specimens (Fig 2A), and its upregulation in BPH may be a response to neutralize the enhanced proliferative activity of this disease. NELL2 is a neuronal protein containing EGF-like domains with similarities to thrombospondin, and has not previously been identified in prostate. Kuroda et al. 1999 recently showed abundant expression of NELL2 mRNA in colorectal adenocarcinoma SW480 cells, suggesting that NELL2 participates in the growth and differentiation of cancer cell lines. NELL2 mRNA is predominantly expressed in the epithelial basal cell layer in TURP specimens (Fig 2B). EGF is also abundantly expressed in the basal cells of BPH and may overstimulate epithelial cell proliferation (De Miguel et al. 1999 ). Altered expression of other growth factors has been implicated in the disruption of molecular mechanisms that regulate cell death and proliferation in the aging and diseased prostate (Gomella and Godwin 1997 ). We have identified NELL2 as a putative growth factor regulator of prostate epithelial cells. When upregulated, this protein may act synergistically with other growth factors contributing to alterations in epithelial–stromal homeostasis in BPH. Analysis of prostate specimens from an expanded patient population is in progress.

View larger version (64K): [in this window] [in a new window]   Figure 2. ISH of TURP specimens with antisense probes for lactoferrin (A) and NELL2 (B) mRNA.

	Footnotes

Presented in part at the Joint Meeting of the Histochemical Society and the International Society for Analytical and Molecular Morphology, Santa Fe, NM, February 2–7, 2001.

Received for publication December 6, 2000; accepted February 16, 2001.

	Literature Cited

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Damiens E, Mazurier J, Yazidi IE, Masson M, Duthille I, Spik G, Boilly–Marer Y (1998) Effects of human lactoferrin on NK cell cytotoxicity against hematopoietic and epithelial tumour cells. Biochim Biophys Acta 1402:277-287[Medline]

De Miguel MP, Royuela M, Bethencourt FR, Ruiz A, Fraile B, Paniagua R (1999) Immunohistochemical comparative analysis of transforming growth factor , epidermal growth factor, and epidermal growth factor receptor in normal, hyperplastic and neoplastic human prostates. Cytokine 11:722-727[Medline]

Gomella LG, Godwin BW (1997) Apoptosis and benign prostatic hypertrophy. J Urol 158:2-3[Medline]

Hubank M, Schatz DG (1994) Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res 22:5640-5648[Abstract/Free Full Text]

Kuroda S, Oyasu M, Kawakami M, Kanayama N, Tanizawa K, Saito N, Abe T, Matsuhashi S, Ting K (1999) Biochemical characterization and expression analysis of neural thrombospondin-1-like proteins NELL1 and NELL2. Biochem Biophys Res Commun 265:79-86[Medline]

Reese JH, McNeal JE, Goldenberg SL, Redwine EA, Sellers RG (1992) Distribution of lactoferrin in the normal and inflamed prostate: an immunohistochemical study. Prostate 20:73-85[Medline]

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