The Journal of Steroid Biochemistry and Molecular Biology
Molecular activity of 1,25-dihydroxyvitamin D3 in primary cultures of human prostatic epithelial cells revealed by cDNA microarray analysis
Introduction
Prostate cancer remains the most common non-cutaneous malignancy in U.S. males with over 230,000 cases diagnosed annually. An estimated 28,900 men die each year of the disease, making it the second leading cause of male cancer deaths in the United States [1]. Unfortunately, despite increased screening for prostate cancer, many patients still develop extraprostatic or metastatic disease that is not amenable to conventional surgical or medical therapy. Thus, new emphasis is being placed on identifying methods to prevent this devastating disease. Several promising agents may act by either preventing initiation of malignancy or slowing progression of established disease [2].
The active metabolite of Vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], has emerged as a promising preventive or therapeutic agent. In addition to its well-characterized roles in calcium and phosphate regulation and bone metabolism, 1,25(OH)2D3 promotes cellular differentiation and inhibits proliferation in a variety of tissues by binding to the Vitamin D receptor (VDR) [3]. A large body of work, including our own, suggests that 1,25(OH)2D3 has potent growth inhibitory and pro-differentiating effects on prostate cells [4], [5], [6].
1,25(OH)2D3 has anti-proliferative effects on prostatic cancer cell lines, including LNCaP and PC-3, as well as on primary prostatic epithelial cell strains [7], [8], [9]. A number of mechanisms have been proposed to account for the anti-proliferative action of 1,25(OH)2D3, including induction of cell cycle arrest, differentiation, and apoptosis. No single molecular signaling pathway, however, explains the effects of 1,25(OH)2D3 in all cell types. For example, the gene encoding the cyclin-dependent protein kinase inhibitor, p21, may be directly induced by 1,25(OH)2D3 in the leukemia cell line U937 [10], yet p21 levels are unchanged in PC-3 cells despite inhibition of growth of these cells by 1,25(OH)2D3 [11].
The molecular basis of action for 1,25(OH)2D3 in prostatic cells remains poorly understood. The well characterized gene 25-hydroxyvitamin D3-24-hydroxylase (CYP24), which encodes the enzyme that initiates the catabolism of 1,25(OH)2D3, is induced by 1,25(OH)2D3 in many types of target cells, including prostate cells [3]. Several other 1,25(OH)2D3-regulated genes have also been identified in prostate cancer cell lines. 1,25(OH)2D3 decreases expression of multiple antiapoptotic proteins in LNCaP cells, leading to activation of the mitochondrial pathway for apoptosis [12], [13]. 1,25(OH)2D3 also upregulates the expression of androgen receptor and prostate-specific antigen (PSA) in LNCaP cells, but inhibition of this phenomenon by cycloheximide suggests that the effect is indirect and mediated by an unidentified protein [14].
The advent of genome-wide screening methods through the use of complementary DNA (cDNA) microarrays offers a new opportunity to understand regulation of gene expression [15], [16]. To better characterize the transcriptional response to 1,25(OH)2D3, we employed cDNA microarrays to assess gene expression profiles of cultured prostatic epithelial cells. Primary cultures derived from normal and malignant prostatic tissues were examined on an array system with 25,000 elements and over 20,000 independent genes and expressed sequence tags (ESTs). Regulation of several candidate molecular targets in additional primary cultures treated with 1,25(OH)2D3 was examined by real-time reverse transcription-polymerase chain reaction (RT-PCR). The functions of the genes regulated by 1,25(OH)2D3 in the primary cultures provoke new ideas about the mechanism of anti-tumor activity of Vitamin D in the prostate.
Section snippets
Isolation, culture, and Vitamin D treatment of primary prostatic epithelial cells
Tissues were derived from radical prostatectomy specimens that were obtained from men undergoing surgery to treat prostate cancer. The normal cell strain used for microarray analysis (E-PZ-10) was derived from peripheral zone tissue with no histological evidence of cancer in adjacent sections. The cancer cell strain used for microarray studies (E-CA-15) was derived from an adenocarcinoma of Gleason grade 3/3. Two other normal cell strains (E-PZ-1 and E-PZ-2) and three additional cancer cell
Normal prostatic epithelial cells: genes upregulated in response to 1,25(OH)2D3
Initial experiments focused on the response of normal prostatic epithelial cells to 1,25(OH)2D3. Semi-confluent cells (E-PZ-10) in standard serum-free growth medium were treated with or without 50 nM 1,25(OH)2D3 for 6 and 24 h in duplicate experiments and gene expression profiles were generated on separate arrays. This dose of 1,25(OH)2D3 was selected because it has been shown to significantly inhibit the growth of these cells [22].
A list of genes upregulated by 1,25(OH)2D3 in normal cells is
Discussion
Microarray technology has proven to be a powerful new tool to identify changes in gene expression caused by factors that regulate cell growth. One emerging tenet is that there is a great deal of cell-type specificity in gene expression profiles generated by a given factor. Furthermore, the genetic background upon which the factor works has a significant impact on the final outcome. Even related cell types can show very different responses to a specific factor. Given that established cell lines
Acknowledgements
This work was supported in part by NIH Training Grant 5-T32-DK07217 (L.N.), the Doris Duke Foundation (T98064) (R.J., J.D.B.), NIH DK42482 (A.V.K., D.F.) and the Cancer Research Fund (D.M.P., E.S.), under Interagency Agreement #97-12013 (University of California, Davis contract #98-00924V) with the Department of Health Services, Cancer Research Section. Mention of trade name, proprietary product or specific equipment does not constitute a guaranty or warranty by the Department of Health
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