G2/M Arrest by 1,25-Dihydroxyvitamin D3 in Ovarian Cancer Cells Mediated through the Induction of GADD45 via an Exonic Enhancer

Abstract

Vitamin D 3 (VD) 1 is a lipophilic hormone essential for normal bone structure and maintenance of serum calcium (1). VD action is mediated through the vitamin D receptor (VDR) (2), a member of the steroid/thyroid receptor superfamily of ligandregulated transcription factors (3-5). This superfamily includes receptors for steroids such as progesterone, androgens, estrogens, glucocorticoids, and mineralocorticoids; receptors for non-steroid hormones like vitamin D, thyroid hormones, alltrans-retinoic acid and 9-cis-retinoic acid; and orphan receptors for which the ligand is unknown. VDR and other non-steroid receptor members of the superfamily form heterodimers with the retinoid X receptor (RXR), the receptor for 9-cis-retinoic acid. In most cases, the VDR⅐RXR heterodimer binds VD response elements (VDREs) to mediate the biological activities of VD through transcriptional activation or repression of target genes containing VDREs in their regulatory sequence. In response to VD activation, the VDR recruits multiple coactivators, including members of the p160 steroid receptor coactivator family (6), which are associated with histone acetyltransferase activity, and the vitamin D receptor interacting proteins complex (7); the latter serves as a mediator between the VDR and RNA polymerase II (Pol II) complex.In addition to its well defined role in calcium homeostasis and bone cell metabolism, the active metabolite of VD, 1,25-dihydroxyvitamin D 3 (1,25VD), modulates cellular proliferation and differentiation of both normal and malignant cells (8). 1,25VD and its synthetic analogues can inhibit carcinogenesis in mouse skin (9, 10), decrease the size of transplanted sarcomas, reduce lung metastasis in mice (11), suppress the growth of human colonic cancer cell-derived xenografts in immunesuppressed mice (12), and increase cell differentiation and decrease proliferation of leukemia (13), breast cancer (14), and prostate cancer (15) cells. Studies in cancer cell lines have shown that 1,25VD causes cancer cells to accumulate in the G 1 phase of the cell cycle (15) or in the G 2 phase (16) or to undergo apoptosis (17,18). Genes that mediate each of these specific activities remain largely unidentified.Similar to breast and prostate cancers, ovarian cancer (OCa) mortality and incident rates are lower in countries within 20°o f the equator (19) where there is a high amount of sunlight. In the United States, women between the ages of 45 and 54 living in the north have 5 times the OCa mortality rate than women living in southern states (20,21). In the epidermis, sunlight controls the first step of 1,25VD synthesis, namely the photoconversion of 7-dehydrocholesterol to pre-vitamin D 3 . Exposure to sunlight, rather than food consumption, is the primary source of 1,25VD (22). The inverse correlation between sunlight exposure and OCa mortality indicates that decreased synthesis of 1,25VD may contribute to OCa initiation and/or progression. VDR has been found in rat ovaries by immunohistochemistry (23) and in hen ovaries by ligand bin...

p27Kip1 Stabilization and G1 Arrest by 1,25-Dihydroxyvitamin D3 in Ovarian Cancer Cells Mediated through Down-regulation of Cyclin E/Cyclin-dependent Kinase 2 and Skp1-Cullin-F-box Protein/Skp2 Ubiquitin Ligase

“…In a separate study (13), G 2 /M arrest was observed in MEFs from another mouse strain. It is thus clear that MEFs from different mouse strains vary in their response to the treatment by 1,25(OH) 2 D 3 .…”

“…1,25(OH) 2 D 3 and its synthetic analogs inhibit carcinogenesis in mouse skin (3,4), decrease the size of transplanted sarcomas, reduce lung metastasis in mice (5), suppress the growth of human colonic cancer cell-derived xenografts in immune-suppressed mice (6), and increase the differentiation and decrease the proliferation of leukemia (7), breast cancer (8), prostate cancer (9), colon cancer (10), and squamous carcinoma (11) cells. Growth analyses have demonstrated that 1,25(OH) 2 D 3 causes cancer cells to accumulate in G 1 (9) or G 2 (12,13) or undergo apoptosis (14,15), suggesting that 1,25(OH) 2 D 3 or its synthetic analogs are potential agents for cancer treatment or chemo-prevention.…”

“…All of the stable clones were obtained through selection in medium containing 100 g/ml G418 for a period of about 4 weeks followed by clonal isolation with glass cylinders as described previously (13).…”

Deficiency in TR4 nuclear receptor abrogates Gadd45a expression and increases cytotoxicity induced by ionizing radiation

“…pRL-TK (TK Renilla luciferase) was purchased from Promega. GADDLuc and GADDLuc-3 reporters are generous gifts from Dr. Wenlong Bai as described in his publication [25]. The pSuperior.retro.puro.…”

The vitamin D receptor in cancer

“…By contrast, p27 (kip1) protein levels appear to be regulated by a range of post-transcriptional mechanisms, such as enhanced mRNA translation, and attenuating degradative mechanisms, often in a cell-type-specific manner (98)(99)(100) . The upregulation of p21 (waf1/cip1) and p27 (kip1) principally mediate G 1 cell cycle arrest, but 1a,25(OH) 2 D 3 has been shown to mediate a G 2 /M cell cycle arrest in a number of cancer cell lines via direct induction of GADD45a (94,101,102) . Again, this regulation appears to combine direct gene transcription and a range of post-transcriptional mechanisms.…”