Cdx2 Polymorphism Affects the Activities of Vitamin D Receptor in Human Breast Cancer Cell Lines and Human Breast Carcinomas

Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). It regulates the action of hormone responsive genes and is involved in cell cycle regulation, differentiation and apoptosis. VDR is a critical component of the vitamin D pathway and different common single nucleotide polymorphisms have been identified. Cdx2 VDR polymorphism can play an important role in breast cancer, modulating the activity of VDR. The objective of this study is to assess the relationship between the Cdx2 VDR polymorphism and the activities of VDR in human breast cancer cell lines and carcinomas breast patients. Cdx2 VDR polymorphism and antiproliferative effects of vitamin D treatment were investigated in a panel of estrogen receptor-positive (MCF7 and T-47D) and estrogen receptor-negative (MDA-MB-231, SUM 159PT, SK-BR-3, BT549, MDA-MB-468, HCC1143, BT20 and HCC1954) human breast cancer cell lines. Furthermore, the potential relationship among Cdx2 VDR polymorphism and a number of biomarkers used in clinical management of breast cancer was assessed in an ad hoc set of breast cancer cases. Vitamin D treatment efficacy was found to be strongly dependent on the Cdx2 VDR status in ER-negative breast cancer cell lines tested. In our series of breast cancer cases, the results indicated that patients with variant homozygote AA were associated with bio-pathological characteristics typical of more aggressive tumours, such as ER negative, HER2 positive and G3. Our results may suggest a potential effect of Cdx2 VDR polymorphism on the efficacy of vitamin D treatment in aggressive breast cancer cells (estrogen receptor negative). These results suggest that Cdx2 polymorphism may be a potential biomarker for vitamin D treatment in breast cancer, independently of the VDR receptor expression.     

Immunocytology with microwave-fixed fibroblasts shows 1 alpha,25- dihydroxyvitamin D3-dependent rapid and estrogen-dependent slow reorganization of vitamin D receptors

Prior studies have given no evidence for regulation of vitamin D receptor (VDR) compartmentalization or subcellular organization. Microwave fixation (9-15 s) and an indirect immunodetection system of avidin-biotin enhancement and phycoerythrin fluorophore resulted in sufficient spatial and temporal resolution to allow analysis of these processes. We studied cultured fibroblasts from normals or from patients with four different types of hereditary defect compromising VDR function (mutant cells). Compartmentalization of VDRs in the absence of 1,25-dihydroxyvitamin D3 (calcitriol) was regulated by serum or estrogen. VDRs were mainly cytoplasmic in cells cultured without serum and phenol red, but VDRs were mainly intranuclear after addition of serum or an estrogen to cells for at least 18 h (slow regulation). Calcitriol initiated a rapid and multistep process (rapid regulation) of reorganization in a portion of VDRs: clumping within 15-45 s, alignment of clumps along fibrils within 30-45 s, perinuclear accumulation of clumps within 45-90 s, and intranuclear accumulation of clumps within 1-3 min. We found similar rapid effects of calcitriol on VDRs in various other types of cultured cells. These sequential VDR pattern changes showed calcitriol dose dependency and calcitriol analogue specificity characteristic for the VDR. In mutant fibroblasts VDR pattern changes after calcitriol were absent or severely disturbed at selected steps. Treatment of normal cells with wheat germ agglutinin, which blocks protein transport through nuclear pores, also blocked calcitriol-dependent translocation of VDRs. We conclude that immunocytology after microwave fixation provides evidence for regulation of VDR organization and localization.     

Change of bone mass in postmenopausal Caucasian women with and without hormone replacement therapy is associated with vitamin D receptor and estrogen receptor genotypes

Our purpose is to assess whether genotypes of the vitamin D receptor (VDR) and estrogen receptor (ER) and their interaction influence changes in bone mass in postmenopausal Caucasian women with and without hormone replacement therapy (HRT). A population of 108 US Mid-West women who participated in a study of low-dose continuous estrogen/progestin was genotyped at the VDR BsmI site and the ER XbaI and PvuII sites. Adequate vitamin D and calcium nutritional intakes were assured in all the study subjects. For the 3.5-year duration of the study, we analyzed changes in bone mineral density (BMD) at the spine, femoral neck, distal radius, and the total body (total body bone mineral content, tbBMC). We adjusted for confounding factors, such as age and weight, in the analysis. We found that VDR and/or ER genotypes and/or their interaction generally had significant effects on the changes in the bone mass measurements in both the placebo and HRT groups. When a significant gene-by-gene interaction exists between VDR and ER genotypes, failure to account for them in analyses may yield nonsignificant results, even if significant genotypic effects exist. The amount of variation in changes in bone mass measurements explained by the total genotypic effects of the VDR and ER loci varies from ∼1.0% (for the tbBMC changes in combined placebo and HRT groups) to ∼18.7% (for the spine BMD changes in the HRT group). These results suggest that individual genotypes are important factors in determining changes in bone mass in the elderly with and without HRT and thus may need to be considered with respect to the treatment to preserve bone mass in elderly Caucasian women. Received: 31 July 1998 / Accepted: 11 September 1998     

Vitamin D3 modulated gene expression patterns in human primary normal and cancer prostate cells

The vitamin D receptor (VDR) is a member of the steroid/retinoid receptor superfamily of nuclear receptors and has potential tumor-suppressive functions in prostate and other cancer types. Vitamin D3 (VD3) exerts its biological actions by binding within cells to VDR. The VDR then interacts with specific regions of the DNA in cells, and triggers changes in the activity of genes involved in cell division, cell survival, and cellular function. Using human primary cultures and the prostate cancer (PCa) cell line, ALVA-31, we examined the effects of VD3 under different culture conditions. Complete G0/G1 arrest of ALVA-31 cells and approximately 50% inhibition of tumor stromal cell growth was observed. To determine changes in gene expression patterns related to VD3 activity, microarray analysis was performed. More than approximately 20,000 genes were evaluated for twofold relative increases and decreases in expression levels. A number of the gene targets that were up- and down-regulated are related to potential mechanisms of prostatic growth regulation. These include estrogen receptor (ER), heat shock proteins: 70 and 90, Apaf1, Her-2/neu, and paxillin. Utilizing antibodies generated against these targets, we were able to confirm the changes at the protein level. These newly reported gene expression patterns provide novel information not only potential markers, but also on the genes involved in VD3 induced apoptosis in PCa.     

Cytochemical detection of estrogen receptors in mammary tumours

The technical details of a cytochemical method for estrogen receptor identification in mammary tumours are presented and applied in 19 carcinomas and six adenofibromas. A fluorescent estradiol conjugate was used as receptor tracer. From the 19 mammary carcinomas, six were interpreted as estrogen receptor-negative (ER-), two were strongly receptor-positive (ER++) and eleven were moderately estrogen receptor-positive (ER+), having cells with a heterogeneous content of estrogen receptors. In fibroadenomas, the proliferating epithelia were moderately estrogen-receptor positive.     

Where is the vitamin D receptor?

The vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and plays a central role in the biological actions of vitamin D. VDR regulates the expression of numerous genes involved in calcium/phosphate homeostasis, cellular proliferation and differentiation, and immune response, largely in a ligand-dependent manner. To understand the global function of the vitamin D system in physiopathological processes, great effort has been devoted to the detection of VDR in various tissues and cells, many of which have been identified as vitamin D targets. This review focuses on the tissue- and cell type-specific distribution of VDR throughout the body.     

Vitamin D3 and androgen receptors in testis and epididymal region of roosters (Gallus domesticus) as affected by epididymal lithiasis

Epididymal lithiasis is a dysfunction characterized by formation of calcium-rich stones in the epididymal region of roosters, associated with decreased serum testosterone and loss of fertility. The segment most affected by the lithiasis is the efferent ductules, which, in birds, are responsible for reabsorption of calcium and luminal fluid. Therefore, we postulated that epididymal lithiasis could result from local impairment of calcium or fluid homeostasis, culminating in initiation of stone formation. Transepithelial calcium transport depends on vitamin D3 and vitamin D3 receptor (VDR). Based on the fact that VDR are present in efferent ductules, possible changes in the pattern of VDR in roosters affected by the epididymal lithiasis was investigated, to start to gain an understanding of the molecular mechanisms involved in the development of calcium stones. To evaluate the potential impact of androgen reduction, changes in androgen receptor (AR) were also investigated. Both VDR and AR were increased in specific segments of the epididymal region, whereas no alterations were found in the testes of affected animals. The increase in VDR was most likely due to an increase in the number of VDR-positive mononuclear leukocyte infiltrates found in the connective tissue followed by an increase in epithelial receptors. The AR were increased, however, mainly in the epididymal duct epithelium. These results suggest that the vitamin D3 and androgen responsive system may be directly/indirectly involved in the development of the disease.     

Expression of vitamin D receptors in the superior cervical ganglia of rats

We investigated the role of vitamin D in the sympathetic nervous system including the distribution of vitamin D receptors (VDR), 1α-hydroxylase and 24-hydroxylase (CYP24) in neuronal subpopulations and satellite glia in the superior cervical ganglia (SCGs) of rats using immunohistochemistry. VDR immunoreactivity was observed in the cytoplasm and nucleus of nearly all neurons in the SCG. Intensity of VDR fluorescence was significantly greater in the cytoplasm of neuropeptide Y (NPY) negative somata compared to NPY positive neurons. Immunoreactivity for 1α-hydroxylase also was observed in the cytoplasm of all neurons of the SCG, but the intensity of fluorescence was less in the nuclei. To the contrary, the immunoreactivity for CYP24 was stronger in the nuclei, although it was present at lower intensity also in the cytoplasm of neurons. VDR and 1α-hydroxylase immunofluorescence was observed in many non-neuron cells, except satellite glial cells, which exhibited weak CYP24 immunofluorescence. Expression of VDRs and key metabolizing enzymes indicated the importance of vitamin D in the autonomic nervous system and the ability of sympathetic neurons to activate and deactivate vitamin D for its autocrine and paracrine roles.     

Histochemical localization of estrogen and progesterone receptors: evaluation of a method

A histochemical method for the detection of estrogen (ER) and progesterone (PR) receptors in human endometrium, using estrogen and progesterone derivatives linked to fluorochrome-labeled bovine serum albumin (E2-BSA-fluorescein isothiocyanate (FITC) and progesterone-BSA-tetramethylrhodamine isothiocyanate (TMRITC], has been evaluated. The fluorochrome-labeled steroids were bound to the cytoplasm--preferably in glandular epithelial cells but to a lesser extent also to stromal cells. The steroid specificity of the observed binding was studied by preincubating the sections with a series of unlabled steroids and nonsteroidal, hormonally active compounds (estradiol-17 beta, diethylstilbestrol, tamoxifen, 5 alpha-dihydrotestosterone and R 1881 for ER and ORG 2058, R 5020, dexamethasone, cortisol and 5 alpha-dihydrotestosterone for PR). The inhibition studies indicated that E2-BSA-FITC and progesterone-BSA-TMRITC bind to ER and PR in human endometrium with a reasonable degree of specificity. The method was reproducible and various procedural steps were tested, showing satisfactory technical stability. The method is applicable to small tissue samples, and is a valuable complement to quantitative biochemical receptor assays, as it localizes the receptors in tissue slices.