The iroquois homeobox gene 5 is regulated by 1,25-dihydroxyvitamin D3 in human prostate cancer and regulates apoptosis and the cell cycle in LNCaP prostate cancer cells.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the most active metabolite of vitamin D3, has significant antitumor activity in a broad range of preclinical models of cancer. In this study, we show that the Iroquois homeobox gene 5 (Irx5) is down-regulated by 1,25(OH)2D3 in human prostate cancer samples from patients randomly assigned to receive weekly high-dose 1,25(OH)2D3 or placebo before radical prostatectomy. Down-regulation of Irx5 by 1,25(OH)2D3 was also shown in the human androgen-sensitive prostate cancer cell line LNCaP and in estrogen-sensitive MCF-7 breast cancer cells. Knockdown of Irx5 by RNA interference showed a significant reduction in LNCaP cell viability, which was accompanied by an increase in p21 protein expression, G2-M arrest, and an increase in apoptosis. The induced apoptosis was partially mediated by p53, and p53 protein expression was increased as a result of Irx5 knockdown. Cell survival was similarly reduced by Irx5 knockdown in the colon cancer cell line HCT 116 and in MCF-7 breast cancer cells, each being derived from clinical tumor types that seem to be inhibited by 1,25(OH)2D3. Overexpression of Irx5 led to a reduction of p21 and p53 expression. This is the first report that Irx5 is regulated by 1,25(OH)2D3 in humans and the first report to show that Irx5 is involved in the regulation of both the cell cycle and apoptosis in human prostate cancer cells. Irx5 may be a promising new therapeutic target in cancer treatment.     

Vitamin D receptor and p21/WAF1 are targets of genistein and 1,25-dihydroxyvitamin D3 in human prostate cancer cells

We investigated mechanisms by which genistein and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] act synergistically to inhibit the growth of the human prostate cancer cell line LNCaP. We demonstrate that 1,25(OH)(2)D(3) and genistein cooperate to up-regulate the vitamin D receptor protein by increasing the stability of the vitamin D receptor. Genistein and 1,25(OH)(2)D(3) also cooperate to up-regulate the levels of p21/WAF1 (p21). Small interfering RNA-mediated knockdown of p21 expression showed that p21 is essential for significant growth inhibition of LNCaP cells in response to either compound or their combination. We conclude that one mechanism of synergism between genistein and 1,25(OH)(2)D(3) is through genistein modulation of vitamin D signaling.     

Expression of a novel factor, com1, is regulated by 1,25-dihydroxyvitamin D3 in breast cancer cells

Tumor cells and their surrounding microenvironment produce a variety of factors that promote tumor growth and metastasis. We recently identified a nuclear factor, termed com1, that is up-regulated in human breast carcinoma cells on formation of experimental metastatic tumors and is assumed to act as a growth-promoting factor in breast cancer. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is a potent inhibitor of growth in breast cancer both in vitro and in vivo. We compared the growth-regulatory mechanisms of nontumorigenic and estrogen-dependent MCF-7 cells with those of the tumorigenic and tamoxifen-resistant subline MCF7/ LCC2 in the presence of 1,25(OH)2D3. Proliferation of MCF7/LCC2 cells, which revealed constitutive com1 expression, was inhibited by 1,25(OH)2D3 (10(-7) M). This was strongly associated with cell cycle arrest in G1 phase, consistent with accumulation of the hypophosphorylated form of the retinoblastoma protein as well as the induction of the cyclin-dependent kinase inhibitor p21. These cell cycle events were preceded by a transient up-regulation (5-8-fold) of com1 mRNA. Furthermore, clonal growth of the MCF7/LCC2 cells was also inhibited by 1,25(OH)2D3 (10(-7) M), and when the com1-negative MCF-7 cells were stably transfected with com1, the resulting MCF7/com1 cells showed a significant decrease in colony formation. These results seem to indicate that rather than promoting growth, com1 may participate in the regulatory pathway involved in cellular growth inhibition when recruited by inhibitory signals.     

The combined treatment of 1,25-dihydroxyvitamin D3 and a non-steroid anti-inflammatory drug is highly effective in suppressing prostate cancer cell line (LNCaP) growth

BACKGROUND: The active metabolite of vitamin D3, 1,25(OH)2D3, is known to possess anti-proliferative and pro-differentiative activities in prostate cancer (PCa) cells. However, its clinical use is limited because of the risk of hypercalcemia. Concurrent administration of lower doses of 1,25(OH)2D3 together with other anticancer drugs may help to overcome this obstacle and lead to an effective and tolerable therapy. In the present in vitro study, we investigated the combined anti-cancer effect of 1,25(OH)2D3 and ibuprofen, a well-known non-steroidal anti-inflammatory drug (NSAID) that is also recognized for its ability to reduce prostate cancer development. MATERIALS AND METHODS: An androgen-sensitive prostate cancer cell line (LNCaP), grown in medium containing androgen (5alpha-dihydrotestosterone (DHT)) or without it, was treated with 1,25(OH)2D3 or ibuprofen alone or with a combination of both drugs. The effects of the treatments on LNCaP cell proliferation, cell cycle and apoptosis were evaluated by the thymidine incorporation method, the propidium iodide method and ELISA, respectively. The unpaired t-test was used for statistical analysis. RESULTS: Simultaneous treatment of LNCaP cells grown without DHT with 10 nM 1,25(OH)2D3 and 0.2 mM ibuprofen decreased cell growth by 42% (p<0.001, compared to the control cells). This effect was found to be additive since each single drug reduced cell proliferation by only 24%. On the other hand, highly significant synergistic cell growth inhibition (67%, p<0.001) was achieved by combined treatment of 1,25(OH)2D3 and ibuprofen in DHT-stimulated LNCaP cells. This combined treatment was also found to be effective in decreasing the cell transition from G1- to S-phase (p<0.003) and effective in enhancing apoptosis. CONCLUSION: Although both 1,25(OH)2D3 and ibuprofen demonstrate in vitro anti-carcinogenic activities as a single drug treatment, the present results showed that the combined use of 1,25(OH)2D3 with ibuprofen is superior to treatment with a single drug.     

Regulation of epidermal growth factor receptor levels by 1,25-dihydroxyvitamin D3 in human breast cancer cells

Specific, high affinity receptors for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] have been demonstrated in human breast cancer cells. In addition, 1,25-(OH)2D3 has been shown to inhibit replication in some human breast cancer cell lines, although the mechanism(s) of this anti-tumor activity remain undefined. There is currently considerable interest in the role of autocrine growth factors in the control of breast cancer cell proliferation and the effects of steroid hormones on their production, receptor binding, and action. Since the epidermal growth factor (EGF) receptor mediates the effects of both EGF and the autocrine growth factor, alpha-transforming growth factor, we investigated the effect of 1,25-(OH)2D3 on EGF receptor levels in several human breast cancer cell lines. Preincubation of T-47D cells with 1,25-(OH)2D3 for 24 h resulted in a significant concentration-dependent decline in the specific binding of [125I]EGF. The effect was observed when EGF binding was assayed at either 0 or 37 degrees C, both before and after treatment with acid to remove receptor bound endogenous ligand. This indicated that the effect on [125I]-EGF binding was not due to effects of 1,25-(OH)2D3 on receptor internalization and degradation or receptor occupancy. The half-maximal inhibitory concentration of 1,25-(OH)2D3 was approximately 2 nM. The decrease in EGF binding was due to a decrease in receptor number from 2,900 sites/cell in control cultures to 2,330 and 1,730 sites/cell in cells treated for 24 h with 10(-8) and 10(-6) M 1,25-(OH)2D3, respectively. There was no change in the affinity of the receptor for EGF following treatment with 1,25-(OH)2D3 [Kd = 0.075 +/- 0.006 nM (+/- SEM) for control and Kd = 0.083 +/- 0.004 nM for treated cells]. Decreased EGF receptor levels were also achieved with a number of analogues of 1,25-(OH)2D3 in accordance with their affinities for the 1,25-(OH)2D3 receptor, i.e., potencies for decreasing EGF binding in T-47D cells were in the order: 1,25-(OH)2D3 greater than 1,24,25-trihydroxyvitamin D3 greater than 1,25,26-trihydroxyvitamin D3 greater than 24,25-dihydroxyvitamin D3 greater than or equal to 25-hydroxyvitamin D3. Specific, saturable EGF binding to MCF-7 cells was also reduced by 1,25-(OH)2D3 while binding to BT-20 and HBL-100 cells was unaffected by this treatment.(ABSTRACT TRUNCATED AT 400 WORDS)     

1,25-Dihydroxyvitamin D3 enhances the susceptibility of breast cancer cells to doxorubicin-induced oxidative damage

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the hormonal form of vitamin D, has anticancer activity in vivo and in vitro. Doxorubicin exerts its cytotoxic effect on tumor cells mainly by two mechanisms: (a) generation of reactive oxygen species (ROS); and (b) inhibition of topoisomerase II. We studied the combined cytotoxic action of 1,25(OH)2D3 and doxorubicin on MCF-7 breast cancer cells. Pretreatement with 1,25(OH)2D3 resulted in enhanced cytotoxicity of doxorubicin. The average enhancing effect after a 72-h pretreatment with 1,25(OH)2D3 (10 nM) followed by a 24-h treatment with 1 microg/ml doxorubicin was 74+/-9% (mean +/- SE). Under these experimental conditions, 1,25(OH)2D3 on its own did not affect cell number or viability. 1,25(OH)2D3 also enhanced the cytotoxic activity of another ROS generating quinone, menadione, but did not affect cytotoxicity induced by the topoisomerase inhibitor etoposide. The antioxidant N-acetylcysteine slightly reduced the cytotoxic activity of doxorubicin but had a marked protective effect against the combined action of 1,25(OH)2D3 and doxorubicin. These results indicate that ROS are involved in the interaction between 1,25(OH)2D3 and doxorubicin. 1,25(OH)2D3 also increased doxorubicin cytotoxicity in primary cultures of rat cardiomyocytes. Treatment of MCF-7 cells with 1,25(OH)2D3 alone markedly reduced the activity, protein, and mRNA levels of the cytoplasmic antioxidant enzyme Cu/Zn superoxide dismutase, which indicated that the hormone inhibits its biosynthesis. This reduction in the antioxidant capacity of the cells could account for the synergistic interaction between 1,25(OH)2D3 and doxorubicin and may also suggest increased efficacy of 1,25(OH)2D3 or its analogues in combination with other ROS-generating anticancer therapeutic modalities.     

1,25-Dihydroxyvitamin D3 and all-trans-retinoic acid sensitize breast cancer cells to chemotherapy-induced cell death

We investigated the capacity of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and all-trans-retinoic acid (ATRA) to sensitize three breast cancer cell lines to the cell killing effects of paclitaxel (Taxol) and Adriamycin, two chemotherapeutic agents commonly used in the treatment of breast cancer. In tissue culture colony assays, 1,25(OH)2D3 and ATRA were synergistic in inhibiting the clonogenicity of MCF-7 and T-47D cells that expressed estrogen receptor; vitamin D receptor; retinoic acid receptors (RARs) alpha, beta, and gamma; and retinoid X receptors alpha, beta, and gamma but were not additive in MDA-MB-231 cells that lacked expression of estrogen receptor, RARalpha, and RARbeta. The hormones used individually or in combination induced up to 40-50% cell death by a trypan blue exclusion assay in a dose-dependent manner up to concentrations of 10(-7) M in MCF-7 and T-47D cells, more modestly in MDA-MB-231 cells, and not at all in MCF-10 and MCF-12 nontransformed mammary epithelial cells. Pretreating the cancer cell lines with 1,25(OH)2D3 and ATRA individually or in combination for 3 days prior to a 1-h incubation with paclitaxel or Adriamycin decreased the ED50 for inhibition of colony formation or for cell death by trypan blue by up to 2 logs for paclitaxel and up to 1 log for Adriamycin in all three cell lines but had no effect on chemotherapy-induced MCF-12 cell death. The effects of the hormones were synergistic with those of the chemotherapy agents in all of the breast cancer cell lines, generally at the higher concentrations. Cell death took place by apoptosis. To determine one potential reason for the greater potentiation of the effects of paclitaxel than those of Adriamycin, we determined the effects of preincubation of MCF-7 cells on paclitaxel-induced phosphorylation of Bcl-2. Pretreatment of MCF-7 cells with either 1,25(OH)2D3 or ATRA increased the phosphorylation of Bcl-2 by variable concentrations of paclitaxel. These data suggest that pretreatment of breast cancer with 1,25(OH)2D3 or ATRA lowers the threshold for cell killing by chemotherapy agents and may provide a novel treatment option for this disease.     

Two novel 14-Epi-analogues of 1,25-dihydroxyvitamin D3 inhibit the growth of human breast cancer cells in vitro and in vivo

The biological activity of two novel 14-epi-analogues of 1,25(OH)2D3, 19-nor-14-epi-23-yne-1,25(OH)2D3 (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)2D3 (TX 527), is described. Both analogues were at least 10 times more potent than 1,25(OH)2D3 in inhibiting in vitro cell proliferation and had much lower in vivo calcemic effects than 1,25(OH)2D3. Treatment with 1,25(OH)2D3, TX 522, or TX 527 in vitro was accompanied by an accumulation of cells in the G1 phase of the cell cycle. Protein levels of cyclin C and cyclin D1 in in vitro cultures of MCF-7 cells were down-regulated to 50 and 30%, respectively, of control levels at 72 and 120 h after stimulation. Protein levels of p21 and p27 at 72 h were significantly enhanced by 1,25(OH)2D3 and TX 522 but surprisingly not by TX 527. The inability of TX 527 to up-regulate p21 seemed to be cell type specific because p21 was induced in other cell types. Diminished phosphorylation of the retinoblastoma protein after treatment with 1,25(OH)2D3, TX 522, or TX 527 may ultimately contribute to the growth inhibition caused by these compounds. According to the data presented, the induction of apoptosis seemed not to be a major mechanism responsible for the growth-inhibitory effect of 1,25(OH)2D3 and analogues. Both 14-epianalogues significantly retarded tumor progression (40% reduced compared with control mice) in an in vivo model of MCF-7 breast cancer cells established in nude mice. In conclusion, these novel analogues have the eligible profile to be tested as therapeutic agents for the treatment of hyperproliferative diseases such as breast cancer.     

Androgen signaling is required for the vitamin D-mediated growth inhibition in human prostate cancer cells

Epidemiological data on prostate cancer incidence has suggested that vitamin D deficiency may be a risk factor for prostate cancer. The antiproliferative activity of 1alpha, 25-dihydroxyvitamin D3 (1,25-VD) and its analogues has been demonstrated in many prostate cancer models, yet the detailed mechanisms underlying this protective effect of vitamin D remain to be determined. Here, we demonstrate that two androgen receptor (AR)-positive prostate cancer cell lines, LNCaP and CWR22R, are more sensitive to the growth inhibitory effects of 1,25-VD compared to the AR-negative prostate cancer cell lines, PC-3 and DU 145. 1,25-VD treatment inhibited cyclin-dependent kinase 2 (cdk2) activity and induced G0/G1 arrest. Interestingly, we also found that 1,25-VD treatment induced the expression of AR, and that the onset of the G0/G1 arrest in LNCaP and CWR22R cells is correlated with the onset of increasing expression of AR. This implies that the antiproliferative actions of 1,25-VD in AR-positive prostate cancer might be mediated through AR. Furthermore, a reduction in 1,25-VD-mediated growth inhibition was observed when AR signaling was blocked by antiandrogens, AR RNA interference, or targeted disruption of AR. Taken together, our data suggest that the androgen/AR signaling plays an important role in the antiproliferative effects of 1,25-VD and restoration of androgen responsiveness by 1,25-VD might be beneficial for the treatment of hormone-refractory prostate cancer patients.     

Reduced 1alpha-hydroxylase activity in human prostate cancer cells correlates with decreased susceptibility to 25-hydroxyvitamin D3-induced growth inhibition

Evidence from epidemiological, molecular, and genetic studies suggests a role for vitamin D in the development and/or progression of prostate cancer. In experimental models and clinical trials, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was shown to exert antiproliferative, prodifferentiating, and antimetastatic/invasive effects on prostatic epithelial cells. Because the direct clinical application of 1,25(OH)2D3 is limited by the major side effect of hypercalcemia, we investigated the potential therapeutic utility of its less calcemic precursor, 25-hydroxyvitamin D3 [25(OH)D3], which is converted locally within the prostate to 1,25(OH)2D3 by 1alpha-hydroxylase. Quantification of 1alpha-hydroxylase activity in human prostatic epithelial cells by enzyme-substrate reaction analyses revealed a significantly decreased activity in cells derived from adenocarcinomas compared with cells derived from normal tissues or benign prostatic hyperplasia (BPH). In growth assays, we found that 25(OH)D3 inhibited growth of normal or BPH cells similarly to 1,25(OH)2D3. In contrast, in primary cultures of cancer cells and established cell lines, the antiproliferative action of 25(OH)D3 was significantly less pronounced than that of 1,25(OH)2D3. Our results indicate that growth inhibition by 25(OH)D3 depends on endogenous 1alpha-hydroxylase activity, and that this activity is deficient in prostate cancer cells. This finding has ramifications for both the prevention and therapy of prostate cancer with vitamin D compounds.     

25-Hydroxyvitamin D3 1alpha-hydroxylase splice variants in breast cell lines MCF-7 and MCF-10

BACKGROUND: It is known that 25(OH)D3 can be metabolized to 1,25(OH)2 D3 by 1alpha-OHase in breast tissue. This tissue-specific expression of 1alpha-OHase may act as the pivotal link between vitamin D status (25(OH)D3 levels) and the anticancer effects of 1,25(OH)2 D3. Alternative splicing frequently occurs in breast cancer cells; different splice variants of a given protein can display different biological functions and may cause tissue-specific variations. With this study it is the first time that expression and alternative splicing of 1alpha-OHase in the human breast cancer cell line MCF-7 and thebenign breast cell line MCF-10A are described. MATERIALS AND METHODS: Expression of 1alpha-OHase RNA and protein was assessed using a real-time polymerase chain reaction (RT-PCR). The expression of 1alpha-OHase splice variants was detected by a highly specific PCR that combines nested and touchdown PCR. To determine which variants are translated in protein western blot analysis was carried out. RESULTS: The expression of 1alpha-OHase was found to be 1.25-fold higher in MCF-7 compared to MCF-10A cells. In MCF-10A cells, at least 6 splice variants were detected whereas MCF-7 showed no or marginal expression levels of these variants. In MCF-7 cells the antibody detected a signal at 56 kDa corresponding to the size of normal 1alpha-OHase protein. In MCF-10A cells this signal was weaker. In western blot analysis at least two smaller variants at 45 kDa were found in MCF-7 cells. In MCF-10A cells at least 6 proteins between 37 and 56 kDa were detected with an only faint signal. CONCLUSION: We propose that alternative splicing of 1alpha-OHase can regulate the level of active enzyme. Splice variants may lead to a reduction of the protein. The significance of the smaller variants in MCF-7 cells has not been clarified either, but it is known that they are not able to use 25(OH)D3 as a substrate to generate 1,25(OH)D3. In MCF10A cells, more splice variants were identified, it may be that malignant cells contain inactive variants. How far they show a reduced activity remains unclear as no activity measurements were performed.     

Influence of 1 alpha,25-dihydroxyvitamin D-3 on growth regulation through epidermal growth factor receptor in human breast cancer cell lines

We investigated the relationship between epidermal growth factor (EGF) dependent cell growth and antiproliferative effects of 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) in hormone responsive breast cancer cell lines in vitro. MCF-7 breast cancer cells and GMC-M, which is a serum-independent, hormone receptor-positive subtype derived from MCF-7, were used in this study. EGF stimulated the growth Of both cell lines, and 1,25(OH)(2)D-3 inhibited the EGF-stimulated cell growth in a dose dependent fashion. But treatment with 1,25(OH)(2)D-3 did not change the EGF receptor (EGFR) level significantly in either cell line. GMC-M had a higher level of EGFR and was more sensitive to EGF than MCF-7. These results suggest that other mechanisms of action, which are different from EGFR modulation, concern with the growth inhibitory effect of 1,25(OH)(2)D-3, and that 1,25(OH)(2)D-3 will be a new effective treatment for breast cancer irrespective of EGFR.     

The G gamma / T-15 transgenic mouse model of androgen-independent prostate cancer: target cells of carcinogenesis and the effect of the vitamin D analogue EB 1089.

Transgenic mouse models of prostate cancer provide unique opportunities to understand the molecular events in prostate carcinogenesis and for the preclinical testing of new therapies. We studied the G gamma T-15 transgenic mouse line, which contains the human fetal globin promoter linked to SV40 T antigen (Tag) and which develops androgen-independent prostate cancer. Using the immunohistochemistry of normal mouse prostates before tumor formation, we showed that the target cells of carcinogenesis in G gamma T-15 mice are located in the basal epithelial layer. We tested the efficacy of the 1,25(OH)(2)D(3) analogue, EB 1089, to chemoprevent prostate cancer in these transgenic mice. Compared with treatment with placebo, treatment with EB 1089 at three different time points before the onset of prostate tumors in mice did not prevent or delay tumor onset. However, EB 1089 significantly inhibited prostate tumor growth. At the highest dose, EB 1089 inhibited prostate tumor growth by 60% (P = 0.0003) and the growth in the number of metastases, although this dose also caused significant hypercalcemia and weight loss. We conducted several in vitro experiments to explore why EB 1089 did not prevent the occurrence of the primary tumors. EB 1089 significantly inhibited the growth of a Tag-expressing human prostate epithelial cell line, BPH-1, and an androgen-insensitive subline of LNCaP cells [which was not inhibited by 1,25(OH)(2)D(3)]. Thus, neither Tag expression nor androgen insensitivity explain the absence of chemopreventive effect. Conversely, neither 1,25(OH)(2)D(3) nor EB 1089 inhibited the growth of the normal rat prostate basal epithelial cell line NRP-152. It is likely that EB 1089 was not effective in delaying the growth of the primary tumor in G gamma T-15 transgenic mice because the target cells of carcinogenesis in these mice are located in the basal epithelial layer. We conclude that G gamma T-15 transgenic mice are a useful model for testing vitamin D-based therapies in androgen-insensitive prostate cancer but are not suitable for studies of vitamin D-based chemoprevention. The superiority of EB 1089 over 1,25(OH)(2)D(3) in the growth suppression of androgen-insensitive prostate cancer cells supports the use of EB 1089 in androgen-insensitive prostate cancer.     

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, E.C. 1.2.1.12.) gene expression in two malignant human mammary epithelial cell lines: BT-20 and MCF-7. Regulation of gene expression by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3).

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the control of glycolysis. Its gene expression was analyzed in two breast cancer cell lines of human origin, BT-20 and MCF-7. We used a cDNA probe of 1.3 kb for Northern blot hybridization. It is found that GAPDH mRNA is overexpressed only in the poorly differentiated BT-20 cell line and that treatment of these cells by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) stimulates GAPDH mRNA expression in a dose-and time-dependent manner. The present investigation on the BT-20 cells indicates that the expression of GAPDH is sensitive to 1,25-(OH)2D3 and up-regulated by low doses of this steroid.     

Comparative effects of 1,25(OH)2D3 and EB1089 on cell cycle kinetics and apoptosis in MCF-7 breast cancer cells

1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], the active metabolite of vitamin D, inhibits breast cancer cell growth both in vivo and in vitro. In addition to its anti-proliferative effects, 1,25(OH)₂D₃ induces morphological and biochemical markers of apoptosis in MCF-7 cells. In the studies reported here, we compared the effects of 1,25(OH)₂D₃ and EB1089, a low calcemic vitamin D analog, on cell cycle kinetics and apoptosis in MCF-7 cells. Both vitamin D compounds reduced viable MCF-7 cell number in a time and dose dependent manner, with EB1089 approximately 50 fold more potent than 1,25(OH)₂D₃. Flow cytometric analysis indicated that both agents induced cell cycle arrest in G₀/G₁ which was associated with accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein. MCF-7 cells treated with either 1,25(OH)₂D₃ or EB1089 for 48 h exhibited characteristics of apoptosis, including cytoplasmic condensation, pyknotic nuclei, condensed chromatin and DNA fragmentation. Cells treated with either agent exhibited up regulation of proteins associated with mammary gland regression (clusterin and cathepsin B) and down regulation of the anti-apoptotic protein bcl-2. These studies demonstrate that, despite its lower calcemic activity in vivo, the vitamin D analog EB1089 induces effects that are indistinguishable from those of 1,25(OH)₂D₃ on cell number, cell cycle and indices of apoptosis in MCF-7 cells in vitro. In addition, since both agents rapidly down regulate estrogen receptor, disruption of estrogen dependent signalling may play a role in the induction of apoptosis by vitamin D compounds in MCF-7 cells.