Yin Yang 1 promotes mTORC2-mediated AKT phosphorylation.

Yin Yang 1 (YY1) regulates both gene expression and protein modifications, and has shown a proliferative role in cancers. In this study, we demonstrate that YY1 promotes AKT phosphorylation at S473, a marker of AKT activation. YY1 expression positively correlated with AKT(S473) phosphorylation in a tissue microarray and cultured cells of breast cancer, but negatively associated with the distant metastasis-free survival of 166 breast cancer patients. YY1 promotes AKT phosphorylation at S473 through direct interaction with AKT, and the AKT-binding site is mapped to the residues G201-S226 on YY1. These residues are also involved in YY1 interaction with Mdm2, Ezh2, and E1A, and thus are designated as the oncogene protein binding (OPB) domain. YY1-promoted AKT phosphorylation relies on the OPB domain but is independent of either transcriptional activity of YY1 or the activity of phosphoinositide-3-kinases. We also determine that YY1-promoted mTORC2 access to AKT leads to its phosphorylation at S473. Importantly, a peptide based on the OPB domain blocks YY1 interaction with AKT and reduces AKT phosphorylation and cell proliferation. Thus, we demonstrate for the first time that YY1 promotes mTORC2-mediated AKT activation and disrupting YY1-AKT interaction by OPB domain-based peptide may represent a potential strategy for cancer therapy.

Understanding key assay parameters that affect measurements of trastuzumab-mediated ADCC against Her2 positive breast cancer cells.

Use of the antibody trastuzumab to kill HER2+ breast cancer cells is an attractive therapy because of its specificity and minimal adverse effects. However, a large fraction of HER2+ positive patients are or will become resistant to this treatment. No other markers are used to determine sensitivity to trastuzumab other than HER2 status.Using the xCELLigence platform and flow cytometry, we have compared the ability of mononuclear cells (MNCs) from normal and breast cancer patients to kill different breast cancer cell lines in the presence (i.e., ADCC) or absence of trastuzumab. Image analysis and cell separation procedures were used to determine the differential contribution of immune cell subsets to ADCC activity. The assay demonstrated that ADCC activity is dependent on the presence of trastuzumab, the level of HER2 expression on the target, and the ratio of MNCs to tumor cells. There is a wide range of ADCC activity among normal individuals and breast cancer patients for high and low HER2-expressing tumor targets. Fresh MNCs display higher ADCC levels compared with cryopreserved cells. Natural killer cells display the highest ADCC followed by monocytes. T cells and B cells were ineffective in killing. A major mechanism of killing of tumor cells involves insertion of granzyme B and caspase enzymes via the antibody attached MNCs.

Yin Yang 1 plays an essential role in breast cancer and negatively regulates p27.

Yin Yang 1 (YY1) is highly expressed in various types of cancers and regulates tumorigenesis through multiple pathways. In the present study, we evaluated YY1 expression levels in breast cancer cell lines, a breast cancer TMA, and two gene arrays. We observed that, compared with normal samples, YY1 is generally overexpressed in breast cancer cells and tissues. In functional studies, depletion of YY1 inhibited the clonogenicity, migration, invasion, and tumor formation of breast cancer cells, but did not affect the clonogenicity of nontumorigenic cells. Conversely, ectopically expressed YY1 enhanced the migration and invasion of nontumorigenic MCF-10A breast cells. In both a monolayer culture condition and a three-dimensional Matrigel system, silenced YY1 expression changed the architecture of breast cancer MCF-7 cells to that resembling MCF-10A cells, whereas ectopically expressed YY1 in MCF-10A cells had the opposite effect. Furthermore, we detected an inverse correlation between YY1 and p27 expression in both breast cancer cells and xenograft tumors with manipulated YY1 expression. Counteracting the changes in p27 expression attenuated the effects of YY1 alterations on these cells. In addition, YY1 promoted p27 ubiquitination and physically interacted with p27. In conclusion, our data suggest that YY1 is an oncogene and identify p27 as a new target of YY1.

Inhibition of DNA Synthesis by a Platinum-Acridine Hybrid Agent Leads to Potent Cell Kill in Non-Small Cell Lung Cancer.

The platinum-acridine anti-cancer agent [PtCl(en)(LH)](NO(3))(2) (1) (en = ethane-1,2-diamine, LH = N-(2-(acridin-9-ylamino)ethyl)-N-methylpropionimidamide, acridinium cation) and the clinical drug cisplatin were studied in chemoresistant non-small cell lung cancer (NSCLC) cell lines for their cytotoxic potency and cell-kill mechanisms. In the three cell lines tested (NCI-H460, NCI-H522, and NCI-H1435) compound 1 shows a pronounced cytotoxic enhancement of 40-200-fold compared to cisplatin at inhibitory concentrations reaching the low-nanomolar range. Based on changes in cell adhesion and cell morphology, monitored in real time by impedance measurements, compound 1 kills NCI-H460 cells significantly more efficiently than cisplatin at equitoxic concentrations. Flow cytometry analysis of NCI-H460 cells reveals a robust S-phase arrest of cells treated with compound 1, whereas cells treated with cisplatin progress to G2/M of the cell cycle. A pronounced inhibition of DNA replication in 75% of viable cells is observed in NCI-H460 cells treated with compound 1 at an IC(90) molar concentration for 48 h, based on the reduced incorporation of the fluorophore-clickable nucleoside analogue 5-ethynyl-2´-deoxyuridine (EdU) into newly synthesized DNA. The distinct cell-cycle perturbations and cell-kill potential of compound 1 are discussed in the light of the DNA interactions of this agent and its potential to overcome cisplatin resistance in NSCLC.

SOSTDC1 differentially modulates Smad and beta-catenin activation and is down-regulated in breast cancer.

Sclerostin domain containing 1 (SOSTDC1) protein regulates processes from development to cancer by modulating activity of bone morphogenetic protein (BMP) and wingless/int (Wnt) signaling pathways. As dysregulation of both BMP and Wnt signaling has been observed in breast cancer, we investigated whether disruption of SOSTDC1 signaling occurs in breast cancer. SOSTDC1 mRNA expression levels in breast tissue were examined using a dot blot. Affymetrix microarray data on SOSTDC1 levels were correlated with breast cancer patient survival using Kaplan-Meier plots. Correlations between SOSTDC1 protein levels and clinical parameters were assessed by immunohistochemistry of a breast cancer tissue microarray. SOSTDC1 secretion and BMP and Wnt signaling were investigated using immunoblotting. We found that SOSTDC1 is expressed in normal breast tissue and this expression is reduced in breast cancer. High levels of SOSTDC1 mRNA correlated with increased patient survival; conversely, SOSTDC1 protein levels decreased as tumor size and disease stage increased. Treatment of breast cancer cells with recombinant SOSTDC1 or Wise, a SOSTDC1 orthologue, demonstrated that SOSTDC1 selectively blocks BMP-7-induced Smad phosphorylation without diminishing BMP-2 or Wnt3a-induced signaling. In conclusion, SOSTDC1 mRNA and protein are reduced in breast cancer. High SOSTDC1 mRNA levels correlate with increased distant metastasis-free survival in breast cancer patients. SOSTDC1 differentially affects Wnt3a, BMP-2, and BMP-7 signaling in breast cancer cells. These results identify SOSTDC1 as a clinically important extracellular regulator of multiple signaling pathways in breast cancer.

Breast tumor cells isolated from in vitro resistance to trastuzumab remain sensitive to trastuzumab anti-tumor effects in vivo and to ADCC killing.

An understanding of model systems of trastuzumab (Herceptin) resistance is of great importance since the humanized monoclonal antibody is now used as first line therapy with paclitaxel in patients with metastatic Her2 overexpressing breast cancer, and the majority of their tumors has innate resistance or develops acquired resistance to the treatment. Previously, we selected trastuzumab-resistant clonal cell lines in vitro from trastuzumab-sensitive parental BT-474 cells and showed that cloned trastuzumab-resistant cell lines maintain similar levels of the extracellular Her2 receptor, bind trastuzumab as efficiently as the parental cells, but continue to grow in the presence of trastuzumab and display cell cycle profiles and growth rates comparable to parental cells grown in the absence of trastuzumab (Kute et al. in Cytometry A 57:86-93, 2004). We now show that trastuzumab-resistant and trastuzumab-sensitive cells both surprisingly display trastuzumab-mediated growth inhibition in athymic nude mice. This demonstrates that resistance developed in vitro is not predictive of resistance in vivo. The observation that in vitro resistant cells are sensitive to trastuzumab in vivo could be explained by antibody dependent cellular cytotoxicity (ADCC). Therefore, both parental and trastuzumab-resistant cells were assayed for ADCC in real time on electroplates with and without trastuzumab in the presence of a natural killer cell line (NK-92), and granulocyte or mononuclear cellular fractions isolated from human peripheral blood. Mononuclear cells and NK-92 cells were more effective in killing both parental and trastuzumab-resistant cells in the presence of trastuzumab. Both trastuzumab-resistant cells and trastuzumab-sensitive cells showed similar susceptibility to ADCC despite displaying divergent growth responses to trastuzumab. The granulocyte fraction was able to kill these cells with equal efficacy in the presence or absence of trastuzumab. These results support a model of trastuzumab tumor cell killing in vivo mediated primarily by ADCC from the mononuclear fraction of innate immune cells and suggest that in the clinical setting not only should changes in signaling transduction pathways be studied in acquired tumor resistance to trastuzumab, but also mechanisms by which tumors impede immune function should be evaluated.

Measurement of PTEN expression using tissue microarrays to determine a race-specific prognostic marker in breast cancer.

CONTEXT: African American women with breast cancer have worse prognoses than non-African Americans and might benefit with a race-specific prognostic marker such as PTEN (phosphatase and tensin homologue), a tumor suppressor protein. Reduced PTEN expression is associated with worse outcomes and resistance to trastuzumab in human epidermal growth factor receptor 2-positive breast cancers. Standardized PTEN evaluation is therefore needed. OBJECTIVE: To assess PTEN as a race-specific prognostic marker in breast cancer by using a novel semiquantitative score and a percent staining assessment. DESIGN: Between 1991 and 1996, 146 patients with invasive ductal adenocarcinoma were grouped by race and recurrence; there was a median follow-up of 7.2 years with 63 recurrences. Immunostaining of PTEN in tissue microarrays was correlated with race, recurrence, node positivity, stage, size, age, estrogen/progesterone receptor status, grade, and DNA ploidy. RESULTS: No significant racial difference was detected in mean PTEN values using either the semiquantitative score (P = .46) or the percent staining (P = .54). Unrelated to race, the percentage of tumor cells with positive PTEN expression correlated with longer time to recurrence (P = .047), positive estrogen receptor status (P = .009), and lower tumor grade (P = .005). The semiquantitative score correlated with positive estrogen receptor status (P = .01) and lower tumor grade (P = .001). CONCLUSIONS: PTEN expression is not a race-specific biologic prognostic marker for invasive ductal adenocarcinoma. Increased PTEN expression correlates with longer time to recurrence, positive estrogen receptor status, and lower tumor grade. The novel semiquantitative score may be used to evaluate PTEN expression, but the approximate percentage of tumor cells with any PTEN staining may be the most useful measure of PTEN expression.

Multidrug resistance protein 1 (MRP1, ABCC1) mediates resistance to mitoxantrone via glutathione-dependent drug efflux.

Based upon several previous reports, no consistent relationship between multidrug resistance protein 1 (MRP1, ABCC1) expression and cellular sensitivity to mitoxantrone (MX) toxicity can be ascertained; thus, the role of MRP1 in MX resistance remains controversial. The present study, using paired parental, MRP1-poor, and transduced MRP1-overexpressing MCF7 cells, unequivocally demonstrates that MRP1 confers resistance to MX cytotoxicity and that resistance is associated with reduced cellular accumulation of MX. This MRP1-associated reduced accumulation of MX was partially reversed by treatment of cells with 50 microM MK571 [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid]-an MRP inhibitor that increased MX accumulation in MRP1-expressing MCF7 cells but had no effect on MRP-poor MCF7 cells. Moreover, in vitro experiments using inside-out membrane vesicles show that MRP1 supports ATP-dependent, osmotically sensitive uptake of MX. Unlike ABCG2 (breast cancer resistance protein, mitoxantrone-resistant protein), MRP1-mediated MX transport is dependent upon the presence of glutathione or its S-methyl analog. In addition, MX stimulates transport of [3H]glutathione. Together, these data are consistent with the interpretation that MX efflux by MRP1 involves cotransport of MX and glutathione. The results suggest that MRP1-like the alternative MX transporters ABCG2 and ABCB1 (MDR1, P-glycoprotein)-can significantly influence tumor cell sensitivity to and pharmacological disposition of MX.

Measurement of glut-1 expression using tissue microarrays to determine a race specific prognostic marker for breast cancer.

BACKGROUND: African-American women (AAW) have more adverse tumor characteristics than non-African American women (non-AAW) and there is a need for a specific predictive marker for AAW recurrence. Tumors with higher grade and proliferative activity are associated with overexpression of the glucose transporter (Glut-1). An examination of Glut-1 expression relative to recurrence and no recurrence was conducted on both groups to determine if it predicts prognosis and could predict a race specific prognosis. METHODS: A breast cancer data set containing clinical information including race and biological characteristics was generated between 1991 and 1996. Tissue samples were selected from this group with similar characteristics in both racial groups for a retrospective analysis of Glut-1 expression using tissue microarrays (TMAs). Mean Glut-1 expression for intensity and percent of tumor cells staining was determined using standard immunohistochemistry. RESULTS: Clinical and biological differences were noted in the original data set between AAW and non-AAW. No significant difference between races was noted in mean Glut-1 values using a subset which had similar characteristics. The mean Glut-1 did not predict recurrence but a rounded score did indicate that higher levels of Glut-1 expression was indicative of a lower disease free survival (p = 0.063). The actual average mean for AAW with no recurrence was significantly lower than any other group (p = 0.04). Conclusions. TMA analysis for Glut-1 expression may be useful to predict disease free survival but it does not predict race specific recurrence.

5-Oxo-ETE analogs and the proliferation of cancer cells.

MDA-MB-231, MCF7, and SKOV3 cancer cells, but not HEK-293 cells, expressed mRNA for the leukocyte G protein-coupled 5-oxo-eicosatetraenoate (ETE) OXE receptor. 5-Oxo-ETE, 5-oxo-15-OH-ETE, and 5-HETE stimulated the cancer cell lines but not HEK-293 cells to mount pertussis toxin-sensitive proliferation responses. Their potencies in eliciting this response were similar to their known potencies in activating leukocytes and OXE receptor-transfected cells. However, high concentrations of 5-oxo-ETE and 5-oxo-15-OH-ETE, but not 5-HETE, arrested growth and caused apoptosis in all four cell lines; these responses were pertussis toxin-resistant. The same high concentrations of the oxo-ETEs but again not 5-HETE also activated peroxisome proliferator-activated receptor (PPAR)-gamma. Pharmacological studies indicated that this activation did not mediate their effects on proliferation. These results are the first to implicate the OXE receptor in malignant cell growth and to show that 5-oxo-ETEs activate cell death programs as well as PPARgamma independently of this receptor.

Tachpyridine, a metal chelator, induces G2 cell-cycle arrest, activates checkpoint kinases, and sensitizes cells to ionizing radiation.

Iron is critical for cell growth and proliferation. Iron chelators are being explored for a number of clinical applications, including the treatment of neurodegenerative disorders, heart disease, and cancer. To uncover mechanisms of action of tachpyridine, a chelator currently undergoing preclinical evaluation as an anticancer agent, cell-cycle analysis was performed. Tachpyridine arrested cells at G2, a radiosensitive phase of the cell cycle, and enhanced the sensitivity of cancer cells but not nontransformed cells to ionizing radiation. G2 arrest was p53 independent and was accompanied by activation of the checkpoint kinases CHK1 and CHK2. G2 arrest was blocked by UCN-01, a CHK1 inhibitor, but proceeded in CHK2 knock-out cells, indicating a critical role for CHK1 in G2 arrest. Tachpyridine-induced cell-cycle arrest was abrogated in cells treated with caffeine, an inhibitor of the ataxia-telangiectasia mutated/ataxia-telangiectasia-mutated and Rad3-related (ATM/ATR) kinases. Further, G2 arrest proceeded in ATM-deficient cells but was blocked in ATR-deficient cells, implicating ATR as the proximal kinase in tachpyridine-mediated G2 arrest. Collectively, our results suggest that iron chelators may function as antitumor and radioenhancing agents and uncover a previously unexplored activity of iron chelators in activation of ATR and checkpoint kinases.

Polyploid formation via chromosome duplication induced by CTP:phosphocholine cytidylyltransferase deficiency and Bcl-2 overexpression: identification of two novel endogenous factors.

Polyploidy is a profound phenotype found in tumors and its mechanism is unknown. We report here that when B-cell lymphoma gene-2 (Bcl-2) was overexpressed in a Chinese hamster ovary cell line that was deficient in CTP:phosphocholine cytidylyltransferase (CT), cellular DNA content doubled. The higher DNA content was due to a permanent conversion from diploid cells to tetraploid cells. The mechanism of polyploid formation could be attributed to the duplication of 18 parental chromosomes. The rate of conversion from diploid to tetraploid was Bcl-2 dose dependent. The diploid genome was not affected by Bcl-2 expression or by CT deficiency alone. Endogenous CT or expression of recombinant rat liver CTalpha prior to Bcl-2 expression prevented the formation of polyploid cells. This conversion was irreversible even when both initiating factors were removed. In this study, we have identified Bcl-2 as a positive regulator and CTalpha as a negative regulator of polyploid formation.

19-Nor-1alpha,25-dihydroxyvitamin D2 (paricalcitol) exerts anticancer activity against HL-60 cells in vitro at clinically achievable concentrations.

19-Nor-1alpha,25-dihydroxyvitamin D2 (paricalcitol) is an analogue of 1,25(OH)2D3 with reduced calcemic effects that is approved in the United States for the suppression of parathyroid hormone in chronic renal failure. Paricalcitol has anticancer activity in prostate cancer cells. We tested the effects of paricalcitol on the HL-60 leukemia cells, studying cellular differentiation, cell cycle changes, apoptosis and cellular proliferation. Paricalcitol at 10(-8)M concentration induced the maturation of HL-60 cells in a time-dependent manner, as shown by increased expression of CD11b differentiation surface antigen. The ability of HL-60 cells to reduce nitroblue tetrazolium (NBT) was markedly increased after exposure to paricalcitol at 10(-8)M for 72 h. Paricalcitol inhibited colony formation of HL-60 cells in a soft agar semisolid media after 10-day incubation (estimated IC50 of 5 x 10(-9) M. Exposure to 10(-8)M paricalcitol for 72 h increased the number of cells in G0/G1 phase, and decreased the number of cells in S phase, and significantly increased the number of HL-60 cells undergoing apoptosis. The concentration required to achieve inhibition of growth of HL-60 cells is comparable to clinically achievable levels. These findings support the clinical evaluation of paricalcitol as an antileukemia agent.

Development of Herceptin resistance in breast cancer cells.

BACKGROUND: Herceptin, a humanized antibody to HER-2, is now utilized in the clinic for metastatic breast cancer treatment. The response rate for HER-2+ patients is only 30% and little is known as to mechanisms of resistance. The mechanism of Herceptin action is also unknown but has been related to cell cycle inhibition. METHODS: The effects of Herceptin and other antibody treatments were determined by cell counting and cell cycle analysis. HER-2 and p27 expression levels were analyzed by flow cytometry and levels of activated AKT were compared by Western blot analysis. Cellular HER-2 and p27 expression was measured by immunofluorescence. RESULTS: Herceptin treatment of BT-474 cells results in inhibition of cell growth and arrest in the G1 phase. The efficacy of growth arrest was not directly correlated to the binding affinity of antibodies to Her-2. Our laboratory has developed cell lines that are resistant to Herceptin treatment. In resistant cell lines, binding of antibodies is not hindered. However, Herceptin has completely lost the ability to inhibit cell proliferation. Yet, the mouse isotype 4D5 maintains significant inhibitory activity upon Herceptin-resistant clones. CONCLUSIONS: Herceptin binds effectively to Her-2 on the cell surface of Herceptin-resistant cell lines and the level of Her-2 expression on the cell surface is not downregulated. Herceptin resistance is not due to downregulation of levels of AKT protein expression, although, phosphorylation of AKT is enhanced in resistant lines and could have a role in resistance. Resistance appears to correlate with the loss of nuclear expression of the cyclin-dependent kinase inhibitor, p27, as defined by immunofluorescence and flow cytometry studies and cdk-2 binding studies.

Role of multidrug resistance protein 2 (MRP2, ABCC2) in alkylating agent detoxification: MRP2 potentiates glutathione S-transferase A1-1-mediated resistance to chlorambucil cytotoxicity.

Our previous studies have shown that the glutathione S-transferases (GSTs) can operate in synergy with the efflux transporter multidrug resistance protein 1 (MRP1, ABCC1) to confer resistance to the cyto- and genotoxicities of some anticancer drugs and carcinogens. The current study was designed to determine whether the alternative efflux transporter, MRP2 (ABCC2), can also potentiate GST-mediated detoxifications in HepG2 cells. HepG2 cells, which express high-level MRP2 but not MRP1, were stably transduced with GST expression vectors under tetracycline-repressible transcriptional control. MRP2 was able to support GSTA1-1-mediated resistance to chlorambucil (CHB) cytotoxicity in HepG2 cells. Resistance was GST isozyme-specific in that GSTP1a-1a and GSTM1a-1a failed to confer protection from CHB toxicity. Moreover, inhibition of MRP2 with sulfinpyrazone completely reversed GSTA1-1-associated resistance, indicating that MRP2-efflux function is required to potentiate GSTA1-1-mediated resistance. Relative transport by MRP1 versus MRP2 of monoglutathionyl-CHB (CHB-SG) was examined using inside-out plasma membrane vesicles derived from MCF7 cells transduced with MRP1 or MRP2 expression vectors. Both MRP1 and MRP2 transported CHB-SG efficiently, at the levels of protein expressed, with similar Vmax and with Km of 0.39 and 10 microM, respectively. We conclude that detoxification of CHB by GSTA1-1 requires the removal of the glutathione conjugate formed and that either MRP1 or MRP2 can serve this efflux function. These findings have implications for the role of MRP2 in detoxification of alkylating agents in the apical epithelium of liver and kidney where it is highly expressed as well as the role of MRP2 in the emergence of alkylating drug resistance in cancer cells.

Intrabody-based strategies for inhibition of vascular endothelial growth factor receptor-2: effects on apoptosis, cell growth, and angiogenesis.

VEGF, an endothelial-specific mitogen, is an important tumor angiogenesis growth factor. The major receptor for VEGF on endothelial cells is KDR. We hypothesized that an intrabody could bind newly synthesized KDR and block receptor transport to the cell surface, thereby inhibiting important VEGF effects. We expressed a single chain antibody (p3S5) to KDR with or without the endoplasmic reticulum (ER) retention signal (KDEL), using either a plasmid (p3S5-HAK) or a tet-off adenoviral system (Ad-HAK). Plasmid-mediated expression of the tethered intrabody significantly reduced KDR expression (from 82.5+/-12.5% to 27.9+/-13.6% of cells; P<0.01) and thymidine incorporation in successfully transfected cells. Ad-HAK infection resulted in intrabody expression in >90% of human umbilical vein endothelial cells (HUVECs), producing marked (80%) apoptosis at 48 h postinfection. The intrabody was essential for these effects, as confirmed by inhibiting its expression with doxycycline or by expressing irrelevant genes (lacZ, GFP). Cell death was dependent on KDR, because Ad-HAK infection of cell lines with minimal or no KDR had little effect on cell viability. Infected HUVECs were unable to form tubes on Engelbreth Holm-Swarm (EHS) tumor gel matrix. These results demonstrate the potential for development of an intrabody-based strategy to block angiogenesis and prevent tumor growth.

19-nor-1alpha,25-dihydroxyvitamin D(2) (paricalcitol): effects on clonal proliferation, differentiation, and apoptosis in human leukemic cell lines.

PURPOSE: 19-Nor-1alpha,25-dihydroxyvitamin D(2) (paricalcitol) is an analogue of 1,25(OH)(2)D(3) with reduced calcemic effects that is approved for the suppression of parathyroid hormone in chronic renal failure. Paricalcitol has recently been reported to have anticancer activity in prostate cancer. In order to explore paricalcitol as a potential agent against leukemia, we tested its effects on HL-60 and U937 leukemia cell lines. METHODS: We studied cellular differentiation via expression of CD11b and CD14 surface antigens using flow cytometry, and via the nitroblue tetrazolium (NBT) assay. Cell cycle was analyzed using propidium iodide staining. Apoptosis was assessed with the annexin V assay. Cellular proliferation was determined via colony inhibition on semisolid medium. RESULTS: Paricalcitol induced the maturation of HL-60 and U937 cells, as shown by increased expression of CD11b differentiation surface antigen. CD14 showed increased expression in HL-60 but not in U937 cells. After exposure to paricalcitol at 10(-8) M for 72 h, the ability of HL-60 cells to reduce NBT was markedly increased. Conversely, U937 cells were unchanged. Paricalcitol inhibited colony formation of both HL-60 and U937 cell lines in semisolid medium after a 10-day incubation (estimated IC(50) of 3x10(-8) M in HL-60 cells and 4x10(-8) M in U937 cells). Paricalcitol at 10(-8) M and 10(-7) M caused a significant dose- and time-dependent increase of apoptosis in HL-60 cells ( P<0.05). In both HL-60 and U937 cells, exposure to 10(-7) M paricalcitol for 72 h increased the number of cells in G(0)/G(1) phase, and decreased the number of cells in S phase. CONCLUSIONS: Paricalcitol inhibits colony formation, induces maturation and causes cell cycle arrest in HL-60 and U937 cells. Additionally, paricalcitol induces apoptosis in HL-60 cells. These findings support the further evaluation of paricalcitol as an antileukemia agent.

Genistein and vitamin D synergistically inhibit human prostatic epithelial cell growth.

We performed studies to test synergism between the growth inhibitory effects of genistein and vitamin D compounds on prostatic epithelial cells. Isobolographic analysis demonstrated that genistein, in combination with the hormonally active form of cholecalciferol, 1alpha,25-dihydroxycholecalciferol, synergistically inhibited the growth of primary human prostatic epithelial cells (HPEC) and prostate cancer cells. Synergistic growth inhibition of HPEC was also observed between genistein and the low-calcemic vitamin D compound 25-hydroxycholecalciferol. Flow cytometry with HPEC indicated that genistein induced arrest in the G(2)M phase, whereas 1alpha,25-dihydroxycholecalciferol or 25-hydroxycholecalciferol induced arrest in the G(1/0) phase of the cell cycle. Combining genistein with either vitamin D compound resulted in both G(2)M and G(1/0) arrest in HPEC. In contrast, flow cytometry of prostate cancer cells indicated that both genistein and 1alpha,25-dihydroxycholecalciferol induced a G(1/0) arrest either alone or in combination. These are the first studies that demonstrate synergism between the prostatic cell growth inhibition elicited by genistein and that elicited by vitamin D compounds.