Percentage Of Cells In G1 Research Articles

Culture Of Mobilized Rhesus Macaque CD34+ Cells In Hypoxic Conditions Does Not Improve Lentiviral Transduction Efficiency In Long-Term Repopulating Hematopoietic Stem Cells

Low oxygen levels have been proposed to play a fundamental role in the maintenance of normal hematopoietic stem cells (HSC) function. We investigated whether culture of rhesus macaque mobilized CD34+ cells under low oxygen conditions (1-5% O2) could improve lentiviral transduction efficiency in HSCs compared to culture under atmospheric O2 conditions (21%). Mobilized CD34+ cells from 4 animals were prestimulated for 12 hours in the presence of cytokines and subsequently divided in two equal fractions for transduction with SIN-lentiviral vectors (MOI=100) expressing GFP or TdTomato fluorescent proteins for 48 hours. In 3 animals, cells were transduced with GFP vectors in hypoxia and TdTomato vectors in normoxia. These conditions were reversed in one animal. A portion of the transduced cells was used for phenotypic, cell cycle, clonogenic and apoptosis assays. The remaining cells from both fractions were combined and injected intravenously in lethally irradiated rhesus macaques and in vivo gene marking was measured in peripheral blood granulocytes for up to 21 months after transplantation. The numbers of total live cells and CD34+ cells after 2.5 days in culture were not significantly different compared to baseline for both hypoxic and normoxic conditions (p=0.85 and p=0.84, respectively). Similarly, numbers of CD34+CD38- cells were stable compared to baseline in hypoxia and normoxia (p=0.94). After 2.5 days of culture, the total number of colony-forming cells increased 1.4-fold under both hypoxic and normoxic conditions (p=0.69) compared to freshly isolated CD34+ cells. At baseline, the majority (50.6 + 4.4%) of the CD34+ cells were in the G0 phase of the cell cycle. After 2.5 days in culture under hypoxic or normoxic conditions, the percentages of cells in G0 were 19.9 + 8.8% and 18.6 + 5.9%, respectively (p=0.82). The differences in percentages of cells in the G1 and S/G2/M phases of the cell cycle were also not statistically significant (p=0.175 and p=0.732, respectively). The pO2 had no impact on cell death (18.2 + 5.3% in hypoxia and 16.8 + 4.8% in normoxia, p=0.69) or on the percentages of apoptotic cells (11.1 + 6.7% in hypoxia and 8.4 + 2.3% in normoxia, p=0.47). When considering the bulk of CD34+ cells after transduction with lentiviral vectors, the percentages of GFP-marked cells were consistently higher (range 1.8 to 2.2-fold, mean 2.1-fold) compared to TdTomato-marked cells independently of the transduction conditions used, consistent with a 2.1-fold intrinsic superiority of GFP-based lentiviral vectors compared to TdTomato vectors at equivalent MOI. Taking into account the inherent vector differences, transduction efficiencies were similar in normoxia (70.7 + 11.7%) and hypoxia (68.0 + 16.3%) (p=0.80). When adjusted for the intrinsic superiority of GFP-based vectors, the contribution to long-term in vivo gene marking measured by flow cytometry and quantitative PCR was comparable between cells transduced in 21% pO2 (9.0 + 4.5%) and 5% pO2 conditions (8.4 + 4.9%) (p=0.87). Consistent with these data, GFP and TdTomato-labeled hematopoietic cells were equally visualized, using a confocal/2-photon hybrid microscopy approach, in BM biopsy specimens collected at various time points after transplantation. Given recent evidence suggesting that lower oxygen concentrations (<1.5%) may be required to stabilize HIF-1a, a key sensor of hypoxic conditions, transduction was performed under extreme hypoxic conditions (1% O2) in one animal. While the overall viability of the bulk CD34+ cells after 2.5 days of transduction under extreme hypoxia was comparable to cells cultured in normoxia, their contribution to long-term in vivo marking was negligible (0.3%) compared to cells transduced under normoxic conditions (2.4%), suggesting toxicity of very low oxygen levels on HSCs. Overall, when current methodologies used for the genetic manipulation of HSCs for gene therapy applications were performed under hypoxic conditions, susceptibility to lentiviral transduction of CD34+ cells was not ameliorated compared to cells cultured in 21% O2 conditions in the rhesus macaque transplantation model. The short culture times (2-3 days) used in recent lentivirus-based gene therapy clinical trials and replicated in this study may not be sufficient to impact the phenotype of long-term repopulating HSCs and do not warrant incorporation of hypoxia in current gene therapy protocols. Disclosures:No relevant conflicts of interest to declare.

Pegifna Promotes Proliferation and Myeloid Differentiation In Human Hematopoietic Progenitors In Patients With Polycythemia Vera and Essential Thrombocythemia

The myeloproliferative neoplasms, polycythemia vera (PV) and essential thrombocythemia (ET), are characterized by clonal hematopoiesis that is often associated with a JAK2V617F mutation, although this does not appear to be a disease-initiating event. Treatment of PV and ET with pegylated interferon-alpha (pegInfα) has been shown to lead to hematological remission, a decrease in the JAK2V617F allelic burden in many cases, and even a reversion to polyclonal hematopoiesis. Despite promising therapeutic results, the mechanism of pegInfα-induced remission remains elusive. There are several potential mechanisms through which pegInfα may be acting, which include stimulating the immune system in order to more effectively suppress the aberrant PV clones, enhancing the activation of normal hematopoietic stem cells (HSCs), or by selectively suppressing the mutant clones. It has been previously reported that PV patients on pegInfα have an increased number of CD4+CD25+Foxp3+ T regulatory cells (Tregs) in the peripheral blood as compared to untreated or hydroxyurea treated patients (Riley Blood, 2011), which suggests that PegIFNa maybe altering immunity against the mutated clone. However, we have found that interferon treatment leads to increased proliferation of HSCs and myeloid-specific differentiation in mice (Baldridge Nature, 2010). If this finding is also true in humans, it suggests the return to polyclonality after pegInfα could also involve an increase in normal HSC proliferation.In order to address this question, we are studying the effects of pegInfα treatment on the Tregs and HSCs of PV and EV patients, when compared to hydroxyurea or untreated patients. Previously we showed that pegInfα treatment reduced the JAK2V617F allelic burden in 17 out of 32 patients. Of the 13 female patients for which clonality could be assessed, one developed polyclonal hematopoiesis with three-fold reduction of JAK2V617F allelic burden, but one developed polyclonal hematopoiesis during therapy despite no reduction in the JAK2V617F allelic burden, suggesting that pegInfα treatment is able to affect both pre-JAK2V617F clones and JAK2V617F-positive PV clones.We have now assessed changes in the HSC population in response to pegInfα treatment. Upon analysis of bone marrow samples from these same pegInfα or hydroxyurea treated patients, we found that the number of HSCs (CD45+CD34+CD38-) was increased in patients treated with pegInfα. Further we saw a decrease in the percent of quiescent HSCs in the pegInfα treated samples, measured by the percentage of cells in G0, suggesting a more actively proliferating HSC population. In agreement with these data, our RNA analysis of the HSCs showed an increase in the expression of cell cycle genes in response to short-term pegInfα treatment. In addition to this apparent increase in HSC proliferation, we also saw an increase in the number of colonies formed in methocult media from the bone marrow samples of the pegInfα treated patients, suggesting an increase in myeloid specific differentiation. When we analyzed the RNA of patients who had received long-term pegInfα treatment, we saw a transcriptional profile that was indicative of cell death. Taken together, these data suggest a model in which pegInfα treatment is allowing for a return to polyclonal hematopoiesis by inducing cell division and differentiation of normal HSCs, while suppressing the pre-JAK2V617F or JAK2V617F-positive PV and ET clones, possibly by promoting apoptosis or inducing an immune-mediated cell death. Our findings do not exclude other potential mechanisms for salutary effects of pegInfα for treatment of PV and ET (see accompanying abstract by Swierczek et al). Disclosures:Swierczek:University of Utah: No financial compensation , No financial compensation Patents & Royalties.

Influence of reactive oxygen species on mouse bone marrow hematopoietic stem cell apoptosis

Objective of this study was to investigate the mechanism of the biological function damage resulting from increased ROS in peripheral blood stem cells during peripheral blood stem cell transplantation. Bone marrow hematopoietic stem cells (BMHSC) were cultured at the oxygen concentration imitated according to the bone marrow oxygen concentration (5% O2) including mean venous oxygen concentration (12% O2), mean arterial oxygen concentration (20% O2). The ROS level in BMHSC was detected by using fluorescent probe, the percentage of BM-HSC in cell cycle was determined by flow cytometry, the apoptosis rate was assayed by Annexin V/PI double staining, the expression levels of ATM gene and P21 protein were measured by PCR and Western respectively. The results showed that as compared with control group (5% O2), the ROS levels were lower, the percentage of cells in G1, S,G2/M phase increased (P < 0.01), the apoptosis rate of cells obviously increased (P < 0.01), the expression level of ATM gene obviously decreased (P < 0.01), while the expression level of P21 protein significantly was enhanced (P < 0.01) in 12% O2, 20% O2 and 5%-12%-20% O2 groups. It is concluded that ROS results in the apoptosis of BMHSC through inhibiting the expression of ATM gene and activating P21 protein.

Application of statistical techniques for elucidating flow cytometric data of batch and fed‐batch cultures

The objective of this work is to develop structured, segregated stochastic models for bioprocesses using time-series flow cytometric (FC) data. To this end, mammalian CHO cells were grown in both batch and fed-batch cultures, and their viable cell numbers (VCDs), monoclonal antibody (MAb), cell cycle phases, mitochondria membrane potential/mitochondria mass, Golgi apparatus, and endoplasmic reticulum (ER) were analyzed. For the fed-batch mode, soy hydrolysate was introduced at 24-H intervals. The cytometric data were analyzed for early indicators of growth and productivity by multiple linear regression analysis, which involved taking into account multicollinearity diagnostics, Durbin-Watson statistics, and Houston tests to determine and refine statistically significant correlations between categorical variables (FC parameters) and response variables (yield parameters). The results indicate that the percentage of G1 cells and ER was significantly correlated with VCD and MAb in the case of batch culture, whereas for fed-batch culture, the percentage of G2 cells and ER was correlated significantly. There was a significant difference between cells in the batch and fed-batch cultures in their ER content, suggesting that the increase in protein synthesis as reflected by the ER content and consequent increase in growth rate and MAb productivity both can be monitored at the cellular level by FC analysis of ER content.

Abstract 1863: Differing sensitivity to stage-specific autophagy inhibition.

Abstract Background: Autophagy is an intracellular process that involves the sequestration of cytosolic proteins and organelles into double-membraned vesicles called autophagosomes and their subsequent degradation in the lysosome. This process is induced during cellular stress and is crucial for maintaining proper homeostasis. Recently, autophagy has been identified as a potential mechanism of chemoresistance. Thus, autophagy inhibition has become an attractive anti-cancer therapy. However, autophagy is a multi-step process and it is still unclear if blocking autophagy at one particular stage is more advantageous than another. Therefore, we inhibited autophagy at various points in the pathway using genetic and pharmacologic means in multiple cancer models. Material and Methods: Murine cell lines used were 4T1, mammary carcinoma, B16-F10, melanoma, DLM8, osteosarcoma, and A20, lymphoma. Autophagy was inhibited by multiple methods. The first method was pharmacologic inhibition using 10uM chloroquine (CQ) or 1nM bafilomycin A1 (Baf A1), doses sufficient to inhibit autophagy. The second method was genetic knockdown by decreasing expression of critical autophagy proteins Beclin-1 (Bcn-1) or Atg7 by shRNA lentiviral delivery. Cells were assessed for protein knockdown by western blot and qPCR analysis. Proliferation of cells in the presence or absence of active autophagy was assessed using an Alamar Blue assay. For cell cycle analysis, cells were grown for 48h, stained with propidium iodine and analyzed by flow cytometry. Results and Conclusions: A significant decrease in expression of Bcn-1 and Atg7 was observed after lentiviral transduction. Autophagy was also successfully inhibited as indicated by a failure to induce elevations in autophagy marker LC3 after serum starvation, a positive autophagy control. CQ and Baf A1, which inhibit late stage autophagy, had little effect on the 4T1 cells, but CQ did inhibit the other three lines. Bcn-1 knockdown, preventing nucleation of the autophagosome, was able to inhibit proliferation in all lines at a level that was even more pronounced than CQ inhibition. In addition, the percentage of cells in G1 significantly increased in the Bcn-1 knockdown cells. Continued study with the Atg7 cells and Baf A1 will reveal if the effects of CQ and Bcn-1 knockdown are merely off target effects or truly autophagy inhibition. The inclusion of Vps 34 inhibitors and late stage mediator knockdown lines will also be useful in determining if the early, nucleation stage of autophagy is the critical step(s) to target. Lastly, it will be important to determine if these effects differ when cells experience stress, such as the presence of chemotherapy. These studies suggest that the sensitivity toward autophagy inhibition may depend on the stage of the pathway that is targeted and cancer type. Acknowledgements: Role of Autophagy in Tumor Cell Death National Cancer Institute 1R01CA150925 Citation Format: Rebecca A. Barnard, Paola Maycotte, Ryan J. Hansen, Daniel L. Gustafson, Andrew Thorburn. Differing sensitivity to stage-specific autophagy inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1863. doi:10.1158/1538-7445.AM2013-1863

Non-Canonical Wnt Signaling Regulates Hematopooetic Repopulation Through the Ryk Receptor

Abstract 852Proper regulation of the balance between quiescence and proliferation is implicated in long-term maintenance of hematopoietic stem cell (HSC) function. Our lab has demonstrated that Wnt5a, a member of the Wnt family of ligands, induces HSC quiescence in vitro and enhances short and long-term hematopoietic repopulation (Nemeth, et al. 2007). To determine the mechanisms of Wnt5a signaling in HSCs we sought to identify potential Wnt5a receptors. The Ryk gene encodes a non-canonical Wnt receptor in lineage-negative, Sca-1+, c-kit+ (LSK) cells. Previous research using neural crest cells identified Ryk as a receptor for Wnt5a. We hypothesized that the ability of Wnt5a to enhance HSC quiescence and hematopoietic repopulation required the Ryk receptor. To test this hypothesis, we cultured C57BL/6 LSK cells in serum-free media with 25ng/ml SCF and Flt3, 250ng/ml recombinant Wnt5a (rWnt5a), and 1.0 μg/ml anti-Ryk neutralizing antibody (α-Ryk) for four days. We observed that compared to control treatment, rWnt5a significantly inhibited cellular expansion (control: 5.9 ± 0.1-fold; rWnt5a: 3.1 ± 0.3-fold; n = 4, p <0.01). The effect of Wnt5a on cellular expansion was blocked by the inhibition of Ryk with α-Ryk (α-Ryk + rWnt5a: 3.1 ± 0.3-fold; n = 4, p = 0.01 compared to rWnt5a alone). We confirmed these data using a genetic approach in which we transduced LSK cells with a lentiviral vector expressing a dominant-negative Ryk mutant; LSK cells transduced with this vector did not respond to rWnt5a treatment as measured by cell expansion. We measured the effects of rWnt5a and α-Ryk treatment on the cell cycle. rWnt5a treatment resulted in a modest decrease in the percentage of cells in the S/G2/M phases (control: 35.0 ± 1.9%; rWnt5a: 29.1 ± 0.5%, n=3, p < 0.01). Treatment with α-Ryk partially restored this response (α-Ryk + rWnt5a; 32.2 ± 2.0%, n=3, p = 0.06 compared to rWnt5a). We then tested the effects of rWnt5a and α-Ryk treatment on cell quiescence. As expected, rWnt5a treatment increased the percentage of cells in G0 compared to control (control: 11.3 ± 6.7%; rWnt5a: 26.1 ± 0.9%, n=3, p < 0.01). The effect of rWnt5a on quiescence was blocked by the addition of α-Ryk (α-Ryk + rWnt5a; 21.1 ± 1.4%, n=3, p < 0.05 compared to rWnt5a). To test the effects of inhibiting Ryk on Wnt5a-mediated enhancement of hematopoietic repopulation, we performed competitive bone marrow transplant assays. We cultured C57BL/6 LSK cells for 4 days and transplanted 1 × 104 cells along with 5 × 105 whole bone marrow cells from Ubc-GFP transgenic mice (C57BL/6 background) into lethally irradiated B6.SJL mice. At 4 and 16 weeks, we measured the contribution of cultured LSK cells to hematopoietic repopulation by determining the percentage of peripheral blood cells that were CD45.2+, GFP-. rWnt5a increased the contribution of cultured LSK cells to hematopoietic repopulation after 4 weeks (control: 5.3 ± 1.4%, n = 8, rWnt5a: 19.5 ± 7.1%, n = 6, p < 0.05) and 16 weeks (control: 4.5 ± 1.6% n = 8, rWnt5a: 32.0 ± 13.1%, n = 6, p = 0.01). Inhibiting Ryk blocked the effects of rWnt5a on reconstitution after 4 and 16 weeks (4 weeks: α-Ryk + rWnt5a; 5.8 ± 2.1% n = 6, p < 0.05 compared to rWnt5a; 16 weeks: α-Ryk + rWnt5a; 11.9 ± 7.1%, n = 6, p < 0.05 compared to rWnt5a). These data indicate that the effects of rWnt5a on hematopoietic cell proliferation and HSC engraftment are regulated by the Ryk receptor. To determine a mechanism by which Wnt5a induces quiescence and hematopoietic repopulation, we tested whether Wnt5a regulates production of reactive oxygen species (ROS). Excessive ROS production is linked to decreased HSC quiescence and reduced long-term function. We measured the level of ROS in our sorted LSK cells using the ROS probe dihydrodichlorofluorescein. We observed that treatment of LSK cells with rWnt5a resulted in a 1.6 ± 0.3-fold reduction in ROS levels compared to control (n = 3); this reduction was comparable to that observed using the proto-typical anti-oxidant N-acetyl cysteine. This reduction in ROS production was inhibited by the addition of α-Ryk (1.04 ± 0.1-fold compared to control, n = 3). Together, our data indicate that the Ryk receptor functionally interacts with Wnt5a to regulate hematopoietic stem and progenitor quiescence and function and that regulation of ROS production is a potential mechanism by which Wnt5a regulates HSC function. Disclosures:No relevant conflicts of interest to declare.

Molecular Mechanisms Involved in the Synergistic Interaction of the EZH2 Inhibitor 3-Deazaneplanocin A with Gemcitabine in Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma (PDAC) is characterized by overexpression of enhancer of Zeste homolog-2 (EZH2), which plays a pivotal role in cancer stem cell (CSC) self-renewal through methylation of histone H3 lysine-27 (H3K27me3). Against this background, EZH2 was identified as an attractive target, and we investigated the interaction of the EZH2 inhibitor DZNeP with gemcitabine. EZH2 expression was detected by quantitative PCR in 15 PDAC cells, including seven primary cell cultures, showing that expression values correlated with their originator tumors (Spearman R(2) = 0.89, P = 0.01). EZH2 expression in cancer cells was significantly higher than in normal ductal pancreatic cells and fibroblasts. The 3-deazaneplanocin A (DZNeP; 5 μmol/L, 72-hour exposure) modulated EZH2 and H3K27me3 protein expression and synergistically enhanced the antiproliferative activity of gemcitabine, with combination index values of 0.2 (PANC-1), 0.3 (MIA-PaCa-2), and 0.7 (LPC006). The drug combination reduced the percentages of cells in G(2)-M phase (e.g., from 27% to 19% in PANC-1, P < 0.05) and significantly increased apoptosis compared with gemcitabine alone. Moreover, DZNeP enhanced the mRNA and protein expression of the nucleoside transporters hENT1/hCNT1, possibly because of the significant reduction of deoxynucleotide content (e.g., 25% reduction of deoxycytidine nucleotides in PANC-1), as detected by liquid chromatography/tandem mass spectrometry. DZNeP decreased cell migration, which was additionally reduced by DZNeP/gemcitabine combination (-20% in LPC006, after 8-hour exposure, P < 0.05) and associated with increased E-cadherin mRNA and protein expression. Furthermore, DZNeP and DZNeP/gemcitabine combination significantly reduced the volume of PDAC spheroids growing in CSC-selective medium and decreased the proportion of CD133+ cells. All these molecular mechanisms underlying the synergism of DZNeP/gemcitabine combination support further studies on this novel therapeutic approach for treatment of PDACs.

Abstract 1180: Modulation of p73 isoform expression regulates aggressive behavior in NHL cell line model with 1p36 chromosomal disruption

Abstract Abnormalities of chromosome 1p36 locus are frequently detected in non Hodgkin lymphoma (NHL) and are associated with aggressive clinical behavior. TP73, a gene at 1p36 locus, is a member of the p53 tumor suppressor gene family, and regulates many important cellular pathways. TP73 gene function is controlled by a balance between two isoforms with opposing functions, the full length TAp73 protein (pro-apoptotic) and the NH2-terminally truncated ΔNp73 protein (anti-apoptotic through antagonizing both TAp73 and p53). Our previous work on diagnostic specimens of NHL cases had shown a differential ΔNp73 up-regulation and increase in ΔNp73:TAp73 expression ratio among NHL cases with 1p36 chromosomal disruption as compared to NHL cases without 1p36 disruption, which correlates with decreased apoptosis, increased cellular proliferation and increased angiogenesis. We hypothesized that modulated expression of the p73 isoforms will alter the aggressive behavior of a NHL cell line containing 1p36 disruption. We used a NHL cell line (Granta-519) that has 1p36 disruption as a model. Granta-519 cells express higher levels of ΔNp73 isoform similar to what was observed in NHL cases with 1p36 abnormality. Next, we stably transfected Granta-519 with a TAp73 mammalian expression vector to modulate the ΔNp73:TAp73 ratio. Cell cycle analysis showed an increase in the percentage of G1 cells and decrease in the percentage of S and G2 cells in TAp73-trasnfected Granta-519 cells as compared to control vector-transfected cells. Serum deprivation significantly inhibited the growth of Granta-519-TAp73- cells as compared to control vector-transfected cells. Moreover, reconstitution of TAp73 expression in Granta-519 cells significantly enhanced their response to Doxorubicin. Together, these results provide an insight on modulation of ΔNp73:TAp73 as a novel therapeutic target in NHL with 1p36 abnormalities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1180. doi:1538-7445.AM2012-1180

Establishment of a docetaxel-induced human breast carcinoma drug-resistant cell line MCF7/docetaxel and its biological characteristics

Objective To establish a docetaxel-induced human breast carcinoma drug-resistant cell line MCF7/Docetaxel and study its biological characteristics. Methods The drug-resistant cell line MCF7/Docetaxel was established in culture by exposing MCF7 parent cells to moderate concentration of docetaxel over a period of 11 months. The resistance index to docetaxel was determined by methyl thiazol tetrazolium (MTT) assay. Cell growth curve was drawed and the doubling time was accounted. Flow cytometry (FCM) was performed to determine cell cycle. Fluorescence activated cell analysis (FACS) was employed to determine the concentration of fluorescence dye of rhodamine 123 ( Rh123) in the cells for the evaluation of drug-absorptive and expelling capacity. Results The resistant index of MCF7/Docetaxel was 16. 2. When MCF7/Docetaxel was stored with docetaxel at - 196℃ for 3 months and then went back to the normal environment, its characteristic of anti-tumor drug resistance was still maintained. But after being cultured in RPMI 1640 without docetaxel for a period of 1 month, MCF7/Docetaxel showed a decrease in the resistant index. As compared with the parent cells, MCF7/Docetaxel exhibited a prolonged doubling time [(39. 89 ±0. 43 vs 27. 51 ±0. 55 h,P＜0. 05] and lower growth rate. The percentages of cells in G2 and M phases were significantly increased in MCF7/Docetaxel in comparison with those in MCF7 [(39. 50 ± 3. 83) % vs (23. 59 ± 2. 51) % , P ＜ 0.05]. In soft agar growth test, MCF7/Docetaxel cells formed more cell clones than MCF7 cells (67% vs 23% ,P ＜0. 05). Finally, the concentration of Rh123 in MCF7/Docetaxel was lower than that in MCF7. Conclusion The newly established stable cell line MCF7/Docetaxel possesses typical drug-resistant characteristics and its drug-resistant phenotype is stable. Key words: Docetaxel; Drug resistance; Breast carcinoma

Effects of Homeodomain Protein CDX2 Expression on the Proliferation and Migration of Lovo Colon Cancer Cells

The homeobox gene, CDX2, plays a major role in development, especially in the gut, and also functions as a tumor suppressor in the adult colon. In the present study, we investigated the effects of CDX2 expression on the proliferation, migration, and apoptosis of the human colon cancer cell line, Lovo. Lovo cells exogenously expressing CDX2 exhibited no significant differences in the percentage of cells in G1- and S-phase or in apoptosis, as determined by flow cytometry. MTT assay also confirmed that CDX2 expression had no effect on proliferation in these cells. Interestingly, conditioned medium collected from CDX2-overexpressing Lovo cells showed a significant decrease in secretion of MMP-2 and the invasive potential of these cells was significantly inhibited. Collectively, these data suggest that CDX2 may play a critical role in the migration and metastasis of colon carcinoma and over-expression of CDX2 in colon cancer cells markedly inhibits invasion. Based on these results, exogenous expression of CDX2 might be a promising option in the treatment of colon carcinoma.

Cytotoxic Effect of Dexamethasone Restricted to Noncycling, Early G1 Phase of Melanoma Cells

This study deals with the inhibitory effects of dexamethsone on the proliferation of a human melanoma cells after separation into different fractions according to their position in the cell cycle using a gravity sedimentation chamber. Fractions with high percentage of G1 cells were more susceptible to the action of dexamethsone than those with a lower percentage of G1 cells. Dexamethsone stimulated the rate of incorporation of radioactive precursors into acid-precipitable materials of melanoma M-5A cells starting 24 hours after treatment for one hour. Moreover, dexamethsone treatment markedly increased the volume of the M-5A cells with increasing the possibility of stimulating the transcriptional/translational activity in the cells. This study may hopefully stimulate the development of new approaches in systemic and/or regional chemotherapy with malignant melanoma using dexamethsone with other alkylating agents to get synergestic interaction.

Mesenchymal stem cells isolated from human uterine cervix cancer tissues

In the present study, MSCs (mesenchymal stem cells) were successfully isolated and identified from hUCC (human uterine cervix cancer) tissues. The morphological appearance, immunophenotype, growth curve, cell cycle, cytogenetic features and differentiation potential of these cells were investigated. Results showed that cells isolated from the uterine cervix cancer tissues displayed fibroblast-like morphology and grew into colonies. Immunophenotyping by flow cytometry revealed that the isolated cells were positive for CD13, CD29, CD44, CD105 and HLA-I, while negative for CD10, CD14, CD31, CD34, CD38 and HLA-DR. The cells kept a normal karyotype by chromosome analysis. At the third passage, the percentages of cells in G0-/G1-, 2-/M- and S-phase were 84.94, 8.36 and 6.71%, respectively. Under appropriate induction conditions, these cells can differentiate into osteogenic, adipogenic cells and hepatocytes. Taken together, MSCs were confirmed to exist in hUCC tissues, which may provide a new target for clinical cancer therapy.

Abstract 2702: Renal cell carcinomas lack arginosuccinate synthetase expression and respond to pegylated arginine deiminase treatment

Abstract Biomarkers may be predictive of benefit from therapy. As a result, there is increasing interest in determining new biomarkers and new agents that may target specific pathways that are abnormal in a certain cancer. Normal cells produce arginine internally via the urea cycle. However, certain cancer cells lack one of the urea cycle enzymes, argininosuccinate synthetase (ASS) and become dependent upon an exogenous supply of arginine for growth and survival. For these ASS-deficient cancers, preferential killing of tumor cells induced by arginine deprivation may be effective and safe. ADI-PEG20 is a microbial enzyme, arginine deiminase conjugated with polyethylene gycol. The primary mechanism of action of ADI-PEG20 is arginine deprivation. Thus cancers that are deficient in ASS expression and require arginine could benefit from ADI-PEG20. ADI-PEG20 has been in phase 2 clinical trials for the treatment of hepatocellular carcinoma and metastatic melanoma. Patients whose cancers were deficient in ASS expression have demonstrated medical benefit from ADI-PEG20. Thus at least in these cancers, lack of ASS expression could serve as a biomarker to predict clinical benefit from ADI-PEG20. Other investigators have noted that human renal cell carcinoma (RCC) tissue specimens lack expression of ASS. To confirm these observations and investigate the extent of ASS deficiency in RCC, we analyzed biopsy samples obtained from 31 patients for ASS expression by immunohistochemistry and found all to be ASS negative. We also established three primary RCC cell lines and tested them for response to ADI-PEG20 treatment. Dose dependent growth inhibition was observed after 48 hours for all three cell lines using the MTS proliferation assay. We further tested three ATCC RCC cell lines, 786-O, ACHN and Caki-1 using the MTS assay and observed similar growth inhibition after 48 hours. Cell cycle analysis using the three primary RCC lines revealed that G1 arrest was apparent in samples treated with ADI-PEG20. After 48 hours of ADI-PEG20 treatment, the percentage of cells in G1 increased from 33% to 62%. These results suggest that ADI-PEG20 can potentially be an effective therapeutic agent for the treatment of RCC. They also suggest the potential utility of ASS expression as a biomarker in identifying patients that are most likely to benefit from the ADI-PEG20 treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2702.

Abstract 1004: Investigating the role of Cdc42 in the developing mouse mammary gland

Abstract Rho GTPases coordinate and transmit extracellular signals to control downstream effectors. The Rho GTPase cell division cycle 42 (Cdc42) is overexpressed and hyperactivated in human breast cancers. Cdc42 regulates cell polarity, mitosis, adhesion, migration, and stem cell function. These same cellular processes also function in normal mammary gland development. Exactly which functions of Cdc42 are crucial for normal mammary gland (MG) development and its precise role in mammary tumor formation and progression remain unknown. We hypothesize that Cdc42 plays critical and pleiotropic roles in normal MG development and tumorigenesis through regulation of the signal transduction pathways that are involved in cell cycle progression, cell polarity determination, cell motility, and cell adhesion. To investigate the molecular mechanisms by which Cdc42 regulates MG development, we studied the effects of Cdc42 deletion on morphogenesis of primary mammary epithelial cells (MECs). Adeno-cre viral transduction was used to introduce cre recombinase into Cdc42fl/fl primary MECs, and effects on morphogenesis were assessed using a three-dimensional mammary morphogenesis assay. Transduction efficiency ranged from 50-80% and ∼70% percent knockdown of Cdc42 expression was achieved. Control Adeno-GFP transduced MECs developed into polarized mammary acini whereas morphogenesis was markedly perturbed in the Cdc42 deficient MECs. Cdc42 knockout MECs formed small (30 µm in diameter compared to 45 µm in the control structures, p &amp;lt; .001, n = 28), disorganized structures lacking polarity and lumens (99% vs. 8% in the controls, n &amp;gt; 30). Markers of normal development including polarity markers, pERM and α6; adhesion, ZO-1; and downstream signaling, pkc-ζ, displayed aberrant expression patterns in the knockout structures. Interestingly, Cdc42 deficient structures contained invasive cells, which were rarely seen in control acini (41% vs 7%, n=80), suggesting that loss of Cdc42 induces aberrant migration. Cdk4 levels were also reduced in Cdc42 deficient cells and cell cycle analysis by flow cytometry demonstrated an increase in the percentage of cells in G1, suggesting that loss of Cdc42 inhibits MEC proliferation by blocking the transition from G1 to S phase. In addition, analysis of an S phase marker, phospho-histone H3, was performed at an early time point (3 days) in morphogenesis. Only 35% of the transduced knockout structures expressed phospho-histone H3, compared to 100% in the controls (n&amp;gt;20). These studies demonstrate a vital role for Cdc42 in the processes of growth, adherence, and polarity establishment during mammary gland development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1004.

A Specialized Polarized Membrane Domain Found On Normal but Not Leukemic Hematopoietic Progenitor Cells Is Required for Homing to the Bone Marrow Microenvironment.

Abstract 82Hematopoietic stem/progenitor cells (HSPCs) reside in a bone marrow niche, where adhesive interactions with osteoblasts provide essential cues for their proliferation and survival. In co-cultures of osteoblasts with primary human normal CD34+ cells, CD34+CD38- cells, or the KG1a progenitor cell line, we previously showed, using live cell imaging approaches, that HSPCs made prolonged contact with the osteoblast surface via a specialized membrane domain enriched in prominin 1 (CD133), the very late antigen-4 (VLA-4), the phosphatidylethanolamine (PE) analogue rhodamine-PE, and the tetraspanins CD63 and CD81. At the contact site, portions of the specialized domain of the CD34+ cells containing these molecules were taken up by osteoblasts and internalized into signaling endosomes within the osteoblasts. This caused the osteoblasts to downregulate Smad signaling and to increase their production of stromal-derived factor-1 (SDF-1), a chemokine responsible for HSPC homing to bone marrow (Gillette J. et al, Nat. Cell Biol. 11(3): 303–311, 2009). We have now evaluated the functional significance of these specialized membrane domains for in vivo homing of normal HSPCs to the bone marrow microenvironment. G-CSF mobilized human peripheral blood CD34+ cells from two normal donors were treated for 30 minutes with the cholesterol sequestration agent methyl-β-cyclodextrin (MβCD). This treatment resulted in disruption of the CD34+ cell membrane domains but had no effect on cell viability, proliferation or colony forming capacity in vitro. However, in two independent experiments, we observed a three-fold decrease in homing of MβCD-treated CD34+ cells to the bone marrow of NOD/SCID IL2rψcnull mice 16 hours after transplantation as compared to mock-treated CD34+ cells (p=0.0002). In contrast to homing studies, long-term human cell engraftment determined by CD45 cell surface expression 2 months after transplantation in two independent experiments was not significantly different in mice transplanted with MβCD-treated CD34+ cells compared to mock-treated CD34+ cells (p=0.13). Rapid repolarization of the membrane domain after transplantation may have resulted in engraftment of MβCD-treated CD34+ cells at levels similar to those observed with mock-treated CD34+ cells. Given the known homing/engraftment defect of actively cycling HSPC, we compared membrane domains on quiescent and cycling CD34+ cells from two normal donors. At baseline, 55–68% of CD34+ cells were in the G0 phase of the cell cycle as measured by Hoechst/Pyronin Y staining and specialized membrane domains were detected on a similar percentage of CD34+ cells for both donors. After culture for 4 days in the presence of stimulatory cytokines (SCF, TPO and Flt3), less than 10% of the CD34+ cells remained in G0 and, similarly, less than 10% of the cells analyzed by microscopy had a specialized membrane domain. After 4 days, cells were continued in culture for 2 days under non-stimulatory conditions (SCF alone). Under these conditions, for one donor, the percentage of cells in G0 increased from 9% to 18% and the percentage of cells with membrane domains increased similarly from 6% to 11%. For the other donor, the percentage of cells in G0 and with membrane domains both remained unchanged after 2 days in non-stimulatory culture. Surprisingly, the polarized membrane domains detected on normal CD34+ cells were not found on peripheral blood blast cells from patients diagnosed with relapsed AML (n=2 patients) or CML (n=1 patient). Accordingly, in a preliminary experiment, no difference in homing was observed 16 hours after transplantation of MβCD- or mock-treated peripheral blood cells from an AML patient in NOD/SCID IL2rψcnull mice. Additional homing and engraftment studies using cells from leukemic patients are ongoing. In combination, these findings indicate that specialized membrane domains are found on normal but not leukemic HSPCs and that these domains are required for homing to the bone marrow microenvironment. Disruption of these domains in actively cycling progenitor cells may provide an explanation for the previously demonstrated homing/engraftment defect of cycling cells compared to quiescent cells in the G0 phase of the cell cycle. Disclosures:No relevant conflicts of interest to declare.

NDRG4 Is Required for Cell Cycle Progression and Survival in Glioblastoma Cells

NDRG4 is a largely unstudied member of the predominantly tumor suppressive N-Myc downstream-regulated gene (NDRG) family. Unlike its family members NDRG1-3, which are ubiquitously expressed, NDRG4 is expressed almost exclusively in the heart and brain. Given this tissue-specific expression pattern and the established tumor suppressive roles of the NDRG family in regulating cellular proliferation, we investigated the cellular and biochemical functions of NDRG4 in the context of astrocytes and glioblastoma multiforme (GBM) cells. We show that, in contrast to NDRG2, NDRG4 expression is elevated in GBM and NDRG4 is required for the viability of primary astrocytes, established GBM cell lines, and both CD133(+) (cancer stem cell (CSC)-enriched) and CD133(-) primary GBM xenograft cells. While NDRG4 overexpression has no effect on cell viability, NDRG4 knockdown causes G(1) cell cycle arrest followed by apoptosis. The initial G(1) arrest is associated with a decrease in cyclin D1 expression and an increase in p27(Kip1) expression, and the subsequent apoptosis is associated with a decrease in the expression of XIAP and survivin. As a result of these effects on cell cycle progression and survival, NDRG4 knockdown decreases the tumorigenic capacity of established GBM cell lines and GBM CSC-enriched cells that have been implanted intracranially into immunocompromised mice. Collectively, these data indicate that NDRG4 is required for cell cycle progression and survival, thereby diverging in function from its tumor suppressive family member NDRG2 in astrocytes and GBM cells.

Prostaglandin E2 (PGE2) Treatment Rapidly Increases the Fraction of Non-Dividing Hematopoietic Stem Cells (HSCs)

HSCs are pluripotent cells responsible for the establishment and renewal of the entire hematopoietic system. Our group and others have established that osteoblastic cells in the bone marrow microenvironment regulate HSC cell fate decisions. Specifically, Parathyroid hormone (PTH) expands HSCs by activating osteoblasts in the HSC niche. However, the molecular mechanisms for this increase are unknown. PTH increases local production of prostaglandin E2 (PGE2) in osteoblasts by stimulating cyclo-oxygenase 2 (Cox-2). We also recently found that treatment of osteoblastic MC3T3 cells with PTH (10−7 M) rapidly induces PGE2 Synthase expression. Therefore, we hypothesized that PGE2 may act as a mediator of the PTH effect on HSCs. We have shown that in vivo PGE2 treatment caused a 2.75-fold increase in lineage− Sca-1+ c-kit+ (LSK) cells within the bone marrow compared with vehicle treated mice (p=0.0061, n=8/group). Bone marrow mononuclear cells (BMMC) from mice treated with PGE2 also demonstrated superior lymphomyeloid reconstitution in competitive repopulation analyses, suggesting that HSCs are being expanded or modulated to more efficiently reconstitute the hematopoietic system in the recipients. It is known that HSCs that reside in the G0 phase of the cell cycle have increased ability to reconstitute myeloablated recipient mice. Since PGE2 treatment resulted in superior reconstitution, we hypothesized that PGE2 may increase the percentage of HSCs residing in G0. To test this hypothesis, we treated BMMC from male C57b/6 mice with 10−6 M PGE2 or vehicle for 90 minutes. The percentage of cells in G0 vs. G1 was determined by flow-cytometric analysis using the RNA and DNA dyes, Pyronin-Y and Hoechst 33342 respectively. As we predicted, PGE2 treatment increased the percentage of wild-type LSK cells in G0 1.85 fold over vehicle-treated LSK cells (23.63% in vehicle-treated, n=4 vs. 43.7% in PGE2-treated, n=6). Since the PTH-dependent increase in HSCs is Protein Kinase A (PKA) mediated and the PGE2 receptors EP2 and EP4 signal via PKA, we assayed the effect of PGE2 on the percentage of cells in G0 in mice lacking the EP2 receptor (EP2−/− mice). Interestingly, there was no enrichment for HSC in G0 when BMMC from EP2−/− mice were treated with PGE2 (55.25% in vehicle-treated, n=4 vs. 56.06% in PGE2-treated, n=5). These findings suggest that PGE2-dependent regulation of HSC activity may involve increasing the percentage of HSCs that reside in G0 by activation of EP2, thereby augmenting their ability to reconstitute the hematopoietic system of a myeloablated recipient. 5-bromo-2-deoxyuridine (BrdU) incorporation was also used to investigate the effect of PGE2 on cell cycling of HSCs. Male 6–8 week old C57b/6 mice were injected intraperitoneally with 1 mg BrdU and PGE2 (6 mg/kg) or vehicle. After 30, 60, 90 or 120 minutes, mice were sacrificed and BMMC were subjected to flow cytometric analysis for incorporation of BrdU and DNA content in HSCs. As expected for the highly quiescent HSC population, only a small fraction of HSCs incorporated BrdU. After 30 and 60 minutes of treatment, there was no difference in the percentage of cells that incorporated BrdU between vehicle and PGE2-treated mice. However, at the 90 and 120 minute time points, there were significantly less HSCs cycling in the bone marrow from the PGE2 treated mice (12.1% vs. 5.3% at 90 min, n=2 per group; 11.1% vs. 1.8% at 120 min, n=5 per group, p=0.0060), suggesting that fewer PGE2-treated cells were synthesizing DNA. Taken together, the increase in the percentage of HSCs in G0 and the decrease in cycling HSCs after PGE2 treatment indicate that PGE2 could improve engraftment and reconstitution of the hematopoietic system by enriching for HSCs in G0. These results suggest that PGE2 may exert its beneficial effect on bone marrow reconstitution by altering cell cycle dynamics in HSCs. Identification of the molecular events mediating this novel PGE2 action on HSC could provide additional targets for HSC manipulation in clinical situations requiring rapid and efficient bone marrow reconstitution, such as recovery from iatrogenic or pathologic myeloablative injury.

Culture of Mobilized Human CD34+ Cells in Hypoxic Conditions Improves Lentiviral Transduction Efficiency in SCID-Repopulating Cells

Under normal physiological conditions, hematopoietic stem cells (HSC) are sequestered in a hypoxic microenvironment in the bone marrow (BM), suggesting that low oxygen levels may play a fundamental role in the maintenance of normal stem cell function and protect these cells from the damaging effects of reactive oxygen species (ROS). In vitro culture of human BM CD34+ cells under hypoxic conditions has been shown to result in expansion of SCID-repopulating cells (SRC) as compared to culture under normoxic conditions (JCI 112 (1); 126, 2003). We investigated whether culture of human mobilized CD34+ cells under low oxygen conditions (5% O2) could improve lentiviral transduction efficiency in SRC compared with culture under atmospheric O2 conditions (21%). G-CSF mobilized CD34+ cells from 4 healthy volunteers were prestimulated for 48 hours in the presence of cytokines (SCF, Flt-3 ligand and thrombopoietin) and subsequently transduced in fibronectin coated plates for 24 hours with SIN-lentiviral vectors carrying the GFP gene under the control of an EF1α promoter. In 3 experiments, cells were used for in vitro assays, including ROS, phenotypic, cell cycle, clonogenic and apoptosis assays. In one experiment, cells were injected intravenously in the tail vein of sublethally irradiated NOD/SCID IL2rγ −/− mice after transduction. Intracellular ROS levels increased more significantly in human CD34+ cells cultured for 3 days in 21% O2 compared with cells cultured in 5% O2. When cultures were maintained more than 3 days, ROS levels were similar between the 2 conditions. The levels of expansion of CD34+ cells compared with baseline were similar in hypoxia (3.9-fold) and normoxia (3.5-fold) (p=0.47). In contrast, the expansion of CD34+CD38− cells, a subpopulation enriched in HSCs, was greater in hypoxia (3.8-fold) than in normoxia (2.2-fold) (p=0.02). After 3 days of culture, the total number of colony-forming cells (CFC) increased 1.1-fold and 1.3-fold under hypoxic and normoxic conditions, respectively (p=0.32) compared with freshly isolated CD34+ cells. The level of O2 had no significant effect on lineage commitment of the CFC. At baseline, the majority (59.5%) of the CD34+ cells were in the G0 phase of the cell cycle. After 3 days in culture under hypoxic or normoxic conditions, the percentages of cells in G0 were 5.5% and 3.5%, respectively (p=0.03). The differences in percentages of cells in the G1 and G2/S/M phases of the cell cycle were not statistically different. The percentages of CD34+ apoptotic cells were similar between hypoxic (32.8%) and normoxic (29.5%) conditions (p=0.18). The pO2 also had no impact on CD34+ cell death (12.2% at 5% O2 and 11.7% at 21% O2, p=0.9). When considering the bulk of CD34+ cells after transduction with GFP-lentiviral vectors, there was no statistically significant difference in the percentages of GFP+ cells under hypoxia (22.3%) or normoxia (21%) (p=0.88). In contrast, when CD34+ cells cultured under hypoxia were injected into NOD/SCID IL2rγ −/− mice at the end of the transduction period, improved human cell engraftment and lentiviral transduction efficiency were detected 2 months after transplantation compared with CD34+ cells cultured under normoxia. Human cell engraftment in the mouse BM, as determined by flow cytometry using a human specific CD45 antibody, was 84% in the hypoxic group (n=4) and 54% in the normoxic group (n=4) (p=0.04). The level of O2 had no significant impact on the lineage commitment of the SRC, with a majority of CD45+CD15+ granulocytes in both groups. The percentage of GFP+CD45+ cells was 54% (hypoxia) and 43% (normoxia) (p=0.02), indicating an improved transduction efficiency of SRC under hypoxic conditions. Overall, these data indicate that human CD34+ cells cultured under low oxygen conditions maintain a more primitive phenotype and have an increased susceptibility to lentiviral transduction compared with cells cultured in 21% O2 conditions. Improved engraftment and transduction efficiency do not appear to be related to decreased apoptosis in lower O2 concentrations; instead, increased ROS production in higher O2 concentrations could lead to increased cell signaling and differentiation. Use of low O2 levels for in vitro transduction of human CD34+ cells could have important clinical implications in gene therapy.

Effects of 3-aminobenzamide on poly (ADP-ribose) polymerase expression, apoptosis and cell cycle progression of HeLa cells after X-ray irradiation

The aim of this paper is to study the changes of apoptosis and cell cycle progression in HeLa cells after the poly (ADP-ribose) polymerase (PARP) was inhibited by its inhibitor 3-aminobenzamide (3-AB) and the mechanisms of PARP action on HeLa cells damaged by irradiation. Flow cytometry (FCM) was used to examine the PARP expression and the percentage of apoptotic cells and cell cycle progression. The percentage of HeLa cells with positive expression of PARP protein 2, 4, 8 and 12 h after administrated with 3-AB was significantly lower than that of the control (P < 0.01). The percentages of apoptotic cells in the 3-AB plus irradiation group at the time points of 2, 8, 12 and 24 h after 2 Gy irradiation were higher than that in the irradiation group (P < 0.01 or P < 0.05) and the percentage of G2 cells decreased significantly (P < 0.01 or P < 0.05). It indicates that 3-AB can rapidly inhibit PARP expression of HeLa cells, promote cell apoptosis and block G2 arrest induced by irradiation.

High Dose 17 β-Estradiol and the α-Estrogen Agonist PPT Trigger Apoptosis in Human Adrenal Carcinoma Cells but the β-Estrogen Agonist DPN Does Not

Previous studies have shown that high dose 17beta-estradiol (10 (-5) M) has a G2/M blocking effect in SW-13 human adrenal carcinoma cultures and strongly enhances apoptosis. To examine the differential effects of estrogen alpha and beta-receptors in this system, we incubated SW-13 cells with specific alpha- and beta-estrogen receptor agonists, PPT [4,4',4''-(propyl-[ (1)H]-pyrazole-1,3,5-triyl) trisphenol] and DPN [2,3-bis (4-hydroxyphenyl) propionitrile], respectively (each at 10 (-5) M). Flow cytometry was used to analyze the percentages of cells in various phases of the cell cycle [sub-G1 (apoptosis), G1, S, and G2/M] in each experimental condition. Exposure to 17 beta-estradiol for 48 hours increased apoptosis more than 5-fold (from 3.6+/-0.5 to 20+/-2.2% of cells; p<0.01). The alpha-estrogen agonist PPT had a similar effect, increasing apoptosis to 22+/-1.7% (p<0.01), but the beta-agonist DPN caused no change (3.6+/-0.5 vs. 3.9+/-0.8%). While estrogen and the alpha-estrogen agonist decrease apoptosis in this system, both of these compounds decreased the percentage of cells in G1 (from 59+/-1.4% for control to 34+/-2.3% for estrogen and 40+/-2.0% for PPT; p<0.01 for both agents relative to control); the beta-agonist again had no effect. Estrogen was also found to block the cell cycle in G2/M, increasing it from 15+/-0.4 to 21+/-1.0% of cells (p<0.01), but neither the alpha- nor beta-estrogen agonists had any effect at this point in the cell cycle, indicating that the influence of estrogen was not likely to be either alpha- or beta-receptor mediated. There was no apparent effect of any of these agents on DNA synthesis, as indicated by unchanged percentages of cells in S phase. These studies suggest that induction of apoptosis by estrogen in SW-13 human adrenal cortical carcinoma cultures is mediated by the alpha-receptor, but the G2/M blocking effect of estrogen is not likely to be related to either alpha or beta mechanisms.