Association between pelvic inflammatory disease, infertility, ectopic pregnancy and the development of ovarian serous borderline tumor, mucinous borderline tumor and low-grade serous carcinoma.

OBJECTIVE: Risk factors for ovarian borderline tumors and low-grade serous carcinoma (LGSC) are poorly understood. The aim of this study was to examine the association between infertility, pelvic inflammatory disease (PID), endometriosis, ectopic pregnancy, hysterectomy, tubal ligation and parity and the risk of serous borderline tumor (SBT), mucinous borderline tumor (MBT) and LGSC. METHODS: This was a population-based cohort study using linked administrative and hospital data. Participants were 441,382 women born between 1945 and 1975 who had been admitted to hospital in Western Australia between 1 January 1980 and 30 June 2014. We used Cox regression to estimate hazard ratios (HRs). RESULTS: We observed an increased rate of SBT associated with infertility, PID and ectopic pregnancy (HRs and 95% CIs were, respectively, 1.98 (1.20-3.26); 1.95 (1.22-3.10) and 2.44 (1.20-4.96)). We did not detect an association between any of the factors under study and the rate of MBT. A diagnosis of PID was associated with an increased rate of LGSC (HR 2.90, 95% CI 1.21-6.94). CONCLUSIONS: The association with PID supports the hypothesis that inflammatory processes within the upper gynaecological tract and/or peritoneum may predispose to the development of SBT and LGSC.

Risk of persistent or recurrent cervical neoplasia in patients with 'pure' adenocarcinoma-in-situ (AIS) or mixed AIS and high-grade cervical squamous neoplasia (cervical intra-epithelial neoplasia grades 2 and 3 (CIN 2/3)): a population-based study.

OBJECTIVE: To compare outcomes of patients with pure adenocarcinoma-in-situ (AIS) and mixed AIS/CIN 2/3 lesions including the incidence of AIS persistence, recurrence and progression to adenocarcinoma. DESIGN: Retrospective cohort study. SETTING: Statewide population in Western Australia. POPULATION: Women diagnosed with AIS between 2001 and 2012. METHODS: We conducted a retrospective, population-based cohort study. MAIN OUTCOME MEASURES: De-identified linked data were utilised to ascertain the association between patient age at excisional treatment, margin status, lesion type, lesion size, and risk of persistent AIS (defined as the presence of AIS <12 months from treatment), recurrent AIS (≥12 months post-treatment), and adenocarcinoma. RESULTS: 636 patients were eligible for analysis. The mean age was 32.3 years and median follow-up interval was 2.5 years. Within the study cohort, 266 patients (41.8%) had pure AIS and 370 (58.2%) had mixed AIS/CIN 2/3. Overall, 47 patients (7.4%) had AIS persistence/recurrence and 12 (1.9%) had adenocarcinoma. Factors associated with persistence/recurrence were pure AIS (hazard ratio (HR) 2.3; 95%CI 1.28-3.94; P = 0.005), age >30 years (HR 2.1; 95%CI 1.16-3.81; P = 0.015), positive endocervical margins (HR 5.8; 95%CI 3.05-10.92; P = <0.001) and AIS lesions >8 mm (HR 2.5; 95%CI 1.00-6.20; P = 0.049). A histologically positive AIS ectocervical margin was not associated with persistence/recurrence. CONCLUSION: In this study, pure AIS was associated with greater risk of persistence/recurrence than was mixed AIS/CIN 2/3. AIS lesions >8 mm and positive endocervical margins were significant predictors for persistent or recurrent disease. TWEETABLE ABSTRACT: Pure cervical adenocarcinoma-in-situ (AIS) may have greater risk of recurrence than AIS co-existing with CIN 2/3.

GCIG-Consensus guideline for Long-term survivorship in gynecologic Cancer: A position paper from the gynecologic cancer Intergroup (GCIG) symptom benefit committee.

INTRODUCTION: Long-term survivors of gynecological cancers may be cured but still have ongoing health concerns and long-term side effects following cancer treatment. The aim of this brainstorming meeting was to develop recommendations for long-term follow-up for survivors from gynecologic cancer. METHODS: International experts, representing each member group within the Gynecologic Cancer InterGroup (GCIG), met to define long-term survival, propose guidelines for long term follow-up and propose ways to implement long term survivorship follow-up in clinical trials involving gynecological cancers. RESULTS: Long-term survival with/from gynecological cancers was defined as survival of at least five years from diagnosis, irrespective of disease recurrences. Review of the literature showed that more than 50% of cancer survivors with gynecological cancer still experienced health concerns/long-term side effects. Main side effects included neurologic symptoms, sleep disturbance, fatigue, sexual dysfunction, bowel and urinary problems and lymphedema. In this article, long-term side effects are discussed in detail and treatment options are proposed. Screening for second primary cancers and lifestyle counselling (nutrition, physical activity, mental health) may improve quality of life and overall health status, as well as prevent cardiovascular events. Clinical trials should address cancer survivorship and report patient reported outcome measures (PROMs) for cancer survivors. CONCLUSION: Long-term survivors after gynecological cancer have unique longer term challenges that need to be addressed systematically by care givers. Follow-up after completing treatment for primary gynecological cancer should be offered lifelong. Survivorship care plans may help to summarize cancer history, long-term side effects and to give information on health promotion and prevention.

A20/TNFAIP3, a new estrogen-regulated gene that confers tamoxifen resistance in breast cancer cells.

The zinc-finger protein A20/TNFAIP3, an inhibitor of nuclear factor-kappaB (NF-kappaB) activation, has been shown to protect MCF-7 breast carcinoma cells from TNFalpha-induced apoptosis. As estrogen receptor (ER) status is an important parameter in the development and progression of breast cancer, we analysed the effect of 17beta-estradiol (E2) treatment on the expression of A20. We found that A20 is a new E2-regulated gene, whose expression correlates with ER expression in both cell lines and tumor samples. With the aim of investigating the impact of A20 expression on MCF-7 cells in response to ER ligands, we established stably transfected-MCF-7 cells overexpressing A20 (MCF-7-A20). These cells exhibited a phenotype of resistance to the 4-hydroxy-tamoxifen cytostatic and pro-apoptotic actions and of hyper-response to E2. Dysregulations in bax, bcl2, bak, phospho-bad, cyclin D1, cyclin E2, cyclin D2 and cyclin A2 proteins expression were shown to be related to the resistant phenotype developed by the MCF-7-A20 cells. Interestingly, we found that A20 was also overexpressed in MVLN and VP tamoxifen-resistant cell lines. Furthermore, high A20 expression levels were observed in more aggressive breast tumors (ER-negative, progesterone receptor-negative and high histological grade). These overall findings strongly suggest that A20 is a key protein involved in tamoxifen resistance, and thus represents both a new breast cancer marker and a promising target for developing new strategies to prevent the emergence of acquired mechanisms of drug resistance in breast cancer.

Macrophage scavenger receptor CD36 is the major receptor for LDL modified by monocyte-generated reactive nitrogen species.

The oxidative conversion of LDL into an atherogenic form is considered a pivotal event in the development of cardiovascular disease. Recent studies have identified reactive nitrogen species generated by monocytes by way of the myeloperoxidase-hydrogen peroxide-nitrite (MPO-H(2)O(2)-NO(2)(-)) system as a novel mechanism for converting LDL into a high-uptake form (NO(2)-LDL) for macrophages. We now identify the scavenger receptor CD36 as the major receptor responsible for high-affinity and saturable cellular recognition of NO(2)-LDL by murine and human macrophages. Using cells stably transfected with CD36, CD36-specific blocking mAbs, and CD36-null macrophages, we demonstrated CD36-dependent binding, cholesterol loading, and macrophage foam cell formation after exposure to NO(2)-LDL. Modification of LDL by the MPO-H(2)O(2)-NO(2)(-) system in the presence of up to 80% lipoprotein-deficient serum (LPDS) still resulted in the conversion of the lipoprotein into a high-uptake form for macrophages, whereas addition of less than 5% LPDS totally blocked Cu(2+)-catalyzed LDL oxidation and conversion into a ligand for CD36. Competition studies demonstrated that lipid oxidation products derived from 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine can serve as essential moieties on NO(2)-LDL recognized by CD36. Collectively, these results suggest that MPO-dependent conversion of LDL into a ligand for CD36 is a likely pathway for generating foam cells in vivo. MPO secreted from activated phagocytes may also tag phospholipid-containing targets for removal by CD36-positive cells.

Formation of nitric oxide-derived oxidants by myeloperoxidase in monocytes: pathways for monocyte-mediated protein nitration and lipid peroxidation In vivo.

Protein nitration and lipid peroxidation are implicated in the pathogenesis of atherosclerosis; however, neither the cellular mediators nor the reaction pathways for these events in vivo are established. In the present study, we examined the chemical pathways available to monocytes for generating reactive nitrogen species and explored their potential contribution to the protein nitration and lipid peroxidation of biological targets. Isolated human monocytes activated in media containing physiologically relevant levels of nitrite (NO(2)(-)), a major end product of nitric oxide ((*)NO) metabolism, nitrate apolipoprotein B-100 tyrosine residues and initiate LDL lipid peroxidation. LDL nitration (assessed by gas chromatography-mass spectrometry quantification of nitrotyrosine) and lipid peroxidation (assessed by high-performance liquid chromatography with online tandem mass spectrometric quantification of distinct products) required cell activation and NO(2)(-); occurred in the presence of metal chelators, superoxide dismutase (SOD), and scavengers of hypohalous acids; and was blocked by myeloperoxidase (MPO) inhibitors and catalase. Monocytes activated in the presence of the exogenous (*)NO generator PAPA NONOate (Z-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2- diolate) promoted LDL protein nitration and lipid peroxidation by a combination of pathways. At low rates of (*)NO flux, both protein nitration and lipid peroxidation were inhibited by catalase and peroxidase inhibitors but not SOD, suggesting a role for MPO. As rates of (*)NO flux increased, both nitrotyrosine formation and 9-hydroxy-10,12-octadecadienoate/9-hydroperoxy-10,12-octadecadieno ic acid production by monocytes became insensitive to the presence of catalase or peroxidase inhibitors, but they were increasingly inhibited by SOD and methionine, suggesting a role for peroxynitrite. Collectively, these results demonstrate that monocytes use distinct mechanisms for generating (*)NO-derived oxidants, and they identify MPO as a source of nitrating intermediates in monocytes.

CD4+ T-cells from mice immunized to syngeneic sarcomas recognize distinct, non-shared tumor antigens.

We have utilized a newly developed culture system to study the properties of antitumor CD4+ T-cells relevant to the rejection of syngeneic methylcholanthrene sarcomas. Fresh syngeneic dendritic cells prepared from spleen, then pulsed with crude lysates of methylcholanthrene sarcomas, evoke antigen-specific proliferation by CD4+ but not by CD8+ T-cells from tumor-immune mice. Unfractionated splenocytes display similar antigen presenting capacity if they are not irradiated before the pulse with tumor lysate. CD4+ T-cells from mice immunized to individual methylcholanthrene sarcomas proliferate cross-reactively to dendritic cells pulsed with fresh tumor digests, but not to dendritic cells pulsed with cultured tumor cells. This apparent shared recognition of sarcoma lysates was demonstrated to be a result of sensitization to bacterial collagenase during the immunization procedure. Therefore, the murine CD4+ T-cell response to tumor immunization is similar to the CD8+ response in that sensitization occurs predominantly to tumor specific transplantation antigens rather than to shared tumor antigens. Strategies to avoid artefactual tumor cross-recognition by CD4+ T-cells are discussed.

Characterization of a new intrabody directed against the N-terminal region of human p53.

Genes encoding the rearranged immunoglobulin heavy and light chain variable regions of DO-1, a monoclonal antibody directed against human p53, have been used to construct a single-chain antibody. DO-1 recognizes an N-terminal epitope in the region involved in the transactivation function of p53 and the binding of Mdm2. The DO-1 single chain scFv expressed in the periplasm of E. coli or at the surface of the filamentous phage M13 retained the immunological specificity and affinity of the full length antibody. Furthermore, the DO-1 recombinant antibody was able to inhibit the in vitro binding of Hdm2, and was shown to be a powerful protecting agent of p53's DNA binding activity at 37 degrees C. The DO-1 single-chain antibody has been used to construct single-chain intracellular antibodies (intrabodies) for expression in the cytoplasm and the nucleus of mammalian cells. These anti-p53 intrabodies were additionally modified by addition of a Ckappa domain to increase cytoplasmic and nuclear stability. Here we show that expression of the DO-1 single-chain antibody in the H1299 cell line results in an inhibition of p53's transactivation function. The DO-1 intrabody is a useful tool to study those functions of p53 driven by the N-terminal region of the protein.

Molecular changes associated with the agonist activity of hydroxy-tamoxifen and the hyper-response to estradiol in hydroxy-tamoxifen-resistant breast cancer cell lines.

The aim of this study was to explore the pharmacological response to 4-hydroxy-tamoxifen (OH-Tam) and to estradiol (E2) in three cell lines: MVLN, a human breast carcinoma cell line derived from MCF-7, and two MVLN-derived OH-Tam-resistant (OTR) cell lines, called CL6.8 and CL6.32. The OH-Tam response in the OTR cells was associated with the development of both an agonist activity of the drug on cell proliferation and the resistance of the cells to OH-Tam-induced apoptosis. The OTR cells also developed an increased sensitivity to the E2 growth-stimulating activity. To delineate the genes that determine such responses, we combined a mini-array-based gene-selection approach and an extensive real-time quantitative PCR exploration in the MVLN and OTR cell lines exposed to three pharmacological conditions: a 4-day treatment with E2, OH-Tam or both E2 and OH-Tam. Compiled data revealed a hyper-response to E2 and a modification of the OH-Tam pharmacological response (loss of antagonist action and agonist activity) at the gene-expression level. The proteins encoded by the genes selected in this study have been reported to be involved in the regulation of cell proliferation, cell transformation, DNA repair and apoptosis, or belong to the ErbB/epidermal growth factor receptor-driven pathway. Our data also provide evidence of changes in transcriptional co-regulator expression, elevated mitogen-activated protein kinase activity and increase in the phosphorylation status of estrogen receptor alpha on serine residue 118 in the OTR cell lines, suggesting the possible involvement of such mechanisms in the agonist activity of OH-Tam and/or the hyper-response of cells to E2. Taken together, our study should enhance our knowledge of the multifactorial events associated with the development of Tam resistance in two independent cell lines issued from the same selection process and should help in the identification of potential molecular targets for diagnosis or therapy.

CD4+ T cells in adoptive immunotherapy and the indirect mechanism of tumor rejection.

Tumor-specific CD4+ effector T cells often play a decisive role in immunologic tumor rejection, in some cases without evident co-participation of CD8+ T cells. During such CD4+ T-cell-mediated rejection there is often no detectable direct contact between T cells and tumor cells. Optimally prepared, adoptively transferred CD4+ T cells can reject established tumors with great efficiency even when targeted tumor cells express no MHC Class II molecules, implying that recognition of tumor antigen (Ag) occurs via MHC Class II-expressing host antigen-presenting cells (APC) within the tumor. Because consequent rejection also excludes Ag-specific contact between CD4+ T cells and MHC Class IIneg tumor cells, the most critical CD4+ T-cell-mediated event is likely cytokine release, resulting in an accumulation and activation of accessory cells such as tumoricidal macrophages and lymphokine-activated killer cells. Although such an indirect rejection mechanism may appear antithetical to popular strategies centered on CD8+ cytotoxic T cell (CTL), current evidence suggest that even CD8+ T-cell-mediated recognition/rejection often bypasses direct tumor cell contact and is largely cytokine mediated. While CTL are likely to participate prominently in many models of tumor rejection, indirect mechanisms of recognition/rejection have the theoretical advantage of remaining operative even when individual tumor cells evade direct contact by down-regulating MHC and/or Ag expression.

Diverse functional activity of CD83+ monocyte-derived dendritic cells and the implications for cancer vaccines.

Antigen-loaded dendritic cells (DCs) provide key regulatory signals to T cells during a developing antitumor response. In addition to providing costimulation, mature DC provides cytokine and chemokine signals that can define the T1 vs T2 nature of the antitumor T-cell response as well as whether T cells engage in direct interactions with tumor cells. In serum-free culture conditions that hasten the differentiation of monocytes into mature DCs, certain agents, such as CD40L, accelerate phenotypic maturation (e.g., CD83 and costimulatory molecule expression) without influencing the acquisition of Dc1/Dc2 characteristics. In contrast, exposure to serum-free medium and interferon-gamma (IFN-gamma) rapidly influences CD83+ DCs to secrete high levels of IL-12, IL-6, and MIP-1beta, and promotes Dcl differentiation. In contrast, CD83+ DCs matured in serum-free medium in the absence of IFN-gamma, or in the presence of calcium signaling agents, prostaglandin-E2, or IFN-alpha, produce no IL-12, scant IL-6, and prodigious IL-8, MDC, and TARC, and promote Dc2 differentiation. T cells sensitized via IL-12-secreting, peptide-pulsed DCs secrete cytokines when subsequently exposed to relevant peptide-pulsed antigen-presenting cells (APCs) or to HLA-compatible tumor cells endogenously expressing the peptide. In contrast, T cells sensitized via IL-12 nonsecreting DC were limited to antigenic reactivation through APC contact rather than tumor cell contact. Therefore, the development of antitumor responses can be dramatically influenced not only by costimulation, but also by the cytokine and chemokine production of DCs, which must be considered in the development of cancer vaccines.

CD14+ monocytes as dendritic cell precursors: diverse maturation-inducing pathways lead to common activation of NF-kappab/RelB.

Dendritic cells are extremely potent antigen-presenting cells that are primarily responsible for the sensitization of naïve T cells to protein antigen in vivo. For this reason, dendritic cells are the focus of intense study. Despite this interest, relatively little information is available on the signal transduction pathways that regulate the development and activity of these cells. The last several years, however, have seen a steady accumulation of data regarding methods to cultivate large numbers of DC, the characterization of attendant signals that drive DC development from various precursor cells, and the induction of nuclear transcription factors that presumably direct alterations in gene expression that regulate aspects of DC development. In this review, we briefly summarize some of these findings, with emphasis on monocyte-derived dendritic cells and a discussion of two distinct types of signaling pathways that appear to regulate the final maturation of DC: one pathway calcium-dependent and cyclosporine A-sensitive, the other pathway CsA-insensitive. Although evidence suggests these signaling pathways are quite divergent in their upstream components, they both appear to activate NF-kappaB nuclear factors, particularly RelB.

T-cell adoptive therapy of tumors: mechanisms of improved therapeutic performance.

The T cells of many cancer patients are naturally sensitized to tumor-associated antigens (Ag), or they can readily be sensitized with vaccine maneuvers. In melanoma patients, the adoptive transfer of such T cells can often be causally linked to the objective regression of established tumors. So far, few patients have shown sustained clinical benefit from such therapy, but preclinical mouse studies have now clearly delineated the hurdles that must be overcome to render T-cell-based antitumor therapy effective. Contrary to earlier expectations, it is now established that remarkably potent CD4+ and CD8+ pre-effector T cells are naturally sensitized even in mice bearing progressive, weakly immunogenic tumors. However, such T cells often display signal transduction impairments as a consequence of the tumor environment, which limit their acquisition of optimal effector function. Extracorporealization and culture of these tumor-sensitized T cells with appropriate activation stimuli not only restores normal signal transduction, but also confers resolute effector activity that can often sustain tumor rejection upon reinfusion. In mouse studies, the L-selectin(low) fraction of T cells in tumor-draining lymph nodes (TDLN) constitutes the potent pre-effector population and comprises both CD4+ and helper-independent CD8+ T cells. Appropriate in vitro activation confers an apparently unrestricted trafficking capacity to this fraction, and even the ability to proliferate within the tumor bed, leading to unprecedented tumor rejection at anatomic sites (e.g., subcutaneous and intracranial) that were historically refractory to such treatment. Such results underscore the surprising capacity of appropriately activated effector T cells to withstand the immunosuppressive, tolerogenic, and apoptotic influences of the typical tumor environment. Given the increasingly appreciated and critical communications between T cells and host Ag-presenting cells (APC), which cross-present tumor Ag, it is likely that dendritic cell-based vaccine maneuvers that promote sensitization of T1-committed L-selectin(low) antitumor T cells will play an increasingly important role in adoptive therapy strategies.

T cell adoptive immunotherapy of newly diagnosed gliomas.

Patients with newly diagnosed gliomas were treated with adoptive transfer of ex vivo activated T lymphocytes, derived from lymph nodes (LNs) draining autologous tumor vaccines, to determine the long-term toxicity of this treatment. Twelve consecutive patients were enrolled: 2 with grade II astrocytoma, 4 with anaplastic gliomas, and 6 with glioblastoma multiforme. Patients were injected intradermally with short-term cultured autologous irradiated tumor cells, admixed with granulocyte macrophage colony-stimulating factor, to stimulate draining LNs. The LN cells were activated with staphylococcal enterotoxin A for 48 h and then cultured in medium containing interleukin 2 for an additional 6-8 days and subsequently transferred i.v. to the patients. The number of cells obtained from the LNs ranged from 9 x 10(7) to 1.1 x 10(9), and the median cell proliferation was 41-fold. The dose of T cells infused ranged from 0.6 to 5.5 x 10(10) with a median of 1.1 x 10(10), the majority of which were CD 4+ (mean, 71%). The entire treatment was performed as outpatient therapy and was associated with a toxicity of grade 2 or less, consisting mainly of fever, nausea, and myalgias during the first 24 h. There were no indications of late adverse events from this treatment even among three patients with follow-up greater than 2 years post T cell transfer. Moreover, four patients demonstrated partial regression of residual tumor. This Phase I clinical trial of adoptive immunotherapy for patients with newly diagnosed malignant gliomas demonstrates feasibility, lack of long-term toxicity, and several objective clinical responses.

[Imaging of pediatric spine injury]. / Traumatismes du rachis chez l'enfant.

Spine injuries in the pediatric population can lead to instability and subsequent neurological damage. Initial work up includes frontal and lateral cross-table spine radiographs, completed by CT and MRI when any doubt exists about possible injury. At the cervical level, injuries frequently involve the first three segments.

Novel BRAF and KRAS Mutations in Papillary Thyroid Carcinoma Arising in Struma Ovarii.

Papillary carcinomas of thyroid type rarely arise within struma ovarii. There are limited data on the immunohistochemical and molecular features of these tumors. Three cases of papillary carcinoma arising in struma ovarii (PCSO) were identified. The clinicopathological features were reviewed and immunohistochemical staining for HBME-1, cytokeratin (CK) 19, and CD56 was performed. Tumor DNA was sequenced for somatic mutations using a panel of 26 oncogenes, with a particular focus on BRAF and KRAS mutations. The patients were aged 22, 48, and 55 years. All cases were FIGO stage IA. Two tumors were of classical histological type, and one was a follicular variant papillary carcinoma. All tumors expressed HBME-1 and two were positive for CK19. CD56 was negative in all three cases. One tumor demonstrated a BRAF G469A mutation in exon 11, and in a second case, a KRAS Q61K double base mutation in exon 3 was detected. These mutations have not been described previously in PCSO. No mutations were detected in the benign follicular components of the tumors adjacent to the malignant papillary tissue. None of the patients had tumor recurrence on clinical follow-up (range 11 months to 8½ years). HBME-1, CK19, and CD56 are useful immunohistochemical markers of PCSO. Novel BRAF and KRAS mutations were identified in two of three tumors suggesting that mutations in PCSO may differ from those commonly identified in papillary carcinoma of the eutopic thyroid. The clinical significance of these mutations is uncertain but follow-up data in this small series support the generally good prognosis of PCSO.

Biochemical characterization of different conformational states of the Sf9 cell-purified p53His175 mutant protein.

In this study, we expressed and purified the p53 mutant encoded by the His175 allele (p53His175) in a baculovirus expression system in order to study the folding and the DNA binding activity of the protein. A two-site ELISA revealed that purified p53His175 protein preferentially displayed a PAb1620 conformation, which appeared to be not sufficient to interact specifically with DNA. The cryptic DNA binding activity of this mutant was then investigated by electrophoretic mobility shift assay in the presence of anti-p53 antibodies, and shown to be refractory to significant activation by PAb421 (a potent allosteric activator of wild-type p53's DNA binding activity). Nevertheless, p53His175 DNA binding was regulated by antibodies targeting the N-terminal region of the protein. Furthermore, while the protein preferentially displayed a PAb1620 conformation, our data suggested the existence of an equilibrium between at least two folding states of the protein (PAb1620 and PAb240 conformations). A model rationalizing the conformation, antibody-interacting ability and DNA binding regulation potential of p53His175 is presented.

Estrogen regulation in human breast cancer cells of new downstream gene targets involved in estrogen metabolism, cell proliferation and cell transformation.

We explored, by cDNA mini-arrays, gene expression measurements of MVLN, a human breast carcinoma cell line derived from MCF-7, after 4 days of exposure to 17beta-estradiol (E(2)) treatment, in order to extend our understanding of the mechanism of the pharmacological action of estrogens. We focused on 22 genes involved in estrogen metabolism, cell proliferation regulation and cell transformation. The specificity of the E(2) response was reinforced by comparison with 4-hydroxytamoxifen (OH-Tam), ICI 182,780 and E(2)+OH-Tam expression profiles. Real-time quantitative PCR (RTQ-PCR) confirmed the variation of expression of known (TFF1, AREG, IRS1, IGFBP4, PCNA, ERBB2, CTSD, MYC) as well as novel (DLEU2, CCNA2, UGT1A1, ABCC3, ABCC5, TACC1, EFNA1, NOV, CSTA, MMP15, ZNF217) genes. The temporal response of these gene expression regulations was then investigated after 6 and 18 h of E(2) treatment and this allowed the identification of different time-course patterns. Cycloheximide treatment studies indicated first that estrogen affected the transcript levels of ABCC3 and ABCC5 through dissimilar pathways, and secondly that protein synthesis was needed for modulation of the expression of the CCNA2 and TACC1 genes by estrogens. Western blot analysis performed on TFF1, IRS1, IGFBP4, amphiregulin, PCNA, cyclin A2, TACC1 and ABCC5 proteins confirmed the mini-array and RTQ-PCR data, even for genes harboring low variations of mRNA expression. Our findings should enhance the understanding of changes induced by E(2) on the transcriptional program of human E(2)-responsive cells and permit the identification of new potential diagnostic/prognostic tools for the monitoring of estrogen-related disease conditions such as breast cancer.

Calcium ionophore activation of chronic myelogenous leukemia progenitor cells into dendritic cells is mediated by calcineurin phosphatase.

We have previously demonstrated that Ph+ myeloid progenitor cells of patients with chronic myeloid leukemia (CML) can acquire characteristics of mature dendritic cells (DC) following calcium mobilization with calcium ionophore (A23187, CI). In this study we characterize the intracellular signaling pathway by which CI induces the acquisition of DC features in these leukemic cells. CI-induced activation of CML cells is attenuated by the calcineurin phosphatase inhibitor cyclosporin A (CsA) as well as the calmodulin (CaM) antagonist W-7. These cause ablation of both the CI-induced immunophenotypic expression of DC markers and immunostimulatory properties in mixed leukocyte responses (MLR). Minimal blocking effect was observed when Ca(2+)/CaM kinase II (281-301) inhibitor was added to the cultures. These findings suggest a Ca(2+)-dependent mechanism for the CI-induced activation of CML cells into antigen-presenting cells (APC), which is primarily mediated through the CaM/calcineurin pathway.