Lassa-VSV chimeric virus targets and destroys human and mouse ovarian cancer by direct oncolytic action and by initiating an anti-tumor response.

Ovarian cancer is the third most common female cancer, with poor survival in later stages of metastatic spread. We test a chimeric virus consisting of genes from Lassa and vesicular stomatitis viruses, LASV-VSV; the native VSV glycoprotein is replaced by the Lassa glycoprotein, greatly reducing neurotropism. Human ovarian cancer cells in immunocompromised nude mice were lethal in controls. Chemotherapeutic paclitaxel and cisplatin showed modest cancer inhibition and survival extension. In contrast, a single intraperitoneal injection of LASV-VSV selectively infected and killed ovarian cancer cells, generating long-term survival. Mice with human ovarian cancer cells in brain showed rapid deterioration; LASV-VSV microinjection into brain blocked cancer growth, and generated long-term survival. Treatment of immunocompetent mice with infected mouse ovarian cancer cells blocked growth of non-infected ovarian cancer cells peritoneally and in brain. These results suggest LASV-VSV is a viable candidate for further study and may be of use in the treatment of ovarian cancer.

p53 protein aggregation promotes platinum resistance in ovarian cancer.

High-grade serous ovarian carcinoma (HGSOC), the most lethal gynecological cancer, often leads to chemoresistant diseases. The p53 protein is a key transcriptional factor regulating cellular homeostasis. A majority of HGSOCs have inactive p53 because of genetic mutations. However, genetic mutation is not the only cause of p53 inactivation. The aggregation of p53 protein has been discovered in different types of cancers and may be responsible for impairing the normal transcriptional activation and pro-apoptotic functions of p53. We demonstrated that in a unique population of HGSOC cancer cells with cancer stem cell properties, p53 protein aggregation is associated with p53 inactivation and platinum resistance. When these cancer stem cells differentiated into their chemosensitive progeny, they lost tumor-initiating capacity and p53 aggregates. In addition to the association of p53 aggregation and chemoresistance in HGSOC cells, we further demonstrated that the overexpression of a p53-positive regulator, p14ARF, inhibited MDM2-mediated p53 degradation and led to the imbalance of p53 turnover that promoted the formation of p53 aggregates. With in vitro and in vivo models, we demonstrated that the inhibition of p14ARF could suppress p53 aggregation and sensitize cancer cells to platinum treatment. Moreover, by two-dimensional gel electrophoresis and mass spectrometry we discovered that the aggregated p53 may function uniquely by interacting with proteins that are critical for cancer cell survival and tumor progression. Our findings help us understand the poor chemoresponse of a subset of HGSOC patients and suggest p53 aggregation as a new marker for chemoresistance. Our findings also suggest that inhibiting p53 aggregation can reactivate p53 pro-apoptotic function. Therefore, p53 aggregation is a potential therapeutic target for reversing chemoresistance. This is paramount for improving ovarian cancer patients' responses to chemotherapy, and thus increasing their survival rate.

Biopsy-induced inflammatory conditions improve endometrial receptivity: the mechanism of action.

A decade ago, we first reported that endometrial biopsy significantly improves the success of pregnancy in IVF patients with recurrent implantation failure, an observation that was later confirmed by others. Recently, we have demonstrated that this treatment elevated the levels of endometrial pro-inflammatory cytokines and increased the abundance of macrophages (Mac) and dendritic cells (DCs). We therefore hypothesised that the biopsy-related successful pregnancy is secondary to an inflammatory response, and aimed at deciphering its mechanism of action. Supporting our hypothesis, we found that the pro-inflammatory TNFα stimulated primary endometrial stromal cells to express cytokines that attracted monocytes and induced their differentiation into DCs. These monocyte-derived DCs stimulated endometrial epithelial cells to express the adhesive molecule SPP1 (osteopontin (OPN)) and its receptors ITGB3 and CD44, whereas MUC16, which interferes with adhesion, was downregulated. Other implantation-associated genes, such as CHST2, CCL4 (MIP1B) and GROA, were upregulated by monocyte-derived Mac. These findings suggest that uterine receptivity is mediated by the expression of molecules associated with inflammation. Such an inflammatory milieu is not generated in some IVF patients with recurrent implantation failure in the absence of local injury provoked by the biopsy treatment.

Constitutive proteasomal degradation of TWIST-1 in epithelial-ovarian cancer stem cells impacts differentiation and metastatic potential.

Epithelial-mesenchymal transition (EMT) is a critical process for embryogenesis but is abnormally activated during cancer metastasis and recurrence. This process enables epithelial cancer cells to acquire mobility and traits associated with stemness. It is unknown whether epithelial stem cells or epithelial cancer stem cells are able to undergo EMT, and what molecular mechanism regulates this process in these specific cell types. We found that epithelial-ovarian cancer stem cells (EOC stem cells) are the source of metastatic progenitor cells through a differentiation process involving EMT and mesenchymal-epithelial transition (MET). We demonstrate both in vivo and in vitro the differentiation of EOC stem cells into mesenchymal spheroid-forming cells (MSFCs) and their capacity to initiate an active carcinomatosis. Furthermore, we demonstrate that human EOC stem cells injected intraperitoneally in mice are able to form ovarian tumors, suggesting that the EOC stem cells have the ability to 'home' to the ovaries and establish tumors. Most interestingly, we found that TWIST-1 is constitutively degraded in EOC stem cells, and that the acquisition of TWIST-1 requires additional signals that will trigger the differentiation process. These findings are relevant for understanding the differentiation and metastasis process in EOC stem cells.

TWISTing stemness, inflammation and proliferation of epithelial ovarian cancer cells through MIR199A2/214.

Cancer stem cells are responsible for sustaining the tumor and giving rise to proliferating and progressively differentiating cells. However, the molecular mechanisms regulating the process of cancer stem cell (CSC) differentiation is not clearly understood. Recently, we reported the isolation of the epithelial ovarian cancer (EOC) stem cells (type I/CD44+). In this study, we show that type I/CD44+ cells are characterized by low levels of both miR-199a and miR-214, whereas mature EOC cells (type II/CD44-) have higher levels of miR-199a and miR-214. Moreover, these two micro RNAs (miRNAs) are regulated as a cluster on pri-miR-199a2 within the human Dnm3os gene (GenBank FJ623959). This study identify Twist1 as a regulator of this unique miRNA cluster responsible for the regulation of the IKKbeta/NF-kappaB and PTEN/AKT pathways and its association of ovarian CSC differentiation. Our data suggest that Twist1 may be an important regulator of 'stemness' in EOC cells. The regulation of MIR199A2/214 expression may be used as a potential therapeutic approach in EOC patients.

The isolation and characterization of a novel telomerase immortalized first trimester trophoblast cell line, Swan 71.

Studies using first trimester trophoblast cells may be limited by the inability to obtain patient samples and/or adequate cell numbers. First trimester trophoblast cell lines have been generated by SV40 transformation or similar methods, however, this approach is known to induce phenotypic and karyotypic abnormalities. The introduction of telomerase has been proposed to be a viable alternative for the immortalization of primary human cells. To investigate whether telomerase-induced immortalization might be a more feasible approach for the generation of first trimester trophoblast cell lines, we isolated primary trophoblast cells from a 7-week normal placenta and infected the cells with human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. Although this hTERT-infected first trimester trophoblast cell line, which we have named Swan 71, has been propagated for more than 100 passages, it still has attributes that are characteristic of primary first trimester trophoblast cells. The Swan 71 cells are positive for the expression of cytokeratin 7, vimentin and HLA-G, but do not express CD45, CD68 or the Fibroblast Specific Antigen (FSA), CD90/Thy-1. In addition, we also demonstrated that the Swan 71 cells secrete fetal fibronectin (FFN) as well as low levels of human Chorionic Gonadotrophin (hCG). Moreover, the Swan 71 cells exhibit a cytokine and growth factor profile that is similar to primary trophoblast cells and are resistant to Fas, but not TNF-alpha-induced apoptosis. This suggests that the Swan 71 cells may represent a valuable model for future in vitro trophoblast studies.

Regulation of IKKbeta by miR-199a affects NF-kappaB activity in ovarian cancer cells.

Cancer progression is an abnormal form of tissue repair characterized by chronic inflammation. IkappaB kinase-beta (IKKbeta) required for nuclear factor-kappaB (NF-kappaB) activation plays a critical role in this process. Using EOC cells isolated from malignant ovarian cancer ascites and solid tumors, we identified IKKbeta as a major factor promoting a functional TLR-MyD88-NF-kappaB pathway that confers to EOC cell the capacity to constitutively secrete proinflammatory/protumor cytokines and therefore promoting tumor progression and chemoresistance. Furthermore, we describe for the first time the identification of the microRNA hsa-miR-199a as a regulator of IKKbeta expression. Our study describes the property of ovarian cancer cells to enhance the inflammatory microenvironment as a result of the expression of an active IKKbeta pathway. Identification of these markers in patients' tumor samples may facilitate the adequate selection of treatment and open new venues for the development of effective therapy for chemoresistant ovarian cancers.

Cancers take their Toll--the function and regulation of Toll-like receptors in cancer cells.

Cancer could be deemed as an abnormal and uncontrolled tissue repair process. Therefore, it would not be surprising that factors that function in the tissue repair process, such as cytokines, chemokines, growth factors and Toll-like receptor (TLR) ligands, as well as growth signals for compensatory proliferation, would also be key factors in regulating and enhancing cancer progression. The TLR pathways, which play a critical role in tissue repair, are also key regulators in cancer progression as well as chemoresistance. TLRs serve as cell surface sensors that can initiate pathways leading to proliferation and chemoresistance; as well as mediators that are able to regulate the infiltrating immune cells to provide further support for cancer progression.

Carcinosarcoma of the ovary.

The objective of this study was to evaluate the treatment and outcome in patients with ovarian carcinosarcoma. The Tumor Board Registry was reviewed for patients with ovarian carcinosarcoma treated at our institution from June 1993 to December 2004. The medical records were retrospectively analyzed with emphasis on cytoreduction, cytotoxic regimens, progression-free interval, and survival. Twenty-two patients were identified. All but two presented with advanced stage disease. The median survival for the entire cohort was 38 months. Median survival was 46 months for 18 optimally debulked (<1 cm) patients and 27 months for four suboptimally debulked (>1 cm) patients. Six patients were treated with optimal cytoreduction and adjuvant cisplatin (40 mg/m(2)x 1 day) and ifosfamide (1200 mg/m(2)/day x 4 days) every 28 days. Median progression-free interval in the cisplatin and ifosfamide group was 13 months, and median survival was 51 months. The combination of carboplatin (AUC 5) and taxol (175 mg/m(2)) every 21 days was administered to four patients as first-line chemotherapy following optimal cytoreduction. In the carboplatin and taxol group, median progression-free interval was 6 months and median survival was 38 months. The difference in survival between the cisplatin and ifosfamide group and the carboplatin and taxol group was not statistically significant (P= 0.48). In conclusion, patients with ovarian carcinosarcoma usually present with advanced stage disease. Treatment consists of optimal cytoreduction and chemotherapy. The most effective cytotoxic regimen remains to be determined. First-line cisplatin and ifosfamide or carboplatin and taxol can achieve survival rates observed in epithelial ovarian cancer.

Cell type-specific expression and function of toll-like receptors 2 and 4 in human placenta: implications in fetal infection.

Placental infection is associated with adverse fetal outcomes. Toll-like receptors (TLRs) are critical regulators of the innate immune response based on their ability to recognize and respond to pathogen-associated molecular patterns expressed by microbes. To date, cell-type specific expression and regulation of TLR function in human term placenta remains largely unelucidated. The goal of the current study was to examine the in vivo and in vitro patterns of TLR expression and function in major cell types of term placenta. Immunohistochemical analysis of terminal and stem villi localized TLR-2, which recognizes peptidoglycan (PG) from Gram-positive bacteria, to endothelial cells and macrophages, and to a lesser extent to syncytiotrophoblast (SCTs) and fibroblasts (FIBs). Staining for TLR-4, the receptor for Gram-negative bacterial lipopolysaccharide (LPS), was most prominent in SCTs and endothelial cells. Results from Western blotting, conventional, and quantitative PCR (qRTPCR) analyses using protein and mRNA isolated from cultures of SCTs and myofibroblasts (mFIBs) revealed that SCTs expressed TLR-2 and TLR-4, whereas mFIBs expressed only TLR-4. In addition, qRTPCR showed that LPS treatment increased TLR-2 expression in SCTs, indicating that infection with Gram-negative bacteria may enhance innate immune responses in placenta toward a broad range of microorganisms. In addition, treatment with LPS increased IL-8 levels in both SCTs and mFIBs, whereas PG treatment only stimulated IL-8 levels in SCTs. Our results indicate that there exist cell type-specific patterns of TLR function in placenta which likely regulate innate immune response at the maternal-fetal interface.

Expression of Toll-like receptors in the human decidua.

BACKGROUND: Successful trophoblast invasion and transformation of the maternal spiral arteries requires that the pregnant endometrium (i.e., decidua) act in an immunologically paradoxical fashion, accepting the semi-allogenic placenta, while maintaining host defenses against an array of microbial pathogens. In contrast to the growing evidence that the immune surveillance molecules known as Toll-like receptors (TLRs) are expressed by trophoblasts and fetal membranes, to date, no studies have been conducted on the decidua. METHODS: Decidual tissues and cells were obtained from women undergoing first trimester elective terminations or repeat Cesarean sections and analyzed at both the protein and mRNA level. RESULTS: We now demonstrate for the first time that human decidua differentially express TLRs and their downstream signaling molecules as well as TLR stimulated induction of cytokine production in the first and third trimester of pregnancy. CONCLUSIONS: These findings suggest that the decidua is a critical component of the innate immune response in pregnancy. Moreover, the results have implications for the success or failure of compromised pregnancies in early or late gestation.

Trophoblast-macrophage interactions: a regulatory network for the protection of pregnancy.

PROBLEM: Macrophages are one of the first immune cells observed at the implantation site. Their presence has been explained as the result of an immune response toward paternal antigens. The mechanisms regulating monocyte migration and differentiation at the implantation site are largely unknown. In the present study, we demonstrate that trophoblast cells regulate monocyte migration and differentiation. We propose that trophoblast cells 'educate' monocytes/macrophages to create an adequate environment that promote trophoblast survival. METHOD OF STUDY: CD14(+) monocytes were isolated from peripheral blood using magnetic beads. Co-culture experiments were conducted using a two-chamber system. Monocytes were stimulated with lipopolysaccharide (LPS) and cytokine levels were determined using multiplex cytokine detecting assay. RESULTS: Trophoblast cells increase monocyte migration and induce a significant increase in the secretion and production of the pro-inflammatory cytokines [interleukin-6 (IL-6), IL-8, tumor necrosis factor-alpha] and chemokines (growth-related oncogen-alpha, monocyte chemoattractant protein-1, macrophage inflammatory protein-1beta, RANTES). Furthermore, the response of monocytes to LPS was different in monocytes pre-exposed to trophoblast cells. CONCLUSION: The results of this study suggest that trophoblast cells are able to recruit and successfully educate monocytes to produce and secrete a pro-inflammatory cytokine and chemokine profile supporting its growth and survival. Furthermore we demonstrate that trophoblast cells can modulate monocytes response to bacterial stimuli.

Dermatitis as a component of the fetal inflammatory response syndrome is associated with activation of Toll-like receptors in epidermal keratinocytes.

AIMS: Microbial invasion of the amniotic cavity (MIAC) elicits a fetal inflammatory response such as funisitis and chorionic vasculitis. However, little is known about the changes of fetal skin during MIAC. Toll-like receptors recognize microbial products and initiate an immune response. The aims of this study were to examine histopathological features of fetal skin exposed to MIAC and to assess the changes in Toll-like receptor (TLR)-2 and TLR-4 expression. METHODS AND RESULTS: Skin samples were obtained from fetal autopsies (n = 12). The cases were classified according to the presence (n = 8) or absence (n = 4) of acute chorioamnionitis and analysed by immunohistochemistry using a panel of antibodies. Leucocytic infiltrates into the superficial dermis were observed in cases with chorioamnionitis; the majority of inflammatory cells were neutrophils, lymphocytes and histiocytes. TLR-2 immunoreactivity in the skin was stronger in fetuses with chorioamnionitis than in those without this condition. However, immunoreactivity of TLR-4 in the fetal skin was constitutively expressed, regardless of the presence or absence of chorioamnionitis. CONCLUSIONS: This study demonstrates for the first time that fetal dermatitis can be detected and is part of the fetal inflammatory response syndrome (FIRS). We propose that this 'FIRS-associated fetal dermatitis' is a fetal counterpart of chorioamnionitis.

Toll-like receptors and their role in the trophoblast.

The aim of this review is to discuss the role of Toll-like receptors at the maternal-fetal interface and the capacity of trophoblast cells to initiate innate immune responses towards infection. The maternal-fetal interface represents an immunologically unique site that must promote tolerance to the allogenic fetus, whilst maintaining host defense against a diverse array of possible pathogens. Clinical studies have shown a strong association between certain complications of pregnancy and intrauterine infections. Therefore, innate immune responses against microorganisms at the maternal-fetal interface may have a significant impact on the success of a pregnancy. There is growing evidence that trophoblast cells are able to recognize and respond to pathogens through the expression of Toll-like receptors, an important part of innate immunity. This review will discuss the role of Toll-like receptors at the maternal-fetal interface, the potential for trophoblast cells to function as components of the innate immune system and the impact TLR-mediated trophoblast responses may have on pregnancy outcome.

Metalloproteinase expression by control and telomerase immortalized human endometrial endothelial cells.

Vascular endothelial cells play a critical role in the maintenance of endometrial homeostasis. Indeed many pathological conditions causing abnormal endometrial bleeding including progestin only contraception, hormone replacement therapy, endometrial polyps, myomas, hyperplasia and cancer are associated with aberrant angiogenesis. Critical to the process of angiogenesis is the breakdown of the surrounding tissues by matrix metalloproteases (MMPs). In addition to the cells surrounding the endometrial endothelial cells, the endothelial cells themselves produce their own panel of MMPs. We now characterize the specific MMPs that are expressed by endothelial cells derived from human endometrium. These include MMP-1, MMP-2 and MMP-10 but not MMP-3. In addition, in order to successfully carry out consistent, homogeneous and sufficient numbers of studies we investigated the in vitro expression of the MMPs with both freshly isolated, early passaged endometrial endothelial cells (HEECs) as well as with newly telomerase immortalized HEECs (T-HEECs). The latter were karyotypically normal and expressed classic endothelial cell endpoints such as tubulogenesis on matrigel and expression of the endothelial cell markers CD-31 (PECAM), von Willebrand's factor, and the Tie-2 receptors. The levels of MMP expression as well as that of the metalloprotease inhibitors TIMP-1 and TIMP-2 were similar in parent and immortalized endothelial cells.

Down-regulation of Bcl-2 enhances estrogen apoptotic action in long-term estradiol-depleted ER(+) breast cancer cells.

Postmenopausal women with estrogen receptor positive (ER(+)) breast cancer frequently respond paradoxically to estrogen administration with tumor regression. Using both LTED and E8CASS cells derived from MCF-7 breast cancer cells by long-term estrogen-deprivation, we previously reported that 17beta -estradiol (estradiol) is a powerful, pro-apoptotic hormone which kills the cancer cells through activation of the Fas/FasL death receptor pathway. We postulated that the mitochondrial interactive protein Bcl-2 might play a role in the regulation of estradiol-induced apoptosis in both LTED and E8CASS cells. In this study, we assessed estradiol effects on cell growth, proliferation and apoptosis. Additionally we investigated the effect of estradiol on caspase activation, NF-KB and Bcl-2 expression. The functional role of Bcl-2 in estradiol-induced apoptosis was further studied by knockdown or decrease of Bcl-2 with siRNA. Our results show that estradiol significantly inhibited cell growth primarily through a pro-apoptotic action involving caspase-7 and 9 activations (p < 0.01). Basal Bcl-2 and NF-KB levels were greatly elevated and estradiol decreased NF-KB, but not Bcl-2 expression. Knockdown of Bcl-2 expression with siRNA decreased the levels of this protein by 9 fold (p < 0.01). This reduction markedly sensitized both LTED and E8CASS cells to the pro-apoptotic action of estradiol, leading to a synergistic induction of apoptosis and a concomitant reduction in cell number (p < 0.01). Therefore, down-regulation of Bcl-2 synergistically enhanced estradiol-induced apoptosis in ER(+) postmenopausal breast cancer cells.

Maternal serum of women with pre-eclampsia reduces trophoblast cell viability: evidence for an increased sensitivity to Fas-mediated apoptosis.

OBJECTIVE: Pre-eclampsia or 'toxemia of pregnancy' has been attributed to the presence of a circulating 'toxin' which disappears from peripheral blood after delivery of the placenta. However, the presence, nature and effects of this toxin have eluded characterization. Increased trophoblast apoptosis has been observed in the placenta of women with pre-eclampsia, and it is possible that this biological phenomenon is important for the genesis of the disease and mediated through a soluble factor(s) present in maternal blood. This study was designed to test the hypothesis that serum from women with pre-eclampsia changes trophoblast viability. Moreover, we sought to examine whether this effect could be mediated through changes in sensitivity to Fas/Fas ligand-mediated apoptosis. STUDY DESIGN: H8 trophoblast cells were cultured with serum obtained from normal pregnant women (n = 48) and patients with pre-eclampsia (n = 12). Cell viability was determined by the Cell Titer 96 assay. Fas sensitivity was determined by treating the cells with an agonist anti-Fas antibody or a blocking anti-Fas ligand antibody. RESULTS: Serum from normal pregnant women did not affect trophoblast cell viability. In contrast, serum from pre-eclamptic women reduced trophoblast viability, and this was enhanced by treatment with an anti-Fas antibody. This effect was reversed by the treatment with a blocking anti-Fas ligand antibody. CONCLUSION: Serum from women with pre-eclampsia induces the cytotoxicity of a first-trimester trophoblast cell line (H8). This effect appears to be related to changes in trophoblast sensitivity to Fas-mediated apoptosis. These findings suggest that a factor present in the maternal blood of patients with pre-eclampsia may have a role in the genesis of the syndrome.

Uncoupling proteins-2 and 3 influence obesity and inflammation in transgenic mice.

OBJECTIVE: To test the hypothesis that either uncoupling protein-2 UCP2 or UCP3 or both together influence obesity and inflammation in transgenic mice. DESIGN: We generated 12 lines of transgenic mice for both human UCP2 and 3 using native promoters from a human bacterial artificial chromosome (BAC) clone. The BAC expresses no genes other than UCP2 and 3. Mice used for experiments are N4 or higher of backcross to C57BL/6J (B6). Each experiment used transgenic mice and their nontransgenic littermates. RESULTS: Northern blots confirmed expression on human UCP2 in adipose and spleen, while human UCP3 expression was detectable in gastrocnemius muscle. Western blots demonstrated a four-fold increase of UCP2 protein in spleens of Line 32 transgenic animals. Heterozygous mice of four lines showing expression of human UCP2 in spleen were examined for obesity phenotypes. There were no significant differences between Lines 1 and 32, but female transgenics of both lines had significantly smaller femoral fat depots than the control (littermate) mice (P=0.015 and 0.005, respectively). In addition, total fat of transgenic females was significantly less in Line 1 (P=0.05) and almost significantly different in Line 32 (P=0.06). Male Line 1 mice were leaner (P=0.04) while male Line 32 mice were almost significantly leaner (P=0.06). Heterozygous mice of Lines 35 and 44 showed no significant differences from the nontransgenic littermate controls. Effects of the UCP2/UCP3 transgene on obesity in Line 32 mice were confirmed by crossing transgenic mice with the B6.Cg-Ay agouti obese mice. B6.Cg-Ay carrying the UCP2/UCP3 transgene from Line 32 were significantly leaner than nontransgenic B6.Cg-Ay mice. Line 32 UCP2/UCP3 transgenics showed increased hypothalamic Neuropeptide (NPY) levels and food intake, with reduced spontaneous physical activity. Transgenic baseline interleukin4 (IL-4) and interleukin6 (IL-6) levels were low with lower or later increases after endotoxin injection compared to wild-type littermates. Endotoxin-induced fever was also diminished in transgenic male animals. Low-density lipoprotein (LDL) cholesterol levels were significantly higher in both Line 1 and 32 transgenics (P=0.05 and 0.001, respectively) after they had been placed on a moderate fat-defined diet containing 32% of calories from fat for 5 weeks. CONCLUSION: Moderate overexpression of UCP2 and 3 reduced fat mass and increased LDL cholesterol in two independent lines of transgenic mice. Thus, the reduced fat mass cannot be due to insertional mutagenesis since virtually identical fat pad weights and masses were observed with the two independent lines. Line 32 mice also have altered inflammation and mitochondrial function. We conclude that UCP2 and/or 3 have small but significant effects on obesity in mice, and that their mechanism of action may include alterations of metabolic rate.

Effect of long-term estrogen deprivation on apoptotic responses of breast cancer cells to 17beta-estradiol.

BACKGROUND: High doses of estrogen can promote tumor regression in postmenopausal women with hormone-dependent breast cancer, but the mechanism is unknown. We investigated the molecular basis of this process by using LTED cells, which were derived by growing MCF-7 breast cancer cells under long-term (6-24 months) estrogen-deprived conditions. METHODS: We treated LTED and MCF-7 cells with various concentrations of 17beta-estradiol (estradiol) and assayed their growth by counting the cells and measured apoptosis by annexin V staining and DNA fragmentation. Using western blot analysis, we also examined the expression of the apoptosis-inducing system of the Fas death receptor protein and its ligand, FasL, in these cells. To assess the involvement of Fas and FasL in the induction of apoptosis in LTED cells, we used activating anti-Fas antibodies and the universal caspase inhibitor Z-VAD. Finally, we examined the expression of Fas protein in E8CASS and BSK3 cells, two other cell lines derived by depriving MCF-7 cells of estrogen long term, and the responses of these cells to high-dose estradiol. All statistical tests were two-sided. RESULTS: High concentrations of estradiol (>or=0.1 nM) resulted in a statistically significant, 60% reduction in the growth of LTED cells (P< .001) and in a sevenfold increase in apoptosis (P< .001) as compared with levels in vehicle-treated cells. Both LTED and MCF-7 cells expressed FasL, but only LTED cells expressed Fas. Treatment of LTED cells with 0.1 nM estradiol increased the expression of FasL. Activating anti-Fas antibodies increased apoptosis of LTED cells, which was further stimulated by estradiol. Z-VAD blocked estradiol-induced apoptosis. E8CASS cells, which express Fas protein, but not BSK3 cells, which do not, also responded to 0.1 nM estradiol by increasing apoptosis. CONCLUSION: Tumor regression induced by high-dose estrogen therapy in postmenopausal woman may result from estrogen activation of Fas-mediated apoptosis.

Plasmid DNA vaccines. Immunology, tolerance, and autoimmunity.

DNA vaccination is a novel approach for inducing an immune response. Purified plasmid DNA containing an antigen's coding sequences and the necessary regulatory elements to express them is introduced into the tissue via intramuscular injection or particle bombardment. Once the DNA reaches the tissue, the antigen is expressed in enough quantity to induce a potent and specific immune response and to confer protection against further infections. The effectiveness of DNA vaccines against viruses, parasites, and cancer cells has been demonstrated in numerous animal models. This new approach comes as an aid for the prevention of infectious diseases for which the conventional vaccines have failed. DNA vaccine research is providing new insights into some of the basic immunological mechanisms of vaccination such as antigen presentation, the role of effector cells, and immunoregulatory factors. In addition, DNA vaccines may enable us to manipulate the immune system in situations where the response to agents is inappropriate or ineffective. The study of the potential deleterious effects of DNA vaccines is furthering our knowledge regarding the relationship between bacterial DNA and the immune system, as well as its potential application for the study of neonatal tolerance and autoimmunity.