Regulation of vascular endothelial growth factor (VEGF) gene transcription by estrogen receptors α and β

Vascular endothelial growth factor (VEGF) mediates angiogenic activity in a variety of estrogen target tissues. To determine whether estrogen has a direct transcriptional effect on VEGF gene expression, we developed a model system by transiently transfecting human VEGF promoter-luciferase reporter constructs into primary human endometrial cells and into Ishikawa cells, derived from a well-differentiated human endometrial adenocarcinoma. In primary endometrial epithelial cells, treatment with 17β-estradiol (E2) resulted in a 3.8-fold increase in luciferase activity, whereas a 3.2-fold induction was demonstrated for stromal cells. Our Ishikawa cells had less than 100 functional estrogen receptors (ER)/cell and were therefore cotransfected with expression vectors encoding either the α- or the β-form of the human ER. In cells cotransfected with ERα, E2 induced 3.2-fold induction in VEGF-promoter luciferase activity. A 2.3-fold increase was observed in cells cotransfected with ERβ. Through specific deletions, the E2 response was restricted to a single 385-bp PvuII-SstI fragment in the 5′ flanking DNA. Cotransfection of this upstream region with a DNA binding domain ER mutant, or site-directed mutagenesis of a variant ERE within this fragment, resulted in the loss of the E2 response. Electromobility shift assays demonstrated that this same ERE sequence specifically binds estradiol-ER complexes. These studies demonstrate that E2-regulated VEGF gene transcription requires a variant ERE located 1.5 kb upstream from the transcriptional start site. Site-directed mutagenesis of this ERE abrogated E2-induced VEGF gene expression.

Induction of tolerance to immunogenic tumor antigens associated with lymphomagenesis in HOX11 transgenic mice

Transgenic mice expressing human HOX11 in B lymphocytes die prematurely from lymphomas that initiate in the spleen and frequently disseminate to distant sites. Preneoplastic hematopoiesis in these mice is unperturbed. We now report that expression of the HOX11 transgene does not affect the ability of dendritic cells (DCs) to process and present foreign peptides and activate antigen-specific T cell responses. We also show that nontransgenic DCs presenting peptides derived from the human HOX11 protein are highly efficient stimulators of autologous T cells, whereas transgenic T cells are nonresponsive to peptides derived from the HOX11 transgene and the murine Meis1 protein. HOX11 transgenic mice thus show normal development of tolerance to immunogenic antigens expressed throughout B cell maturation. DCs pulsed with cell lysates prepared from lymphomas, obtained from HOX11 transgenic mice with terminal lymphoma, activate T cells from nontransgenic and premalignant transgenic mice, whereas T cells isolated from lymphomatous transgenic mice are nonresponsive to autologous tumor cell antigens. These data indicate that HOX11 lymphoma cells express tumor-rejection antigens that are recognized as foreign in healthy transgenic mice and that lymphomagenesis is associated with the induction of anergy to tumor antigen-specific T cells. These findings are highly relevant for the development of immunotherapeutic protocols for the treatment of lymphoma.

Structural requirements for antigen presentation by mouse CD1

The structural basis for the T cell response to glycolipid antigens (Ags) remains poorly understood. T lymphocytes autoreactive for mouse CD1 (mCD1.1) or reactive for the glycosphingolipid αgalactosylceramide (α-GalCer) presented by mCD1.1 have been described previously. In this paper it is shown that mutations at the top of the α helices and in the bottom of the Ag-binding groove can disrupt both mCD1.1 autoreactivity and α-GalCer recognition. The locations of the positions that affect T cell responses indicate that recognition of mCD1.1 is not likely to be unconventional or superantigen-like. Furthermore, the effects of the bottom of the pocket mutation suggest that the autoreactive response could require an autologous ligand, and they indicate that α-GalCer binds to the groove of mCD1.1, most likely with the shorter 18-carbon hydrophobic chain in the A′ pocket. Natural killer T cell hybridomas with identical T cell antigen receptor (TCR) α chains and different β chains respond differently to α-GalCer presented by mCD1.1 mutants. This finding indicates a role for TCR β in defining natural killer T cell specificity, despite the more restricted diversity of the α chains in these cells. Overall, the data are consistent with a mode of lipoglycan recognition similar to that proposed for glycopeptides, in which the TCR α and β chains survey a surface composed of both mCD1.1 and the carbohydrate portion of α-GalCer.

A viral gene that activates lytic cycle expression of Kaposi’s sarcoma-associated herpesvirus

Herpesviruses exist in two states, latency and a lytic productive cycle. Here we identify an immediate-early gene encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus eight (HHV8) that activates lytic cycle gene expression from the latent viral genome. The gene is a homologue of Rta, a transcriptional activator encoded by Epstein–Barr virus (EBV). KSHV/Rta activated KSHV early lytic genes, including virus-encoded interleukin 6 and polyadenylated nuclear RNA, and a late gene, small viral capsid antigen. In cells dually infected with Epstein–Barr virus and KSHV, each Rta activated only autologous lytic cycle genes. Expression of viral cytokines under control of the KSHV/Rta gene is likely to contribute to the pathogenesis of KSHV-associated diseases.

The role of MHC class I glycoproteins in the regulation of induction of cell death in immunocytes by malignant melanoma cells

A deranged expression of MHC class I glycoproteins, characteristic of a variety of malignancies, contributes to the ability of cancer to avoid destruction by T cell-mediated immunity. An abrogation of the metastatic capacity of B16 melanoma cells has been achieved by transfecting an MHC class I-encoding vector into class I-deficient B16 melanoma clones [Gorelik, E., Kim, M., Duty, L. & Galili, U. (1993) Clin. Exp. Metastasis 11, 439–452]. We report here that the deranged expression of class I molecules by B16 melanoma cells is more than a mere acquisition of the capacity to escape immune recognition. Namely, cells of the B16 melanoma prompted splenic lymphocytes to commit death after coculture. However, a class I-expressing and nonmetastatic CL8-2 clone was found to be less potent as an inducer of apoptosis than class I-deficient and metastatic BL9 and BL12 clones. Both Thy1.2+ and Thy1.2− splenocytes underwent cell death when exposed to the class I-deficient BL9 clone. A proportion of CD4+ and CD8+ cells among splenocytes exposed to the BL9 clone was lower than that observed in a coculture with cells of the CL8-2 clone. Consistently, none of the melanoma clones studied produced a ligand to the FAS receptor (FAS-L). Thus, our results provide evidence that (i) the production of FAS-L may not be the sole mechanism by which malignant cells induce apoptosis in immunocytes, and (ii) absence of MHC class I glycoproteins plays an important role in preventing the elimination of potential effector immunocytes by tumor cells.

Role of the arginine-nitric oxide pathway in the regulation of vascular smooth muscle cell proliferation

The objective of this study was to elucidate the mechanisms by which nitric oxide (NO) inhibits rat aortic smooth muscle cell (RASMC) proliferation. Two products of the arginine-NO pathway interfere with cell growth by distinct mechanisms. NG-hydroxyarginine and NO appear to interfere with cell proliferation by inhibiting arginase and ornithine decarboxylase (ODC), respectively. S-nitroso-N-acetylpenicillamine, (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate, and a nitroaspirin derivative (NCX 4016), each of which is a NO donor agent, inhibited RASMC growth at concentrations of 1–3 μM by cGMP-independent mechanisms. The cytostatic action of the NO donor agents as well as α-difluoromethylornithine (DFMO), a known ODC inhibitor, was prevented by addition of putrescine but not ornithine. These observations suggested that NO, like DFMO, may directly inhibit ODC. Experiments with purified, recombinant mammalian ODC revealed that NO inhibits ODC possibly by S-nitrosylation of the active site cysteine in ODC. DFMO, as well as the NO donor agents, interfered with cellular polyamine (putrescine, spermidine, spermine) production. Conversely, increasing the expression and catalytic activity of arginase I in RASMC either by transfection of cells with the arginase I gene or by induction of arginase I mRNA with IL-4 resulted in increased urea and polyamine production as well as cell proliferation. Finally, coculture of rat aortic endothelial cells, which had been pretreated with lipopolysaccharide plus a cytokine mixture to induce NO synthase and promote NO production, caused NO-dependent inhibition of target RASMC proliferation. This study confirms the inhibitory role of the arginine-NO pathway in vascular smooth muscle proliferation and indicates that one mechanism of action of NO is cGMP-independent and attributed to its capacity to inhibit ODC.

CD3-mediated activation of tumor-reactive lymphocytes from patients with advanced cancer

Lymphocytes from blood or tumors of patients with advanced cancer did not proliferate and produced very low levels of tumor necrosis factor and IFN-γ when cultured with autologous tumor cells. Proliferation and lymphokine production dramatically increased in the presence of beads conjugated with mAbs to CD3 plus mAbs to CD28 and/or CD40, and the lymphocytes destroyed the tumor cells. Expression density of CD3 concomitantly increased from low to normal levels. Furthermore, beads providing a CD3 signal (in combination with CD28 or CD28 plus CD40) gave partial protection against the inhibitory effect of transforming growth factor type β1 on lymphocyte proliferation and production of tumor necrosis factor and IFN-γ. MHC class I-restricted cytolytic T cells lysing autologous tumor cells in a 4-h Cr51 release assay were generated when peripheral blood leukocytes were activated in the presence of autologous tumor cells and anti-CD3/CD28 or anti-CD3/CD28/CD40 beads. Experiments performed in a model system using anti-V-β1 or anti-V-β2 mAbs to activate subsets of T cells expressing restricted T cell receptor showed that lymphocytes previously activated by anti-V-β can respond to CD3 stimulation with vigorous proliferation and lymphokine production while retaining their specificity, also in the presence of transforming growth factor type β1. Our results suggest that T lymphocytes from cancer patients can proliferate and form Th1 type lymphokines in the presence of autologous tumor cell when properly activated, and that antigen released from killed tumor cells and presented by antigen-presenting cells in the cultures facilitates the selective expansion of tumor-directed, CD8+ cytolytic T cells.

The type I BMP receptor BmprIB is essential for female reproductive function

Maintenance of female reproductive competence depends on the actions of several hormones and signaling factors. Recent reports suggest roles for bone morphogenetic proteins (BMPs) in early stages of folliculogenesis. A role for the type I BMP receptor BmprIB as a regulator of ovulation rates in sheep has been described recently, but little is known about the roles of BMP signaling pathways in other aspects of reproductive function. We report here that BMPRIB is essential for multiple aspects of female fertility. Mice deficient in BmprIB exhibit irregular estrous cycles and an impaired pseudopregnancy response. BmprIB mutants produce oocytes that can be fertilized in vitro, but defects in cumulus expansion prevent fertilization in vivo. This defect is associated with decreased levels of aromatase production in granulosa cells. Unexpectedly, levels of mRNA for cyclooxygenase 2, an enzyme required for cumulus expansion, are increased. BmprIB mutants also exhibit a failure in endometrial gland formation. The expression of BmprIB in uterine linings suggests that these defects are a direct consequence of loss of BMP signaling in this tissue. In summary, these studies demonstrate the importance of BMP signaling pathways for estrus cyclicity, estradiol biosynthesis, and cumulus cell expansion in vivo and reveal sites of action for BMP signaling pathways in reproductive tissues.

Dual control of LIF expression and LIF receptor function regulate Stat3 activation at the onset of uterine receptivity and embryo implantation

Leukemia inhibitory factor (LIF) expression in the uterus is essential for embryo implantation in mice. Here we describe the spatial and temporal regulation of LIF signaling in vivo by using tissues isolated from uteri on different days over the implantation period. During this time, LIF receptors are expressed predominantly in the luminal epithelium (LE) of the uterus. Isolated epithelium responds to LIF by phosphorylation and nuclear translocation of signal transducer and activator of transcription (Stat) 3, but not by an increase in mitogen-activated protein kinase levels. The related cytokines Il-6, ciliary neurotrophic factor, as well as epidermal growth factor, do not activate Stat3, although epidermal growth factor stimulates mitogen-activated protein kinase. In vivo Stat3 activation is induced by LIF alone, resulting in the localization of Stat3 specifically to the nuclei of the LE coinciding with the onset of uterine receptivity. The responsiveness of the LE to LIF is regulated temporally, with Stat activation being restricted to day 4 of pregnancy despite the presence of constant levels of LIF receptor throughout the preimplantation period. Uterine receptivity is therefore under dual control and is regulated by both the onset of LIF expression in the endometrial glands and the release from inhibition of receptor function in the LE.

Development of HIV vectors for anti-HIV gene therapy.

Current gene therapy protocols for HIV infection use transfection or murine retrovirus mediated transfer of antiviral genes into CD4+ T cells or CD34+ progenitor cells ex vivo, followed by infusion of the gene altered cells into autologous or syngeneic/allogeneic recipients. While these studies are essential for safety and feasibility testing, several limitations remain: long-term reconstitution of the immune system is not effected for lack of access to the macrophage reservoir or the pluripotent stem cell population, which is usually quiescent, and ex vivo manipulation of the target cells will be too expensive and impractical for global application. In these regards, the lentivirus-specific biologic properties of the HIVs, which underlie their pathogenetic mechanisms, are also advantageous as vectors for gene therapy. The ability of HIV to specifically target CD4+ cells, as well as non-cycling cells, makes it a promising candidate for in vivo gene transfer vector on one hand, and for transduction of non-cycling stem cells on the other. Here we report the use of replication-defective vectors and stable vector packaging cell lines derived from both HIV-1 and HIV-2. Both HIV envelopes and vesicular stomatitis virus glycoprotein G were effective in mediating high-titer gene transfer, and an HIV-2 vector could be cross-packaged by HIV-1. Both HIV-1 and HIV-2 vectors were able to transduce primary human macrophages, a property not shared by murine retroviruses. Vesicular stomatitis virus glycoprotein G-pseudotyped HIV vectors have the potential to mediate gene transfer into non-cycling hematopoietic stem cells. If so, HIV or other lentivirus-based vectors will have applications beyond HIV infection.

Improved retroviral gene transfer into murine and Rhesus peripheral blood or bone marrow repopulating cells primed in vivo with stem cell factor and granulocyte colony-stimulating factor.

In previous studies we showed that 5 days of treatment with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) mobilized murine repopulating cells to the peripheral blood (PB) and that these cells could be efficiently transduced with retroviral vectors. We also found that, 7-14 days after cytokine treatment, the repopulating ability of murine bone marrow (BM) increased 10-fold. In this study we examined the efficiency of gene transfer into cytokine-primed murine BM cells and extended our observations to a nonhuman primate autologous transplantation model. G-CSF/SCF-primed murine BM cells collected 7-14 days after cytokine treatment were equivalent to post-5-fluorouracil BM or G-CSF/SCF-mobilized PB cells as targets for retroviral gene transfer. In nonhuman primates, CD34-enriched PB cells collected after 5 days of G-CSF/SCF treatment and CD34-enriched BM cells collected 14 days later were superior targets for retroviral gene transfer. When a clinically approved supernatant infection protocol with low-titer vector preparations was used, monkeys had up to 5% of circulating cells containing the vector for up to a year after transplantation. This relatively high level of gene transfer was confirmed by Southern blot analysis. Engraftment after transplantation using primed BM cells was more rapid than that using steady-state bone marrow, and the fraction of BM cells saving the most primitive CD34+/CD38- or CD34+/CD38dim phenotype increased 3-fold. We conclude that cytokine priming with G-CSF/SCF may allow collection of increased numbers of primitive cells from both the PB and BM that have improved susceptibility to retroviral transduction, with many potential applications in hematopoietic stem cell-directed gene therapy.

In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function.

To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.

Menstrual breakdown of human endometrium can be mimicked in vitro and is selectively and reversibly blocked by inhibitors of matrix metalloproteinases.

The mechanisms underlying the menstrual lysis leading to shedding of the human endometrium and its accompanying bleeding are still largely unknown. In particular, whether breakdown of the endometrial fibrillar extra-cellular matrix that precedes bleeding depends on aspartic-, cysteine-, serine-, or metalloproteinases remains unclear. In the present study, menstrual regression of the human endometrium was mimicked in organ culture. Whereas sex steroids could preserve tissue integrity only in nonperimenstrual explants, matrix breakdown upon sex steroid deprivation was completely and reversibly inhibited at all stages of the menstrual cycle by specific inhibitors of matrix metalloproteinases, but not by inhibitors of the other classes of proteinases. Matrix metalloproteinases are thus identified as the key class of proteinases involved in the initiation of menstruation.

The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation.

Human gene MAGE-1 encodes tumor-specific antigens that are recognized on melanoma cells by autologous cytolytic T lymphocytes. This gene is expressed in a significant proportion of tumors of various histological types, but not in normal tissues except male germ-line cells. We reported previously that reporter genes driven by the MAGE-1 promoter are active not only in the tumor cell lines that express MAGE-1 but also in those that do not. This suggests that the critical factor causing the activation of MAGE-1 in certain tumors is not the presence of the appropriate transcription factors. The two major MAGE-1 promoter elements have an Ets binding site, which contains a CpG dinucleotide. We report here that these CpG are demethylated in the tumor cell lines that express MAGE-1, and are methylated in those that do not express the gene. Methylation of these CpG inhibits the binding of transcription factors, as seen by mobility shift assay. Treatment with the demethylating agent 5-aza-2'-deoxycytidine activated gene MAGE-1 not only in tumor cell lines but also in primary fibroblasts. Finally, the overall level of CpG methylation was evaluated in 20 different tumor cell lines. It was inversely correlated with the expression of MAGE-1. We conclude that the activation of MAGE-1 in cancer cells is due to the demethylation of the promoter. This appears to be a consequence of a genome-wide demethylation process that occurs in many cancers and is correlated with tumor progression.

A melanoma-specific VH antibody cloned from a fusion phage library of a vaccinated melanoma patient.

The human antimelanoma antibody V86 was cloned from a single-chain Fv molecule (scFv) fusion phage library displaying the heavy chain variable domain (VH) and light chain variable domain (VL.) repertoire of a melanoma patient immunized with genetically-modified autologous tumor cells. Previous ELISA tests for binding of the V86 fusion phage to a panel of human metastatic melanoma and carcinoma cell lines and primary cultures of normal melanocytes, endothelial, and fibroblast cells showed that measurable binding occurred only to the melanoma cells. In this communication, the strict specificity of V86 for melanoma cells was confirmed by immunohistochemical staining tests with cultured cells and frozen tissue sections. The V86 fusion phage stained melanoma cell lines but did not stain carcinoma cell lines or cultured normal cells; V86 also stained specifically the melanoma cells in sections of metastatic tissue but did not stain any of the cells in sections from normal skin, lung, and kidney or from metastatic colon and ovarian carcinomas and a benign nevus. An unexpected finding is that V86 contains a complete VH domain but only a short segment of a VL, domain, which terminates before the CDR1 region. This VL deletion resulted from the occurrence in the VL cDNA of a restriction site, which was cleaved during construction of the scFv library. Thus V86 is essentially a VH antibody. The effect of adding a VI. domain to V86 was examined by constructing scFv fusion phage libraries in which V86 was coupled to Vlambda or Vkappa domains from the original scFv library of the melanoma patient and then panning the libraries against melanoma cells to enrich for the highest affinity antibody clones. None of the V86-Vlambda clones showed significant binding to melanoma cells in ELISA tests; although binding occurred with most of the V86-Vkappa clones, it was generally weaker than the binding of V86. These results indicate that most of the VL domains in the original scFv library reduce or eliminate the affinity of V86 for melanoma cells. Accordingly, VH libraries could provide access to anti-tumor antibodies that might not be detected in scFv or Fab libraries because of the incompatibility of most randomly paired VH and VL, domains.