Mullerian Inhibiting Substance (MIS) has been shown to inhibit ovarian cancer cells both in-vitro and in-vivo. Furthermore, recent evidence suggests that MIS may effectively target a putative ovarian cancer progenitor cell population enriched by a panel of CD44+, CD24+, Ep-CAM+, and E-cadherin-cell surface markers. In order to accommodate clinical testing of MIS in ovarian cancer patients, the production of recombinant human MIS must be optimized to increase yield and purity. Here we show that, compared to wild type, the substitution of the MIS leader sequence to that of human serum albumin, combined with a modification of the endogenous cleavage site from RAQR/S to a furin/kex2 RARR/S consensus site results in high expression, increased C-terminus cleavage and a reduction in unwanted cryptic internal cleavage products when produced in CHO cells. Purified MIS containing these alterations retains its capacity to induce regression of the Mullerian duct in fetal rat embryonic urogenital ridge assays.
Verteporfin (VP) was first used in Photodynamic therapy, where a non-thermal laser light (689 nm) in the presence of oxygen activates the drug to produce highly reactive oxygen radicals, resulting in local cell and tissue damage. However, it has also been shown that Verteporfin can have non-photoactivated effects such as interference with the YAP-TEAD complex of the HIPPO pathway, resulting in growth inhibition of several neoplasias. More recently, it was proposed that, another non-light mediated effect of VP is the formation of cross-linked oligomers and high molecular weight protein complexes (HMWC) that are hypothesized to interfere with autophagy and cell growth. Here, in a series of experiments, using human uveal melanoma cells (MEL 270), human embryonic kidney cells (HEK) and breast cancer cells (MCF7) we showed that Verteporfin-induced HMWC require the presence of light. Furthermore, we showed that the mechanism of this cross-linking, which involves both singlet oxygen and radical generation, can occur very efficiently even after lysis of the cells, if the lysate is not protected from ambient light. This work offers a better understanding regarding VP’s mechanisms of action and suggests caution when one studies the non-light mediated actions of this drug.
5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), an analog of AMP, is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy change. We studied the effects of AICAR on the growth of retinoblastoma cell lines (Y79, WERI, and RB143). AICAR inhibited Rb cell growth, induced apoptosis and S-phase cell cycle arrest, and led to activation of AMPK. These effects were abolished by treatment with dypiridamole, an inhibitor that blocks entrance of AICAR into cells. Treatment with the adenosine kinase inhibitor 5-iodotubericidin to inhibit the conversion of AICAR to ZMP (the direct activator of AMPK) reversed most of the growth-inhibiting effects of AICAR, indicating that some of the antiproliferative effects of AICAR are mediated through AMPK activation. In addition, AICAR treatment was associated with inhibition of the mammalian target of rapamycin pathway, decreased phosphorylation of ribosomal protein-S6 and 4E-BP1, down-regulation of cyclins A and E, and decreased expression of p21. Our results indicate that AICAR-induced activation of AMPK inhibits retinoblastoma cell growth. This is one of the first descriptions of a nonchemotherapeutic drug with low toxicity that may be effective in treating Rb patients.
BackgroundType IV collagen is the main component of the basement membrane that gives strength to the blood–gas barrier (BGB). In mammals, the formation of a mature BGB occurs primarily after birth during alveologenesis and requires the formation of septa from the walls of the saccule. In contrast, in avians, the formation of the BGB occurs rapidly and prior to hatching. Mutation in basement membrane components results in an abnormal alveolar phenotype; however, the specific role of type IV collagen in regulating alveologenesis remains unknown.ResultsWe have performed a microarray expression analysis in late chick lung development and found that COL4A1 and COL4A2 were among the most significantly upregulated genes during the formation of the avian BGB. Using mouse models, we discovered that mutations in murine Col4a1 and Col4a2 genes affected the balance between lung epithelial progenitors and differentiated cells. Mutations in Col4a1 derived from the vascular component were sufficient to cause defects in vascular development and the BGB. We also show that Col4a1 and Col4a2 mutants displayed disrupted myofibroblast proliferation, differentiation and migration. Lastly, we revealed that addition of type IV collagen protein induced myofibroblast proliferation and migration in monolayer culture and increased the formation of mesenchymal–epithelial septal-like structures in co-culture.ConclusionsOur study showed that type IV collagen and, therefore the basement membrane, play fundamental roles in coordinating alveolar morphogenesis. In addition to its role in the formation of epithelium and vasculature, type IV collagen appears to be key for alveolar myofibroblast development by inducing their proliferation, differentiation and migration throughout the developing septum.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0281-2) contains supplementary material, which is available to authorized users.
IntroductionHairy cell leukemia (HCL) or leukemic reticuloendotheliosis represents approximately 2% of adult leukemias and is characterized by pancytopenia, hepatomegaly, splenomegaly, leukocytosis, and neoplastic mononuclear cells in the peripheral blood, bone marrow, liver, and spleen. 1 The name of the disease is derived from the presence of broad-based undulating ruffles on the surface of the leukemic cells that appear under the phase contrast microscope as cytoplasmic projections or "hairs." These cells can be derived both from B and T lymphocytes as demonstrated by their expression of B-or T-cell-specific antigens and are characterized biochemically by their abnormal expression of the integrin heterodimer CD11c/ CD18. [2][3][4] Under normal circumstances the gene encoding the CD18 component of this marker is transcribed both in lymphocytes and myeloid cells, whereas the gene encoding the CD11c component is transcribed primarily in cells of the myeloid lineage. 5 The CD11c/ CD18 heterodimer is, therefore, normally largely restricted in its expression to the surface of myeloid cells dictated by the myeloidspecific transcription of the CD11c gene. In hairy cell leukemia the CD11c/CD18 heterodimer is present on not only myeloid cells but also on the neoplastic lymphocytes. Diagnostic of this disease is, therefore, the abnormal regulation of CD11c gene transcription. Consequently, elucidation of the molecular causes of this abnormal regulation are likely to result in insights into the molecular basis of hairy cell leukemia. Using this rationale we isolated the human CD11c gene and identified the cis-acting elements that control its transcription. The most important of these elements interacts with activator protein-1 (AP-1) encoded by the jun and fos families of proto-oncogenes. In hairy cells an AP-1 complex containing JunD exhibits a constitutive pattern of expression, whereas in other cell types it is functionally expressed only upon induction with phorbol ester. The use of dominant-negative mutants in transfection assays demonstrated that both AP-1 and its upstream activator Ras are necessary for CD11c expression in hairy cells. In addition, exogenous expression of a dominant-positive mutant of Ras was able to activate the CD11c promoter in nonhairy cells to a level equivalent to that seen in hairy cells. Taken together, these results indicate that activation of the proto-oncogenes junD and ras underlie the abnormal expression of the CD11c gene characteristic of HCL. Materials and methods Cell cultureThe cell lines HeLa, IM-9, Mo, and U937 were obtained from the American Type Culture Collection (Manassas, VA) and grown according to their specifications. The promegakaryocytic cell line MEG-01 6 was kindly provided by Dr W. S. May (John Hopkins Oncology Center, Baltimore, MD) through permission of Dr H. Saito (Nagoya University School of Medicine, Nagoya, Japan). MEG-01 were cultured in RPMI 1640 medium supplemented with glutamine, 20% fetal calf serum (FCS), aqueous penicillin G (100 U/mL), and streptomycin ...
Inner ear gene therapy using adeno-associated viral vectors (AAV) promises to alleviate hearing and balance disorders. We previously established the benefits of Anc80L65 in targeting inner and outer hair cells in newborn mice. To accelerate translation to humans, we now report the feasibility and efficiency of the surgical approach and vector delivery in a nonhuman primate model. Five rhesus macaques were injected with AAV1 or Anc80L65 expressing eGFP using a transmastoid posterior tympanotomy approach to access the round window membrane after making a small fenestra in the oval window. The procedure was well tolerated. All but one animal showed cochlear eGFP expression 7–14 days following injection. Anc80L65 in 2 animals transduced up to 90% of apical inner hair cells; AAV1 was markedly less efficient at equal dose. Transduction for both vectors declined from apex to base. These data motivate future translational studies to evaluate gene therapy for human hearing disorders.
Objective Given the fact that Mullerian Inhibiting Substance (MIS) causes complex remodeling of the urogenital ridge and regression of the Mullerian ducts during male embryonic development, we examined whether MIS could affect similar cell properties such as migration and invasion that could contribute ultimately to micro-metastasis of cancers arising from Mullerian tissues. MIS receptor expressing cell lines found to be invasive and migratory in vivo are examined in an in vivo assay that is cost effective. Methods We designed in vitro and in vivo experiments to determine if MIS inhibited the movement of cancer lines IGROV-1, HEp3, MDA-MB-231, and HT1080 in cell culture invasion/migration chamber assays and in chick embryo metastasis assays. Results . MIS, at concentrations below those that inhibit cell proliferation, blocked in vitro invasion and in vivo migration of epithelial cancer cells that express the MIS receptor. Conclusions While our laboratory has previously established MIS as an inhibitor of cancer cell proliferation using in vitro assays and in vivo xenografts, we now show that MIS can also inhibit in vivo tumor migration.
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