Human Amnion Cell Line Research Articles

Efficacy of Alternaria alternate NMK1 Secondary Metabolites against some Seed-borne Fungi

AN AGRICULTURAL soil was used to isolate Alternaria alternata. It was molecularly identified and was given an accession number MN645469 at the National Center for Biotechnology Information (NCBI) and a strain identifier to be Alternaria alternata strain NMK1. Four fractions were collected from the acetic acid extract of its filtrate. Fraction no. 2 showed highest antioxidant activity (71 and 64%, at 200 and 150μg/mL) followed by the crude extract (60 % at 200μg/mL). Safety of using the extracted fractions was tested against the normal human amnion (WISH) cell line. Lowest cytotoxicity values were recorded for fraction no. 2 (19%) followed by the crude extract (28%) at 50μg/mL. Aspergillus flavus, Aspergillus niger and Penicillium griseofulvum exhibited highest relative density percentages (28.4, 30.8 and 24.3%) on their respective isolation seeds; wheat, broad bean and kidney bean. Interestingly, fraction no. 2 caused 100% inhibition of these isolated fungal species at 100μg/mL. The minimum fungicidal concentration (MFC) was estimated to be 50μg/mL against A. flavus and 40μg/mL for A. niger and P. griseofulvum. Generally, subjecting each of the tested isolates to fraction no. 2 led to damage in their DNA as shown by the comet assay. Gas chromatography- mass spectroscopy (GC-MS) profiling of fraction no. 2 showed 12 major compounds with diverse possible biological activities being antiinflammatory, insecticide, anticancer, antineoplastic antifungal, and antimicrobial.

Poly(3-hydroxybutyrate)/polyethylene glycol-NiO nanocomposite for NOR delivery: Antibacterial activity and cytotoxic effect against cancer cell lines

Norfloxacin (NOR), a well-known antibacterial agent is also known to have potential antitumor activity. In this study, NOR was immobilized into poly(3-hydroxybutyrate/polyethylene glycol‑nickel oxide (PHB/PEG-NiO) nanocomposites using different NiO contents. The NiO nanoparticles were prepared by sol-gel method and the nanocomposites were prepared by solution cast films. Physicochemical features of nanocomposites were monitored by XRD, FTIR, SEM, TEM and TGA. PHB/PEG-5%NiO nanocomposites showed high NOR loading efficiency (60%) and a long-sustained release in comparison with other carriers. The studies on the adsorption of NOR onto PHB/PEG-NiO nanocomposite revealed that the adsorption process obeyed second order kinetic and Temkin isotherm was applicable to describe the adsorption process. The mechanism of release was studied by using different kinetic equations. The loaded nanocomposites (NOR@PHB/PEG-NiO) showed effective antimicrobial activity towards gram-positive (Staphylococcus aureus), and gram-negative bacteria (E.coli and Klebsiella pneumonia). The in vitro cytotoxicity was conducted on four human cancer cell lines viz., liver Hepatocellular carcinoma, colon cancer cell, prostate cancer cell, and breast adenocarcinoma from human and one normal human amnion cell line using MTT assay. Cytotoxicity results demonstrated that NOR@PHB/PEG-NiO significantly reduced cell viability than free NOR. NOR@ PHB/PEG–NiO has about 2.5-fold more cytotoxicity as compared with free drug with a lack of cytotoxicity against normal cells.

Induction of suppressor of cytokine signaling-3 by herpes simplex virus type 1 contributes to inhibition of the interferon signaling pathway.

We showed previously that herpes simplex virus type 1 (HSV-1) suppresses the interferon (IFN) signaling pathway during the early infection stage in the human amnion cell line FL. HSV-1 inhibits the IFN-induced phosphorylation of Janus kinases (JAK) in infected FL cells. In the present study, we showed that the suppressor of cytokine signaling-3 (SOCS3), a host negative regulator of the JAK/STAT pathway, is rapidly induced in FL cells after HSV-1 infection. Maximal levels of SOCS3 protein were detected at around 1 to 2 h after infection. This is consistent with the occurrence of HSV-1-mediated inhibition of IFN-induced JAK phosphorylation. The HSV-1 wild-type strain VR3 induced SOCS3 more efficiently than did mutants that are defective in UL41 or UL13 and that are hyperresponsive to IFN. Induction of the IRF-7 protein and transcriptional activation of IFN-alpha4, which occur in a JAK/STAT pathway-dependent manner, were poorly induced by VR3 but efficiently induced by the mutant viruses. In contrast, phosphorylation of IRF-3 and transcriptional activation of IFN-beta, which are JAK/STAT pathway-independent process, were equally well induced by the wild-type strain and the mutants. In conclusion, the SOCS3 protein appears to be mainly responsible for the suppression of IFN signaling and IFN production that occurs during HSV-1 infection.

Subcellular localization of prostaglandin H synthase-2 in a human amnion cell line: implications for nuclear localized prostaglandin signaling pathways

We have determined that prostaglandin H synthase-2 localises strongly to the nuclear membrane as well as being found in the endoplasmic reticulum in human amnion-derived WISH cells which have been stimulated with interleukin 1β and phorbol ester. This is consistent with findings in cells of non-reproductive origin. There is strong evidence that prostaglandin J2derivatives, which in other tissues exhibit tumour suppressing, antiproliferative and/or differentiation promoting activities, act through binding of intracellular receptors which then enter the nucleus. In addition, some arachidonic acid derivatives are clearly generated by enzymes at the nuclear envelope and localise to sites in nuclei or bind sites in nuclei. The WISH cell line will make an excellent system for studying these perinuclear intracellular prostanoid signaling mechanisms.

Cytokines regulate prostaglandin H synthase-1 transcription in human amnion-derived cells

We have isolated the prostaglandin H synthase-1 (PGHS-1) promoter from the human amnion cell line WISH by long template PCR. The fragment was 1124 base pairs in length and shared a 96% sequence identity with the sequence in GenBank. The putative transcription start site is located 18 bp upstream of the start codon. The sequence is TATA-less, but contains multiple Sp-1 sites and a GC box at −132. The fragment was subcloned into the promoterless reporter construct pBLCAT3 to produce the promoter reporter construct pPGHS1CAT. pPGHS1CAT expression in amnion-derived AV3 cells was inhibited by the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin 1-beta (IL-1β). PGHS-1 mRNA levels however, were unchanged over a 16-h time course with either treatment. These results suggest that PGHS-1 transcription is regulated in a negative manner by cytokines in human amnion-derived cells.

Presence of integrated DNA sequences of adeno-associated virus type 2 in four cell lines of human embryonic origin.

The human helper virus-dependent parvovirus adeno-associated virus (AAV) has been found in human female genital tissues including material from first trimester miscarriage. In the latter case, AAV type 2 (AAV-2) DNA and viral proteins were detected mainly in the trophoblast cell layer of placenta. In this report, we present evidence that AAV DNA is also present in established human trophoblast cell lines (JEG-3, JAr, BeWo) and in the human amnion cell line FL. In cells of these lines, AAV-2 DNA could be detected both by PCR and Southern blot analysis. Restriction enzyme analysis indicated that AAV DNA was integrated into the host cell genome. Although the cell lines supported AAV replication when infected with AAV-2 and adenovirus type 2 (Ad2) as a helper virus, superinfection with Ad2 alone did not induce replication of AAV DNA, i.e. it failed to rescue AAV from its integrated state. This is probably due to rearrangements within the integrated AAV genome. The presence of AAV DNA in cells derived from human embryonic tissue corroborates the suggestion that human embryonic tissue may be one of the targets of AAV infection.

CA125 phosphorylation is associated with its secretion from the WISH human amnion cell line.

Evidence is presented suggesting that CA125 is a serine and/or threonine phosphoprotein and that its secretion from the human amnion WISH cell line is closely linked to its phosphorylation. It is also indicated that regulation of CA125 secretion requires protein(s) tyrosine phosphorylation. WISH cells treated with a tyrosine phosphatase inhibitor, vanadate/ H2O2, resulted in increased levels of CA125 secretion. Exposure of vanadate-treated cells to epidermal growth factor further enhanced this secretory activity. Immunohistochemistry of vanadate-treated cells resulted in a substantial increase in not only cytoplasmic tyrosine phosphoproteins but also in membrane-associated CA125 when stained with the PY20 anti-phosphotyrosine and M11 anti-CA125 monoclonal antibodies, respectively. M11 immunoprecipitation of CA125 from cells labelled with [32P]-orthophosphate was analyzed by SDS-PAGE and autoradiography. Immunoprecipitates from cell lysates demonstrated that a phosphoprotein of > 200 kD was isolated and immunoreacted with both the OC125 and M11 anti-CA125 monoclonal antibodies by Western blotting. CA125 immunoprecipitated from vanadate-treated cells showed a marked increase in cell-associated CA125 phosphorylation. Although CA125 could be immunoprecipitated from WISH cell media incubated with [32P]-orthophosphate in the presence or absence of vanadate as detected by Western blotting, autoradiographic analysis of the Western blots revealed no [32P]-labelled CA125 co-migrating with the 200-kD plus molecule detected by M11. When the PY20 anti-phosphotyrosine monoclonal antibody was used as the probe, no tyrosine-phosphorylated CA125 was detected in cell lysates.

Isolation and Characterization of a Human Amnion Epithelial Cell Line That Expresses the Pregnancy-Specific β1-Glycoprotein Gene*

Human pregnancy-specific beta 1-glycoprotein (PSG) is a family of closely related glycoproteins of 72K, 64K, 62K, and 54K. Together with the carcinoembryonic antigen, they form new members of the immunoglobulin superfamily. To study the molecular mechanisms that regulate expression of the PSG gene, we established a human amnion cell line, HAA58OD-8C, immortalized with an origin-defective simian virus-40 (SV40) temperature-sensitive A58 mutant virus. HAA58OD-8C cells were temperature sensitive for maintenance of transformation and expressed genes encoding PSG and the alpha- and beta-subunits of hCG. At the permissive temperature (33 C; transformed phenotype), they expressed low levels of PSG, hCG alpha, and hCG beta mRNAs and synthesized low levels of a 48K PSG polypeptide. At the nonpermissive temperature (39.5 C), HAA58OD-8C cells exhibited a differentiated phenotype, expressed increased levels of PSG, hCG alpha, and hCG beta mRNAs, and produced high levels of PSG polypeptides of 72K and 48K. Sodium butyrate induced PSG mRNA expression, and in the presence of butyrate, HAA58OD-8C cells produced high amounts of PSG polypeptides of 72K, 62K, and 48K. Ribonuclease protection analysis indicated that similar PSG transcripts were expressed by HAA58OD-8C cells and human term placenta. However, these amnion cells expressed selectively a certain population of PSG transcripts. Our results show that this amnion cell line provides a suitable model for studies of PSG gene expression and regulation.

Adsorption of fetal surfactant protein SP-B on the human amnion at term and on amniocytes incubated with fetal surfactant in vitro

Fetal surfactant stimulates the synthesis of prostaglandins by slices of human amnion at term and by a human amnion cell line, and these effects are partly dependent upon surfactant apoproteins. In this paper, methods are described for the purification of surfactant from human amniotic fluid and from post-mortem human lung. A procedure is described for the purification of surfactant protein SP-B from human amniotic fluid, and the sequence of 20 amino acids at the N-terminal has been determined. A monoclonal antibody generated against human lung surfactant has been shown to react with SP-B from amniotic-fluid surfactant, and the presence of SP-B on the surface of the amnion at term has been demonstrated by immunohistochemical methods. It has also been shown that SP-B from surfactant is present on the surface of amniocytes incubated with surfactant in vitro.

Evidence of a role for protein kinase C in epidermal growth factor-induced prostaglandin E 2 synthesis in amnion cells

Human amnion cells synthesize and release prostaglandin E2 in response to epidermal growth factor. The protein kinase C activator, phorbol 12-myristate, 13-acetate also stimulates amnion cell prostaglandin E2 synthesis. We used a human amnion cell line (WISH) to conduct in vitro experiments to investigate a potential role of protein kinase C in the signal transduction pathway leading to epidermal growth factor-induced prostaglandin E2 production. Pretreatment of cultured amnion cells with a low, nonstimulating dose of phorbol 12-myristate, 13-acetate potentiated the action of epidermal growth factor in causing prostaglandin E2 production as measured by radioimmunoassay. The protein kinase C-selective inhibitor staurosporine inhibited epidermal growth factor-induced prostaglandin E2 production, further suggesting a role for protein kinase C in epidermal growth factor action. Experiments were conducted in which amnion cells were rendered protein kinase C-deficient by chronic exposure to phorbol ester, which has been shown to down-regulate the enzyme. In these cells, epidermal growth factor caused prostaglandin E2 synthesis at levels comparable to native (non-protein kinase C-deficient) cells. We conclude that protein kinase C plays a more modulatory than direct role in the epidermal growth factor signal transduction cascade that leads to prostaglandin E2 production by amnion cells. We propose a bifurcating transduction scheme in which, under conditions of protein kinase C inactivation, epidermal growth factor alone causes prostaglandin E2 Synthesis. When protein kinase C is activated by as yet unknown endogenous substances, the epidermal growth factor responsiveness of the amnion cells is greatly enhanced. This pathway could have important implications in a feed-forward mechanism regulating the level of prostaglandin E2 production during the onset of labor.

Interferon sensitivity of various cell lines

An economic assay to determine the yield of interferon is necessary for the induction, production and purification of interferon. Using the inhibition of cytopathic effect of vesicular stomatitis virus in a microtitre system, we compared the susceptibility of different cell lines against an internal IFN alpha standard obtained from Sendai virus-induced human leukocytes. Human fibroblasts carrying trisomy G 21 and bovine MDBK cells showed the highest sensitivity followed by normal human fibroblasts. Human RH kidney cells exhibited a susceptibility similar to that of human WISH amnion cells frequently used by others. The human amnion cell line FL and monkey Vero kidney cells, as described here, were unsuitable for the determination of IFN yields.

Heterotransplantation studies with tissue culture cell lines in various animal and in vitro host systems

The human amnion cell line FL was found to be more tumorigenic than HeLa cells when used as a positive control in heterotransplantation assays. FL cells formed significantly larger locally invasive tumors than HeLa cells in both Balb/c/nu/nu mice and ATG-treated newborn Wistar rats. In addition, FL cells resulted in metastatic growths in the lungs of two of 12 mice six weeks and in 10 of 36 rats three weeks after inoculation. HeLa cells did not produce metastases in either mice or rats. Both these cell lines were obtained from the American Type Culture Collection. Heterotransplantation experiments with a variety of animal host systems confirmed previous findings that newborn Wistar rats treated with rat ATG were the most sensitive to tumor growth. Vero and LLC-MK2 continuous monkey kidney cells formed small, non-progressively growing tumors showing tubule formation and occasional mitoses. LLC-MK2 cells were found to be more pleomorphic in appearance and with more mitoses than Vero cells but neither cell line showed any evidence of distant metastatic growth in any of several organs examined. The human lymphoblastoid cell line Namalwa produced large invasive tumors at the inoculation site but no distant metastases. In the chick embryo skin test it was found that MI values (mitotic index--percentage of cells in mitosis) gave more reproducible results and were less time-consuming than counting mean mitoses per section. Significant differences were found between Vero, LLC-MK2, HeLa and FL cells with Vero giving the lowest and FL the highest values. The use of MI values enhanced the sensitivity of the chick embryo skin test which was found to be a rapid and valuable screening test for tumorigenicity.

Decreased colony forming ability of human cells persistently infected with baboon endogenous virus. Brief report.

Plating efficiency (PE), i.e. colony forming ability, of primary human embryonic lung fibroblasts persistently infected with baboon endogenous virus (BaEV) decreased to less than 10 per cent of that of the uninfected cells. A line of human amnion (FL) cells showed a 55 per cent reduction of PE after the establishment of BaEV carrier state. No decrease of PE did occur as a result of BaEV persistent infection in a rhabdomyosarcoma cell line (A204) and in fibroblasts derived from adult human skin. This characteristic reduction of PE in the infected primary embryonic fibroblasts was completely abolished by cultivation of the cells with conditioned medium from confluent either virus-infected or uninfected culture.

The impossibility of defining unequivocally the antiviral activity of human fibroblast interferon in terms of a human leukocyte interferon standard

The antiviral activity titre of human fibroblast interferon expressed in reference units, i.e. determined in comparison to the human leukocyte interferon standard preparation BRS 69/19, depends on the cell type used in the assay. It is about 10-fold higher in human fibroblasts (strains FS4 and VG2S) than in U cells (human amnion cell line). Therefore it is not possible to express unequivocally the antiviral activity of human fibroblast interferon in terms of the British research interferon standard.

Intramitochondrial bodies in a human cell line carrying oncornavirus-like particles.

Electron-dense particles 80-100 nm in diameter were observed in electron micrographs of mitochondria in transformed mouse fibroblasts and in a human amnion cell line infected with supernatants of a human mammary carcinoma cell culture. Intramitochondrial particles were more numerous in the human cell cultures early after subculture. These particles were morphologically similar to those reported in mitochondria of Rous sarcoma virus-infected cells.

Acid phospholipase A 1 and A 2 in the cells, and subcellular redistribution of their activities in the cells infected with measles virus

Presence of soluble acid phospholipase A 1 and A 2 was confirmed in the (lysosomes + mitochondria) fraction of cultured human amnion cell line, FL cells. Activity of these enzymes and acid phosphatase was detected in the cytosol fraction of FL cells harvested at 59 hr after infection with measles virus, indicating that these enzymes in the (lysosomes + mitochondria) fraction were released to the cytosol fraction during the maturation of measles virus in the cells. Further, it was confirmed that the release of acid phospholipase A 1 and A 2 almost paralleled the development of cytopathic effect.

Cellular binding characteristics of human interferon

The binding of human Interferon to a line of human amnion cells was studied by exposing the cells to Interferon and recovering interferon from cell extracts. The bulk of cell-associated interferon was present in the 180 g pellet containing large membrane fragments. Trypsin treatment of cells prior to the exposure to interferon decreased the amount of interferon bound. Cell-bound interferon was resistant to trypsin digestion. In cells continuously exposed to interferon at 37 °, the amount of cell-associated interferon increased up to about 30 min, remaining at this level until about 90 min, and decreasing thereafter to a constant level. This decrease, after 90 min of contact, was not seen when the binding was carried out at 4 ° or in cultures treated with puromycin, UV-irradiation or concanavalin A. Prior exposure to interferon made cells completely refractory to the binding of a subsequent interferon dose. After having bound interferon at 37 °, cells were incubated in interferon-free medium either at 37 ° or at 4 °; release of interferon from the cells into the medium occurred only at 37 °. Binding of human interferon was not demonstrated in mouse L cells which are insensitive to the antiviral action of human interferon.

Mason-Pfizer virus characterization: a similar virus in a human amniotic cell line.

Mason-Pfizer monkey virus (MP-MV) is a RNA virus with an RNA-instructed DNA polymerase first isolated from a rhesus monkey mammary adenocarcinoma in 1970. Until recently, there have been no other isolates. A continuous human amnion cell line, AO, was found to be producing a virus indistinguishable or closely related to the Mason-Pfizer virus as measured by morphological, immunological, and biochemical methods. By thin-section electron microscopy, the extracellular virus particle in AO line is 115 to 130 nm in diameter and has a preformed nucleoid (80 to 90 nm) before budding, properties which are also characteristic of MP-MV. Two proteins of the virus from the AO line were studied. By immunodiffusion, sera which react specifically with MP-MV give a line of identity with virus from the AO line. The AO viral RNA-instructed DNA polymerase purified by phosphocellulose chromatography was specifically inhibited by anti-MP-MV polymerase sera, and the AO cells contained both DNA and RNA sequences related to MP-MV (3)H-DNA. Viruses thus far indistinguishable from MP-MV have also recently been found by others in different human lines, raising again the question of the species of origin of MP-MV. Because the virus in the AO cells cannot be differentiated from MP-MV, we attempted to determine the origin of MP-MV virus by measuring DNA sequences related to MP-MV (3)H-DNA in uninfected human and rhesus monkey cells. The quantity of MP-MV-like DNA sequences in uninfected primate tissues was found to be much lower than the amount of DNA sequences of murine type-B or type-C viruses in uninfected murine tissues. Thus, it was not possible to determine whether the virus produced by AO cells or MP-MV was of human or monkey origin, or both.

Karyotypic changes in mycoplasma-modified lines of FL human amnion cells.

Karyotypes of the mycoplasmainfected and -modified F 138 line of FL human amnion cells and of this line after mycoplasma elimination (F 138 cl) differ distinctly from karyotypes of FL cells. In the hypertriploid FL cells there is an increase in number of chromosomes in all groups as compared to the diploid cell (Denver classification). All FL metaphase plates contain a large submetacentric marker chromosome, sometimes in duplicate or triplicate, and two group C chromosomes with marked secondary constrictions. The hypotriploid F 138 and F 138 cl karyotypes differ from FL by containing fewer chromosomes in groups A, C, and D, but more chromosomes in group B; the two group C chromosomes with pronounced secondary constrictions are absent, but notable new chromosome varieties have appeared. The F 138 cl karyotypes contain lower number of chromosomes in groups E, F, and G, but higher numbers in group B than F 138 karyotypes. There is pronounced variation among karyotypes within each cell line. Attempts are made to correlate differences among karyotypes of the three cell lines to their differences in biological behavior in vitro and in vivo.

Initiation of deoxyribonucleic acid replication at the nuclear membrane in human cells.

Abstract A heteroploid line of human amnion cells was synchronized with excess thymidine and amethopterin so that when exposed to [ 3 H]thymidine the cells would incorporate label for the first 10 minutes of the S period. The demonstration by electron microscope autoradiography that the label in these cells was restricted to the periphery of the nucleus and nucleolus suggests that DNA synthesis in human cells may be initiated at the nuclear membrane.