Comparative susceptibilities of insect cell lines to infection by the occlusion-body derived phenotype of baculoviruses

Twelve insect cell lines from six species were tested for susceptibility to baculovirus infection by occlusion-derived virus (ODV) phenotype through the use of a typical endpoint assay procedure. ODV from three nucleopolyhedroviruses were prepared by alkali treatment (sodium carbonate) of occlusion bodies (OBs) and the virus preparations were titered on various cell lines. More than a four-log difference was realized for each of theses viruses between the various cell lines. The TN368 line from Trichoplusia ni was only marginally susceptible to ODV from each virus, showing only 3-6 infectious units (IU) per million OBs while the gypsy moth line, LdEp was most susceptible, realizing more than 100,000 IU/million OBs. The other lines tested showed various levels of susceptibility between these two extremes and also varied between the three viruses tested. In additional tests, the ODV were treated with trypsin prior to application to the cells. With most cell lines, this treatment increased the infectivity of each virus by 2-10-fold. Exceptions to this trend included the gypsy moth LdEp line, on which the trypsinized ODV from two of the viruses were slightly less infectious than each virus without trypsin, and the TN-368 line, on which the trypsinized ODV was 5,000-75,000 times more infectious. The variable results of trypsinized virus on the different lines are probably due to the levels of endogenous protease activity in the various lines, but the mode of action of the trypsin has not been elucidated. Ultimately, the variable response of cell lines to ODV of different viruses, and the variable effects of trypsin on the ODV may lead to an improved understanding of the infection process of this virus phenotype as well as factors relating to baculovirus host range.     

The establishment of heliothine cell lines and their susceptibility to two baculoviruses

Summary A total of eight cell lines were established from Helicoverpa armigera (3) and H. punctigera (5) embryos and ovaries. Cell lines were established and grown in TC100 and/or TC199-MK containing 10% fetal bovine serum. The serum-free medium ExCell™ 400 was also used, with and without 10% supplemental fetal bovine serum, but failed to generate cell lines from fat bodies, embryos, or ovarian tissues. Cell lines consisted of heterogenous cell types ranging from oval to fibroblast-like. This is the first report on the successful establishment of cell lines from H. punctigera. Cell lines from the two species were distinguishable from each other by DAF-PCR, and noticeable differences in minor bands were observed among cell lines from the same species. All of the established cell lines from both species were susceptible to HzSNPV but did not replicate more virus than that of a H. zea cell line (BCIRL-HZ-AM1-A11). However, an H. punctigera cell line (HP1) replicated AcMNPV to the highest titer (1.0×10⁸ 50% tissue culture infective dose/ml), and only one of the H. armigera cell lines (HA1) was susceptible to this virus.     

Baculovirus infection influences host protein expression in two established insect cell lines

We identified host proteins that changed in response to host cell susceptibility to baculovirus infection. We used three baculovirus-host cell systems utilizing two cell lines derived from pupal ovaries, Hz-AM1 (from Helicoverpa zea) and Hv-AM1 (from Heliothis virescens). Hv-AM1 cells are permissive to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and semi-permissive to H. zea single nucleopolyhedrovirus (HzSNPV). Hz-AM1 cells are non-permissive to AcMNPV. We challenged each cell line with baculovirus infection and after 24 h determined protein identities by MALDI TOF/TOF mass spectrometry. For Hv-AM1 cells, 21 proteins were identified, and for Hz-AM1 cells, 19 proteins were newly identified (with 8 others having been previously identified). In the permissive relationship, 18 of the proteins changed in expression by 70% or more in AcMNPV infected Hv-AM1 cells as compared with non-infected controls; 12 were significantly decreased and 6 cellular proteins were significantly increased. We also identified 3 virus-specific proteins. In the semi-permissive infections, eight proteins decreased by 2-fold or more. Non-permissive interactions did not lead to substantial changes in host cell protein expression. We hypothesize that some of these proteins act in determining host cell specificity for baculoviruses.     

Mass production of HzSNPV baculoviruses in immobilized<Emphasis Type="Italic">Heliothis zea</Emphasis> (HzAM1) insect cell culture

Heliothis zea (HzAM1) insect cells were immobilized in microspheres by sodium-cellulosesulfate (NaCS) and polydiallyldimethylammoniumchloride (PDADMAC). The highest HzAMI cell density was 7.5×10⁷ cells/mL in the microspheres. After infection of the immobilized cells byHeliothis zea single nuclear polyhedrosis virus (HzSNPV), the highest concentration of HzSNPV (polyhedral inclusion bodies: PIBs) produced was 2.87×10¹⁰ PIBs/mL in the microspheres.     

Comparison of nuclear polyhedrosis virus replication in five lepidopteran cell lines

Comparative studies were performed on the replication of the Autographa californica nuclear polyhedrosis virus in cell lines from Estigmene acrea, BTI-EAA; Lymantria dispar, IPLB-LD64BA; Mamestra brassicae, IZD-MB0503; Spodoptera frugiperda, IPLB-SF1254; and Trichoplusia ni, TN-368. Significant differences were observed in the amount of virus obtained from the cell lines, with M. brassicae and T. ni producing more polyhedra than the other lines. These two cell lines also produced nonoccluded virus most rapidly, followed by S. frugiperda, E. acrea, and L. dispar. Sensitivities of the cell lines to infection by the virus, as determined by plaque formation, followed the same pattern, with M. brassicae being most sensitive and L. dispar least so. The T. ni cell line produced polyhedra which were more pathogenic to T. ni larvae than those from the other cells. These differences have important implications in the application of cell cultures in the development of microbial insecticides.     

Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines

In the past decades, a large number of studies in mammalian cells have revealed that processing of glycoproteins is compartmentalized into several subcellular organelles that process N-glycans to generate complex-type oligosaccharides with terminal N -acetlyneuraminic acid. Recent studies also suggested that processing of N-glycans in insect cells appear to follow a similar initial pathway but diverge at subsequent processing steps. N-glycans from insect cell lines are not usually processed to terminally sialylated complex-type structures but are instead modified to paucimannosidic or oligomannose structures. These differences in processing between insect cells and mammalian cells are due to insufficient expression of multiple processing enzymes including glycosyltransferases responsible for generating complex-type structures and metabolic enzymes involved in generating appropriate sugar nucleotides. Recent genomics studies suggest that insects themselves may include many of these complex transferases and metabolic enzymes at certain developmental stages but expression is lost or limited in most lines derived for cell culture. In addition, insect cells include an N -acetylglucosaminidase that removes a terminal N -acetylglucosamine from the N-glycan. The innermost N -acetylglucosamine residue attached to asparagine residue is also modified with alpha(1,3)-linked fucose, a potential allergenic epitope, in some insect cells. In spite of these limitations in N-glycosylation, insect cells have been widely used to express various recombinant proteins with the baculovirus expression vector system, taking advantage of their safety, ease of use, and high productivity. Recently, genetic engineering techniques have been applied successfully to insect cells in order to enable them to produce glycoproteins which include complex-type N-glycans. Modifications to insect N-glycan processing include the expression of missing glycosyltransferases and inclusion of the metabolic enzymes responsible for generating the essential donor sugar nucleotide, CMP- N -acetylneuraminic acid, required for sialylation. Inhibition of N -acetylglucosaminidase has also been applied to alter N-glycan processing in insect cells. This review summarizes current knowledge on N-glycan processing in lepidopteran insect cell lines, and recent progress in glycoengineering lepidopteran insect cells to produce glycoproteins containing complex N-glycans.     

Expression of the green fluorescent protein carried by Autographa Californica multiple nucleopolyhedrovirus in insect cell lines

Summary A recombinant AcMNPV containing the green fluorescent protein (gfp) gene under the polyhedrin promoter (polh) was used to investigate the expression of the gfp gene as well as the production of recombinant extracellular virus in 14 continuous insect cell lines, including Heliothis virescens (BCIRL-HV-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Anticarsia gemmatalis (BCIRL-AG-AM1), Trichoplusia ni (TN-CL1), Spodoptera frugiperda (IPLB-SF21), Spodoptera exigua (BCIRL/AMCY-Se-E1 and BCIRL/AMCY-Se-E5), Bombyx mori (BMN), Sf9 (a clone of IPLB-SF21), and five cell line clones of BCIRL-HV-AM1. The susceptibility of the cell lines to the recombinant virus (AcMNPV.GFP) was ascertained by calculating the mean percentage number of green light-emitting cells as well as by TCID₅₀ titration of extracellular virus with fluorescence as a sign of infection. Of the 14 cell lines tested, all were permissive with varying degrees to Ac-MNPV.GFP, except BCIRL-HV-AMCL2 and BCIRL-HZ-AM1, both grown in serum-containing medium, and BMN, grown in serum-free medium, which were nonpermissive to the virus. Except for BCIRL/AMCY-Se-E1, IPLB-SF21, and four of the five BCIRL-HV-AM1 clones, all the other cell lines (BCIRL-HV-AM1, BCIRL-AG-AM1, TN-CL1, Se-E5, and Sf9) expressed detectable levels of GFP by 48 h postinoculation. The BCIRL/AMCY-Se-E1 and IPLB-SF21 cells, grown in serum-free medium (Ex-Cell 401), expressed detectable levels of GFP at 72 h postinoculation. By contrast, in BCIRL/AMCY-Se-E1 in serum-containing medium (Ex-Cell 401+10% FBS [fetal bovine serum]), GFP was detected at 48 h postinoculation. Furthermore, TN-CL1 cells produced the largest mean percentage number of fluorescent (76.6%) cells in both serum-containing and serum-free medium (64.8%) at 120 h postinoculation. All the BCIRL-HV-AM1 clones showed no GFP expression until 96 h postinoculation, and only then about 1% of the cell population fluoresced. The mean extracellular virus (ECV) production at 120 h postinoculation was highest in BCIRL/AMCY-Se-E5 cells grown in Ex-Cell 401+10% FBS (37.8×10⁶ TCID₅₀/ml) followed by BCIRL-HV-AM1 in TC199-MK (33.4×10⁶ TCID₅₀/ml). Only the BCIRL-HV-AMCL3 clone produced any substantial level of ECV at 120 h postinoculation (16.9×10⁶ TCID₅₀/ml). However, there was no significant correlation between ECV production and the mean percentage number of fluorescent cells. This study provides further information on the susceptibility of 14 insect cell lines to a recombinant AcMNPV containing the green fluorescent protein gene. This information might avail researchers with information to facilitate decisions as to what other cell lines are available for in vitro studies of the gfp gene.     

Production of viral progeny in insect cells undergoing apoptosis induced by a mutant Anticarsia gemmatalis nucleopolyhedrovirus

The Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is the most successful viral biopesticide in use worldwide. We have demonstrated that despite widespread apoptosis and no protein synthesis at 48 h p.i., UFL-AG-286 cells infected with a mutant of AgMNPV (vApAg), produced significant amounts of budded virus (BVs) and viral DNA late in infection. However, a different susceptible cell line (BTI-Tn5B1-4) showed no signs of apoptosis and produced 3.5 times more budded virus when infected with vApAg. A comparison of DNA from AgMNPV and vApAg digested with the same restriction enzymes showed differences in the restriction pattern, indicating that the vApAg phenotype might be due to a mutation in a gene or genes responsible for directly or indirectly inhibiting apoptosis in UFL-AG-286 cells.     

Differences in the expression and localization of human melanotransferrin in lepidopteran and dipteran insect cell lines

The ability of several lepidopteran and dipteran insect cell lines to express human melanotransferrin (p97), a glycosyl phosphatidylinositol (GPI)-anchored, iron-binding sialoglycoprotein, was assessed. Spodoptera frugiperda-derived (Sf9) cell lines, transformed with the p97 gene under control of a baculovirus immediate-early promoter, were able to constitutively express the protein and correctly attach it to the outer cell membrane via a GPI anchor as demonstrated by PI-PLC treatment. In contrast, stable constitutive expression could not be demonstrated with cell lines derived from either Drosophila melanogaster (Kc1 or SL2) or Lymantria dispar (Ld652Y) despite the observation that p97 could be detected in transient expression assays. This may indicate that the long-term expression and accumulation of p97 is inhibitory to Drosophila cells, possibly due to improper localization of the protein and resultant competition for cellular iron. In stably transformed Sf9 cells, p97 was expressed on the cell at a maximal level of 0.18 microg/10(6) cells and was secreted at a maximal rate of 9.03 ng/10(6) cells/h. This level was comparable to the amount expressed with the baculovirus system (0.37 microg/10(6) cells and 31.2 ng/10(6) cells/h) and transformed CHO cells (0.88 microg/10(6) cells and 7.8 ng/10(6) cells/h). Deletion of the GPI cleavage/attachment site resulted in an eightfold increase in the secretion rate of p97, when compared to the intact construct suggesting that the rate-limiting step involves processing of the GPI anchor.     

Production of plasminogen activator by established cell lines of mouse origin

The correlation between malignant transformation and increased plasminogen activator synthesis has been studied in a variety of established cell lines. In contrast to the behavior of secondary mouse embryo cultures, which always show increased fibrinolytic activity when transformed, no such correlation was found within the BALB/c 3T3 line and its transformed derivatives. Cell lines were established from tumors initiated in BALB/c mice by several transformed cell lines. These lines were generally found to contain no more plasminogen activator than the cells used for inoculation. A correlation was found between transformation and plasminogen activator synthesis within Swiss 3T3 cell lines. However, the correlation was not maintained by serum revertants of transformed Swiss 3T3 cells.     

Tooth regeneration from newly established cell lines from a molar tooth germ epithelium

In order to investigate tooth development, several cell lines of the dental epithelium and ectomesenchyme have been established. However, no attempt has been reported to regenerate teeth with cell lines. Here, we have established several clonal cell lines of the dental epithelium from a p53-deficient fetal mouse. They expressed specific markers of the dental epithelium such as ameloblastin and amelogenin. A new method has been developed to bioengineer tooth germs with dental epithelial and mesenchymal cells. Reconstructed tooth germs with cell lines and fetal mesenchymal cells were implanted under kidney capsule. The germs regenerated teeth with well-calcified structures as seen in natural tooth. Germs without the cell lines developed bone. This is the first success to regenerate teeth with dental epithelial cell lines. They are useful models in vitro for investigation of mechanisms in morphogenesis and of cell lineage in differentiation, and for clinical application for tooth regeneration.     

Viral susceptibility of newly established cell lines from the Hawaiian monk seal Monachus schauinslandi

Ten of 11 cell lines, recently established from the snout (MS-SN), periorbital soft tissue (MS-EY), liver (MS-LV), kidney (MS-KD), lung (MS-LG), spleen (MS-SP), heart (MS-HT), thyroid (MS-TY), brain (MS-BR) and urinary bladder (MS-UB) of a juvenile Hawaiian monk seal Monachus schauinslandi, were evaluated in vitro for their susceptibility to 5 mammalian viruses: herpes simplex virus type 1 (HSV-1), vesicular stomatitis virus (VSV), reovirus type 3 (Reo-3), poliovirus type 1 (Polio-1) and vaccinia virus (Vac); 5 fish viruses: channel catfish herpesvirus (CCV), infectious hematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), fish rhabdovirus carpio (RC) and viral hemorrhagic septicemia virus (VHSV); and 2 marine mammal morbilliviruses: phocine distemper virus (PDV) and dolphin distemper virus (DMV). Four well-established continuous cell-lines of nonhuman primate (Vero) and fish (EPC, CHSE-214 and BB) origin served as controls to standardize the virus infectivity assays. Virus yields were quantified as 50% tissue culture infectious dose (TCID50) ml(-1) on Day 7 post-inoculation. Results of the viral challenge assays revealed that the monk seal cell lines shared a similar pattern of susceptibility to the mammalian viruses. Despite their different tissue origins, all monk seal cells were sensitive to HSV-1, Vac, VSV and Reo-3, but were refractory to Polio-1. A characteristic viral-induced cytopathic effect was noted with VSV and Reo-3, and distinct plaques were observed for HSV-1 and Vac. Monk seal cell lines were also susceptible to PDV and DMV, 2 morbilliviruses isolated from seals and dolphins, respectively. By contrast, these cell lines were generally resistant to VHSV, IHNV and IPNV, with varying susceptibility to RC and CCV. The wide range of viral susceptibility of these monk seal cell lines suggests their potential value in studying viruses of monk seals and other marine mammals.     

Replication of an entomopoxvirus in two lepidopteran cell lines

Pseudaletia separata entomopoxvirus replicated in two lepidopteran cell lines, SIE-MSH-805-F and BM-N. Microscopic examination, and the virus passage tests, of infected cultures indicated that the virus replicated more readily in the former cell line. Virus release by exocytosis occurred in both cell lines. A sequence of virus morphogenesis in the cultured cells was described, based on electron microscopic observations of thin sections. The nucleus of infected cells contained spherical inclusions, and the cytoplasm contained virions, immature virus forms, spheroids, and spindles. A portion of the virions in the cytoplasm was occluded within spheroids, which were often associated with crystallogenic matrix. Virions acquired a coat prior to their occlusion.     

Characterization of newly established clonal oviductal cell lines and differential hormonal reculation of gene expression

Summary Oviductal functions have been studied mainly in primary epithelial cell culture and organ culture. However, secretory cells and ciliated cells coexist in the epithelium, and the small size of the oviduct limits the sources of both epithelial and stromal cells. To circumvent the limits, we attempted to establish clonal cell lines from an oviduct of a p53-deficient mouse. An oviduct was enzymatically digested and cultured in medium containing 10% fetal calf serum supplemented with estradiol-17β. Morphologically distinct clones (10 epithelial and 4 fibroblastic clones) were established, and all clones expressed estrogen receptor α and progesterone receptor. Expression of a mouse oviduct-specific glycoprotein gene as a marker of secretory cells was limited in one clone and was stimulated by estrogens and suppressed by progesterone. Expression of helix factor hepatocyte nuclear factor/forkhead homologue-4 gene as a marker of ciliated cells was limited in two clones and was suppressed by estrogens. The two genes were never coexpressed in any clones. The results strongly suggest that the oviductal epithelium consists of two functionally determined populations. To our knowledge, this is the first establishment of functional clonal cell lines of the oviduct and makes it possible to study independently two oviductal functions, secretion and ciliogenesis.     

Prostaglandin actions in established insect cell lines

Prostaglandins (PGs) are oxygenated metabolites of arachidonic acid (AA) and two other C20 polyunsaturated fatty acids that serve as biochemical signals mediating physiological functions. We reported that PGs influence protein expression in insect cell lines, which prompted the question: do PGs influence cell proliferation or viability in insect cell lines? Here, we report on the outcomes of experiments designed to address the question in cell lines from three insect orders: Hemiptera (squash bug, Anasa tristis, BCIRL-AtE-CLG15A), Coleoptera (red flour beetle, Tribolium castaneum, BCIRL-TcA-CLG1), and Lepidoptera (tobacco budworm, Heliothis virescens, BCIRL-HvAM1). Treating the insect cell lines with PGA₁, PGA₂, or PGD₂ led to dose-dependent reductions in cell numbers. All three cell lines were sensitive to PGA₁ and PGA₂ (IC₅₀s = 9.9 to 26.9 μM) and were less sensitive to PGD₂ (IC₅₀s = 31.6 to 104.7 μM). PG treatments also led to cell death at higher concentrations, as seen in mammalian cell lines. PGE₁, PGE₂, and PGF_2α treatments did not influence AtE-CLG15A or HvAM1 cell numbers at lower concentrations, but led to dose-related reductions in TcA-CLG1 cells at higher concentrations. Similar treatments with pharmaceutical inhibitors of PG biosynthesis also led to reduced cell numbers: MAFP (inhibits phospholipase A₂), indomethacin (inhibits PG biosynthesis), and esculetin (inhibits lipoxygenase). Because these pharmaceuticals are used to relieve inflammation and other medical issues in human medicine, they are not toxic to animal cells. We infer PGs are necessary in optimal quantities for ongoing homeostatic functions in established cell lines; in quantities outside the optimal concentrations, PGs are deleterious.     

三株烟草天蛾新细胞系的生长特性及重组蛋白表达

从尚未涉及的昆虫种类中建立新的细胞系能为基础研究和生物技术应用提供重要资源。本实验通过细胞培养技术, 建立了3株来源于鳞翅目昆虫烟草天蛾Manduca sexta卵组织的新细胞系, 分别命名为QB-Ms1-8, QB-Ms2-2和QB-Ms2-7。这3株细胞已经培养在TNM-FH培养基中, 28℃条件下传代培养了约50代, 大部分细胞呈梭形, 细胞群体倍增时间分别为51, 31和49 h。虽然这3株细胞系对苜蓿银纹夜蛾核型多角体病毒（Autographa californica multiple nuclear polyhedrosis virus, AcMNPV）不够敏感, 侵染后96 h感染率在33%~40%之间, 但是QB-Ms2-2细胞与BTI-Tn5B1-4细胞比较, 分泌型碱性磷酸酶（SEAP）活性表达更高。本研究从建立的3株烟草天蛾新细胞系中筛选出SEAP高表达的细胞系QB-Ms2-2, 为进一步细胞克隆和筛选提供了新资源。     

Characterization of the state of differentiation of six newly established human non-small-cell lung cancer cell lines

Six new non-small-cell lung cancer (NSCLC) cell lines were established directly from human tissue or indirectly via nude mouse xenografts in serum-supplemented media with success rates of 8% and 13%, respectively. They comprised one adenocarcinoma (ADLC-5M2), two squamous cell carcinomas (EPLC-32M1, EPLC-65H), two large cell carcinomas (LCLC-97TM1, LCLC-103H), and one malignant biphasic mesothelioma (MSTO-211H). All cell lines grew adherent to culture vessels with population doubling times (PDT) of 16-40 h, formed colonies in soft agarose with efficiencies of 0.1%-5.1%, and all grew in athymic nude mice. Xenograft histologies appeared as follows: (a) undifferentiated carcinomas with feeble resemblance to the original tumors in the case of adenocarcinomas and squamous cell carcinomas; (b) large cell carcinoma with high resemblance to the original tumor; (c) an undifferentiated tumor with predominance of large epithelial cells and few fibrous cells in the case of mesothelioma. Human chorionic gonadotropin (HCG) was found by radioimmunoassay and high-affinity binding sites for epidermal growth factor (EGF) by radio-receptor assay in 4/4 cell lines. A very low activity of L-DOPA decarboxylase (DDC) was detectable only in the adenocarcinoma cell line. All cell lines overexpressed the c-myc protooncogene, and no gene rearrangement or amplification was observed. Chromosome analysis revealed modal chromosome numbers of 70-73 in ADLC-5M2, EPLC-32M1, EPLC-65H, and MSTO-211H. Cell lines derived from large cell carcinoma had modal values of 65 and 170 and a wider chromosome distribution than all other cell lines. A NSCLC specific chromosomal aberration has been undetectable until now. These cell lines may aid in elucidating the biology of NSCLC and its interrelationship to other lung tumors.