Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail

Several continuous tissue culture cell lines were established from methylcholanthrene-induced fibrosarcomas of Japanese quail. The lines consist either of fibroblastic elements, round refractile cells or polygonal cells. They show transformed characteristics in agar colony formation and hexose uptake, and most are tumorigenic. Their cloning efficiency in plastic dishes is not increased over that of normal quail embryo fibroblasts. The quail tumor cell lines do not produce endogenous avian oncoviruses and fail to complement the Bryan high titer strain of Rous sarcoma virus; those tested lack the p27 protein of avian oncoviruses. Most of the cell lines are susceptible to subgroup A avian sarcoma viruses, but are relatively resistant to viruses of subgroups C, E and F as compared to normal quail embryo fibroblasts.     

A novel monoclonal antibody induces the differentiation of monocyte leukemic cells

A cell surface antigen (possibly a receptor) on human myeloid cell lines, that may play a crucial role in the differentiation of promonocytic cells, was detected by a murine monoclonal antibody (MAb 710F). When the myeloid cell lines, HL-60, THP-1 and U937, were cultured with the MAb 710F following pretreatment with phorbol 12-myristate 13-acetate (PMA), they displayed both cellular spreading and a strong adherence to the culture dish substratum. On transforming from their original round shape, the induced cells displayed the well-developed microvilli, spindles, or raffles that are characteristic of macrophages or dendrocytes. Similar morphological changes were not induced by treatment with either PMA or MAb 710F alone. By contrast, the lymphoid cell lines did not respond to these reagents. These results suggested that a signal for cellular adhesion and cytoskeletal reconstitution was triggered by the binding of MAb 710F to an antigen on the PMA-primed cells. Thus, the antigen 710F, which is preferentially expressed on peripheral blood monocytes, may represent a cell surface receptor closely associated with differentiation. The antigen/receptor 710F was shown to be a N-glycosylated protein with Mr. of 35-70k. This MAb may prove useful as a tool for both studies on the differentiation of myeloid lineage cells as well as on monocyte/macrophage adhesion function.     

Nuclear location of the putative transforming protein of avian myelocytomatosis virus

The putative transforming protein of avian myelocytomatosis virus MC29 is a 110,000 dalton (P110gag-myc) polyprotein comprised of sequences derived from both the gag region and the MC29-specific myc region. Two approaches have been taken to determine the location of the MC29 gag-related proteins in transformed cells: subcellular fractionation and immunofluorescence. Analysis of subcellular fractions of MC29-transformed cells by immunoprecipitation indicates that the majority of the gag-myc polyprotein is found in the nuclear fractions of Q8 cells (a nonproducer line of MC29-transformed quail embryo fibroblasts) and nonproducer cells derived from a liver tumor of MC20-infected quail. This is in contrast to the distribution of gag-related helper virus proteins lacking myc, which are found only in nonnuclear fractions of superinfected Q8 cells. The purity of unlabeled nuclei was assessed by electron microscopy and enzyme assays, revealing little contaminating material from other subcellular fractions. Immunofluorescence experiments using monospecific anti-gag serum showed specific, intense immunofluorescence in the nuclei of fixed Q8 cells. In contrast, the majority of P75gag-erb, a candidate transforming protein produced by avian erythroblastosis virus (AEV), is absent from the nuclei of nonproducer AEV-transformed chick embryo fibroblasts. The nuclear association of the MC29 transforming protein may be related to some of the unique properties of MC29-transformed cells.     

Retention or loss of v-mil sequences after propagation of MH2 virus in vivo or in vitro.

During propagation of the defective avian retrovirus MH2 in the presence of replication-competent helper virus, deletion of portions of the viral genome occurred frequently. After transformation of quail cells in vitro, v-mil sequences were lost, leading to populations of MH2 viruses which were highly deficient for mil gene expression but which could transform macrophage and fibroblast cells in vitro with high efficiency. In contrast, after induction of tumors in quail with mil-deficient MH2 viral stocks, a majority of the tumor DNAs contained mil+ proviruses, suggesting that there is selection for retention of the v-mil gene in vivo and that the mil protein may play a role in the oncogenicity of MH2 virus. We also isolated MH2-transformed cell lines which contained deleted proviruses arising from packaging and subsequent integration of the subgenomic v-myc-encoding mRNA. Some of these cell lines produced viruses which encoded abnormal v-myc proteins and had altered in vitro transforming properties. These altered phenotypes may be caused by mutations within the v-myc gene.     

H9N2亚型禽流感病毒基因组的遗传进化分析

2009～2011年从江苏省、湖北省和安徽省等地来源于鸡、鸭、鹌鹑和鸽子的样品中分离鉴定出16株H9N2亚型禽流感病毒。通过反转录聚合酶链式反应(RT-PCR)扩增出分离株的全基因片段,并对其进行测序及遗传进化分析。序列分析显示,16株病毒HA基因裂解位点氨基酸序列为P-S-R/K-S-S-R,符合低致病性禽流感的分子特征;226位均为L,具有与哺乳动物唾液酸α,2-6受体结合的特性。M2基因均出现了对金刚烷胺产生耐药性的N31S突变。不同宿主来源的H9亚型AIV的主要分子特征一致。全基因遗传进化分析表明16株H9N2亚型禽流感病毒全基因发生了3配体重组,即以F98亚系AIV为骨架,HA来源于Y280亚系,PB2和M基因来源于G1亚系,形成了2种新的基因型。因此,要加强对H9N2亚型禽流感病毒的监测,密切关注它的重组趋势。     

MHC-restricted cytotoxic response of chicken T cells: expression, augmentation, and clonal characterization

Major histocompatibility complex (MHC)-restricted cytotoxicity of chicken lymphocytes was studied by using three reticuloendotheliosis virus (REV)-transformed cell lines as targets in 51Cr-release assays. The cell lines, designated RECC-UG5, RECC-UG6, and RECC-UG8, were developed from bone marrow cells of REV-infected line G-B1, line G-B2, and (G-B1 X G-B2)F1 chickens respectively. Effector cells were obtained from spleens of G-B1, G-B2, F1, and F2 chickens 7 days after inoculation of REV. The inbred G-B1 (MHC genotype B13/B13) and G-B2 (MHC genotype B6/B6) lines originate from a common partially inbred line. Initial studies with effector cells from G-B1 and G-B2 chickens showed that significant cytotoxicity occurred only with syngeneic target cells. The degree of cytotoxicity was markedly enhanced by neonatally treating effector cell donors with cyclophosphamide (CY) and delaying virus challenge until the birds were 4 wk old. Augmentation of cytotoxicity was presumed to be due to elimination of bursal-dependent suppressor T cells by CY. The results with spleen cells from REV-inoculated F2 birds clearly showed that cytotoxicity was MHC restricted; i.e., significant lysis only occurred if effector cells and target cells had a common B system antigen. Lysis of RECC-UG5 targets was three to four times higher than lysis of RECC-UG6 targets when effector cells were from heterozygous (B6/B13)F1 and F2 birds. Because these two target cell lines generally showed a similar degree of lysis by effector cells from syngeneic B homozygous birds, the differences obtained with effector cells from B heterozygous birds was most likely due to differences in the number of effector cells with specificity for each target line. Evidence for an additive cytotoxic effect, considered to be due to the lytic activity of two separate T cell clones, was obtained when F1 effector cells were tested with the F1-derived RECC-UG8 targets. The results of other experiments indicated that the effector cells were of T cell lineage and that their activity was probably directed against virus-induced antigens on the transformed target cells.     

Expression of neuronal markers in chick and quail embryo neuroretina cultures infected with Rous sarcoma virus

Cultures of neuroretina (NR) cells from 7-day chick and quail embryos were infected with ts NY-68, a thermosensitive mutant of Rous sarcoma virus (RSV) which transformed NR cells at 36 degrees C. The following differentiation markers for neurones were studied: tetanus toxin-binding sites at the cell surfaces, presence of synapses, and the specific activity of the enzymes choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD). Appearance of synapses and expression of CAT were similar in control and transformed cultures. Tetanus toxin-binding cells were observed in transformed primary cultures and also in quail NR subcultures. GAD-specific activity was markedly stimulated in chick and quail primary cultures transformed by ts NY-68 and further increased in subcultures of ts NY-68-transformed quail NR cells. Stimulation of GAD activity is controlled by the transforming (src) gene of RSV since it was not observed in cultures infected with RAV-1, a leukosis virus which lacks the src gene. These data show that infection of chick and quail NR cultures with RSV results in the transformation of cells with neuronal markers.     

Application of a new human cell line,F2N78, in the transient and stable production of recombinant therapeutics

Host cell lines developed by genetic engineering sometimes show instabilities in maintaining their genetically acquired phenotypes. Previously, a hybrid host cell line, designated as hybrid of kidney and B cells (HKB), capable of retaining selected phenotypes originally existing in the parental cells was developed via fusion of 293 cells and HH514‐16 cells. Although HKB did indeed successfully preserve several favorable phenotypes, the expression of Epstein‐Barr virus (EBV) specific nuclear antigen 1 (EBNA1), which should be constitutively expressed for host cells to utilize oriP expression vector in transient production of therapeutic proteins, was observed to be unstable. Here, in an attempt to obtain stable expression of EBNA1, a cell type that contains an integrated EBV genome, rather than HH514‐16 cells, which harbor an episomal EBV genome, was applied for fusion with 293 cells. Fusion of 293 cells with Namalwa cells led to the creation of a new type of hybrid, F2N, which was able to stably express EBNA1 while not producing EBV particles. One of the F2N clones, F2N78, was observed to maintain EBNA1 expression for more than 1 year under serum‐free suspension culture conditions along with human specific glycosyl phenotypes observed previously in HKB. In addition, F2N78 was demonstrated to be an appropriate host cell line for both the transient and stable production of recombinant therapeutics with the features of safety expected of production cell lines for human use. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 432–440, 2013     

基于N蛋白单克隆抗体的小反刍兽疫病毒抗体检测胶体金试纸条的研制

本研究旨在建立一种简便、快捷、可直观检测小反刍兽疫病毒(peste des petits ruminants virus,PPRV)抗体的检测方法。将pET-32a-N重组质粒转化至大肠杆菌(Escherichia coli) Rosetta(DE3)感受态细胞中进行诱导表达,以纯化的PPRVN蛋白免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,间接酶联免疫吸附试验(enzyme-linked immunosorbent assays, ELISA)筛选及亚克隆,获得了抗PPRV N蛋白的单克隆抗体。将PPRV N蛋白分别作为金标抗原及检测线(T线)包被抗原、单克隆抗体作为质控线(C线)包被抗体,组装成检测PPRVN蛋白抗体的胶体金免疫层析试纸条。结果显示：成功获得1株能稳定分泌抗N蛋白抗体的杂交瘤细胞株,命名为1F1;间接ELISA检测1F1腹水效价为1:128000;亚类鉴定结果为IgG1,轻链为kappa链。Westernblotting结果显示,1F1能与PPRV N蛋白特异性结合;间接免疫荧光(indirect immunofluorescent ass...     

Rous sarcoma virus mutant dlPA105 induces different transformed phenotypes in quail embryonic fibroblasts and neuroretina cells.

dlPA105 is a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion in the N-terminal portion of the v-src gene coding sequence. This virus was isolated on the basis of its ability to induce proliferation of quiescent quail neuroretina cells. The altered v-src gene encodes a phosphoprotein of 45,000 daltons which possesses tyrosine kinase activity. DNA sequencing of the mutant v-src gene has shown that deletion extends from amino acid 33 to 126 of wild-type p60v-src. We investigated the tumorigenic and transforming properties of this mutant virus. dlPA105 induced fibrosarcomas in quails with an incidence identical to that induced by wild-type virus. Quail neuroretina cells infected with the mutant virus were morphologically transformed and formed colonies in soft agar. In contrast, dlPA105 induced only limited morphological alterations in quail fibroblasts and was defective in promoting anchorage-independent growth of these cells. Synthesis and tyrosine kinase activity of the mutant p45v-src were similar in both cell types. These data indicate that the portion of the v-src protein deleted in p45v-src is dispensable for the mitogenic and tumorigenic properties of wild-type p60v-src, whereas it is required for in vitro transformation of fibroblasts. The ability of dlPA105 to induce different transformation phenotypes in quail fibroblasts and quail neuroretina cells is a property unique to this Rous sarcoma virus mutant and provides evidence for the existence of cell-type-specific response to v-src proteins.     

Human cytotoxic T cells stimulated by antigen on dendritic cells recognize the N, SH, F, M, 22K, and 1b proteins of respiratory syncytial virus.

We examined the human cytotoxic T-cell repertoire of nine adults to 9 of the 10 proteins of respiratory syncytial (RS) virus. Peripheral blood mononuclear cells from normal adults were stimulated with RS virus in vitro. The resulting polyclonal cultures were tested for lysis of B-lymphoblastoid cell lines infected with recombinant vaccinia viruses expressing each of nine individual RS virus proteins. The use of peripheral blood dendritic cells to present antigen gave more easily reproducible results over a shorter culture period than conventional methods. The six RS virus proteins most strongly recognized were the nucleoprotein N (nine of nine donors with greater than 10% above background lysis; P = 0.0004), the surface proteins SH (six of nine donors; P = 0.002) and F (five of nine donors; P = 0.008), the matrix proteins M (five of nine donors; P = 0.004) and 22K (three of nine donors; P = 0.01) and the nonstructural protein 1b (six of nine donors; P = 0.004). There was no significant recognition of the major surface glycoprotein G (two of nine donors), the internal phosphoprotein P (one of nine donors), or the nonstructural protein 1c (one of nine donors). Recognition was major histocompatibility complex class I restricted, but no association between major histocompatibility complex phenotype and protein specificity of T cells was seen. Recognition of F and 22K appeared to be associated with recent infection indicated by increased levels of anti-RS virus immunoglobulin G antibody in serum measured by enzyme-linked immunosorbent assay. Since cytotoxic T-cell recognition of RS virus proteins has been demonstrated to be important in the clearance of virus from infected hosts, the N, M, SH, 1b, F, and 22K proteins should be considered potential vaccine components.     

Virogenic Properties of Bromodeoxyuridine-sensitive and Bromodeoxyuridine-resistant Simian Virus 40-transformed Mouse Kidney Cells

When simian virus 40 (SV40)-transformed mouse kidney cells (mKS) were grown in the presence of susceptible indicator cells, SV40 was readily recovered from: (i) 15 transformed cell lines, (ii) transformed cells subcultured 45 times over a 7-month period in medium containing antiviral serum and bromodeoxyuridine (dBU), (iii) 45 of 46 clonal lines isolated in the presence of antiviral serum, (iv) 19 of 19 secondary clones isolated from two clonal lines, and (v) dBU-resistant transformed cell lines. dBU-resistant SV40-transformed mouse kidney cell lines were selected and shown to contain the T antigen and to have normal levels of thymidylate kinase and deoxyribonucleic acid (DNA) polymerase, but to be deficient in thymidine (dT) kinase. Radioautographic and biochemical experiments demonstrated that very little (3)H-dT was incorporated into DNA of dBU-resistant cells during a 6-hr labeling period. After infection of dT kinase-deficient mKS cells with vaccinia virus, high levels of dT kinase were induced. The properties of SV40 recovered from dBU-sensitive and dBU-resistant cells were studied. SV40 recovered from transformed cells was shown to express in CV-1 cells at least six functions characteristic of parental virus: synthesis of capsid antigen, synthesis of T antigen, synthesis of viral DNA, induction of dT kinase, induction of DNA polymerase, and induction of host cell DNA synthesis. In addition, SV40 recovered from the transformed cells induced T antigen, dT kinase, deoxycytidylate deaminase, thymidylate kinase, and DNA polymerase in abortively infected mouse kidney cultures, and the virus was also capable of transforming primary cultures of mouse kidney cells.     

The T/t common region of simian virus 40 large T antigen contains a distinct transformation-governing sequence.

Simian virus 40 large T antigen (T) can transform cultured cells, but the mechanisms by which it functions are not entirely understood. Several lines of evidence have suggested that the amino-terminal approximately 130 residues of T may be sufficient to confer the transforming capability. Oligonucleotide-directed mutagenesis was used to generate a series of deletion and substitution mutants within the amino-terminal 82 residues of T, the segment which is shared with simian virus 40 small t antigen (t). Results of stability and transformation assays of these mutants strongly suggest that the 1-to-82 region of T contains sequences which govern T transforming activity and affect in vivo stability. Instability and a defect in transforming activity could be separated from one another genetically. Thus, the 1-to-82 region appears to contain a specific region that contributes to the transforming function of the protein. This segment operates by means other than the simple binding of pRb and/or p107.     

Malignant transformation of hamster kidney cells by BK virus.

Primary hamster kidney cells were transformed by BK virus, a new human papovavirus. Transformed (HKBK) cell produced BK virus T antigen and induced tumors in hamsters that developed antibodies to BK virus T antigen. BK virus was rescued from HKBK cells by Sendai virus-assisted fusion with permissive cells. One out of six cell lines derived from HKBK cell-induced tumors showed the same characteristics as HKBK cells.     

Simian virus 40 small t antigen activates the carboxyl-terminal transforming p53-binding domain of large T antigen.

Expression of the simian virus 40 large T antigen (large T) in F111 rat fibroblasts generated only minimal transformants (e.g., F5 cells). Interestingly, F111-derived cells expressing only an amino-terminal fragment of large T spanning amino acids 1 to 147 (e.g., FR3 cells), revealed the same minimal transformed phenotype as F111 cells expressing full-length large T. This suggested that in F5 cells the transforming domain of large T contained within the C-terminal half of the large T molecule, and spanning the p53 binding domain, was not active. Progression to a more transformed phenotype by coexpression of small t antigen (small t) could be achieved in F5 cells but not in FR3 cells. Small-t-induced progression of F5 cells correlated with metabolic stabilization of p53 in complex with large T: whereas in F5 cells the half-life of p53 in complex with large T was only slightly elevated compared with that of (uncomplexed) p53 in parental F111 cells or that in FR3 cells, coexpression of small t in F5 cells led to metabolic stabilization and to high-level accumulation of p53 complexed to large T. In contrast, coexpression of small t had no effect on p53 stabilization or accumulation in FR3 cells. This finding strongly supports the assumption that the mere physical interaction of large T with p53, and thus p53 inactivation, in F5 cells expressing large T only does not reflect the main transforming activity of the C-terminal transforming domain of large T. In contrast, we assume that the transforming potential of this domain requires activation by a cellular function(s) which is mediated by small t and correlates with metabolic stabilization of p53.     

A monoclonal antibody specific for avian early myogenic cells and differentiated muscle

A monoclonal antibody raised in mouse in response to homogenates of Remark ganglia and dorsal mesentery of chicken embryos was found to exhibit a unique reactivity towards myogenic cells, heart, striated muscles, and smooth muscles in chicken and quail. Indirect immunofluorescence assays were performed at different stages of chicken and quail embryonic development and, after hatching, on tissue sections and cultured cells. They revealed that the cytoplasmic marker recognized by 13F4 is expressed in early embryonic heart, in somitic myotome (from stage 14 onward), in the skeletal muscles in limbs and trunk, in all muscles in the head and the branchial arches, in the smooth muscles of the digestive tract and blood vessels. In myofibrils of striated muscles, the antigen is localized in the Z lines. The antigenicity of the molecule recognized by 13F4 is not associated with a glycolipid or a glycoprotein. It is of peptidic nature and its molecular weight is 54 kDa. We stress the value of this cell-type-specific marker in studies on ontogenesis and differentiation of all muscular structures, namely, of myocardium and striated muscles, which express 13F4 antigenicity from an early developmental stage.     

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.     

Selective response of gamma delta T-cell hybridomas to orthomyxovirus-infected cells.

A gamma delta T-cell hybridoma established from influenza virus-infected mice responded to a reproducible way when cultured with influenza virus-infected stimulators. Subclones of this line responded to cells infected with influenza viruses A/PR/8/34 (H1N1), X-31 (H3N2), and B/HK/8/73 but not to cells infected with vaccinia virus or Sendai virus. This spectrum of response to both type A and type B orthomyxoviruses has never been recognized for the alpha beta T-cell receptor-positive subsets. There was no response to cells infected with a panel of recombinant vaccinia viruses expressing all individual influenza virus proteins, and so it is unlikely that the stimulating antigen is of viral origin. The alternative is that the antigen is a cellular molecule induced in influenza virus-infected cells. Infectious virus was required for stimulation, and immunofluorescence studies showed increased expression of heat shock protein 60 (Hsp60) in influenza virus- but not Sendai virus- or vaccinia virus-infected cells. Both the hybridoma generated from influenza virus-infected mice and an established hybridoma which uses the same gamma delta T-cell receptor combination responded to recombinant Hsp60. Furthermore, the Hsp60-reactive hybridoma, which was obtained from an uninfected mouse, also responded to influenza virus-infected cells, indicating that Hsp60 may indeed be the target antigen.     

Effect of monensin on the synthesis, maturation and secretion of vesicular stomatitis virus proteins in a monensin-resistant Chinese hamster ovary cell line

We compared the effects of the cationic ionophore, monensin, on the synthesis, maturation and release of vesicular stomatitis virus (VSV) in cultures of Chinese hamster ovary (CHO) cells and the monensin-resistant clone, MonR-31. Our results depended on the dose and time of the addition of monensin to the infected cells, from 1 h prior to VSV infection to 1 h after infection. VSV production was more resistant in MonR-31 than in CHO cells when the ionophore was added 1 h prior to VSV infection. Monensin added 1 h after VSV infection showed the opposite phenomenon; release of virus particles into the medium was 10- to 10(5)-fold less in MonR-31 cells than in CHO cells, and the intracellular virus number in the resistant cells was one-third to one-fourth of that in the parental CHO cells. Syntheses of all virus-associated G, N and M proteins were inhibited in both cell lines by monensin, but especially so in the MonR-31 cells. There were no marked qualitative changes in the biochemical properties of viral glycoprotein G in virus-infected CHO and MonR-31 cells treated with monensin after virus infection. An endoglycosidase H-resistant G with a molecular weight smaller than that of normal G and attachments of palmitate or fucose on the truncated G protein appeared. Alteration of the secretion of as well as the synthesis of the enveloped virus is discussed in relation to the monensin susceptibility of the resistant MonR-31 clone.