Methionine-enkephalin in a porcine endometrial cell line and its responsiveness to potassium depolarization.

Immunoreactive methionine-enkephalin (ir-MENK) has been identified in the porcine uterine fluid and endometrium. Previously, we have established a porcine endometrial cell line of epithelial origin (PE-1) by transfecting primary endometrial cells with temperature sensitive SV40 DNA. The current study was conducted to identify and characterize ir-MENK present in PE-1 cells, and to investigate the effect of KCl depolarization on the kinetics of ir-MENK secretion. PE-1 cells were cultured at 33C until confluency was reached (33C cells), after which they were incubated at 40C for 2 days (40C cells). Ir-MENK in PE-1 cells was analyzed by Sephadex G-15 gel filtration and reverse phase (RP)-HPLC. Analysis of 40C cell extract by Sephadex G-15 and RP-HPLC indicated that the major portion of ir-MENK present in PE-1 cells was eluted at a position similar to that of synthetic MENK. The effect of temperature on ir-MENK synthesis in PE-1 cells was examined by measuring ir-MENK content in 33C and 40C cells over a 14-day culture period. Compared to 33C cells, 40C cells maintained higher and steadier levels of ir-MENK, suggesting that synthesis of ir-MENK is temperature sensitive. KCl stimulated ir-MENK secretion at all concentrations tested (5-60 mM for 60 min), with 30 mM being the optimal concentration. Temporal analysis of ir-MENK secretion showed that incubation for 60 min with 30 mM KCl allowed maximal secretion. Secretion of ir-MENK from PE-1 cells resulted in depletion of ir-MENK in cell content. These results demonstrate that PE-1 cells contain ir-MENK which is biochemically similar to synthetic MENK, PE-1 cells synthesize ir-MENK in a temperature sensitive manner, and these cells secrete ir-MENK upon KCl stimulation.     

Endometrial immunoreactive beta-endorphin increases during mid-estrous cycle and early pregnancy in gilts.

Immunoreactive (ir) beta-endorphin (BEND) was recently identified in porcine uterine fluids. In the study reported here, we examined the hypothesis that porcine endometrium serves as a source of uterine fluid ir-BEND during the estrous cycle and early pregnancy. Endometrial ir-BEND was chromatographically characterized, sites of ir-BEND synthesis were immunocytochemically localized, and concentrations of endometrial ir-BEND during the estrous cycle and early pregnancy were measured. Sephadex G-50 chromatographic profiles of endometrial extracts from Day 15 of the estrous cycle revealed three distinct peaks of ir-BEND, with the first peak occurring near void volume and the second and third peaks coinciding with standard porcine beta-lipotropin and standard porcine BEND, respectively. Reverse-phase HPLC C18 chromatographic profiles indicated that endometrial ir-BEND contained both standard BEND and alpha-N-acetylated BEND. Immunocytochemical studies demonstrated ir-BEND in the surface and glandular epithelial cells of the endometrium, with immunostaining most prominent in the apical portion of epithelial cells. Concentrations of ir-BEND in endometrial tissues were higher on Days 14-15 than on Days 8-12 during the estrous cycle and pregnancy (p less than 0.05); however, values were not different in pregnant and cyclic gilts. Biochemical and immunocytochemical evidence supports our hypothesis that ir-BEND present in uterine fluids is derived from the endometrium. The increase in endometrial ir-BEND concentration during Days 14-15 in cyclic and pregnant gilts indicates that ovarian steroids may influence the synthesis of endometrial ir-BEND.     

Fibronectin synthesis and surface expression is correlated with cell morphology and adhesiveness in a cold-sensitive, G1-defective mutant of CHO cells

A cold-sensitive, G1-defective variant of CHO cells, clone cs4-D3, exhibits a reversible, cell-cycle-dependent change in morphology at the non-permissive temperature (33 °C). We have investigated the role of fibronectin in mediating shape change in these cells, and found them to be defective in synthesis and expression of this molecule and in cell surface/fibronectin interactions at 33 °C. Our results suggest that clone cs4-D3 will provide a valuable in vitro model for investigating the role of cell adhesion in growth control.     

Differential effects of dioctanoylglycerol on fibronectin localization in normal, partially transformed, and malignant human endometrial stromal cells

In this study, we describe the effects of direct activation of PKC by dioctanoylglycerol (DiC8) on cellular morphology and the localization of fibronectin (Fn) in normal, oncogene-transfected, and malignant human endometrial stromal cells. We questioned whether DiC8, an endogenous specific activator of PKC, would function as a second oncogene in partially transformed human endometrial stromal cells (HESC). Cells utilized were (1) normal HESC, (2) HESC transfected with a plasmid containing an origin-defective temperature-sensitive SV40 large T antigen alone or (3) in combination with an EJ ras oncogene, and (4) an endometrial sarcoma cell line (S7). Cell cultures were treated for 1 h with sn-dioctanoylglycerol (DiC8) and stained with a monoclonal fluorescein-labeled anti-Fn antibody. In normal HESC, DiC8 induced cell rounding and caused Fn localization to revert from the perinuclear region to the cell periphery. All experiments in this investigation were performed when cells were maintained at the permissive temperature for SV40 large T antigen function. In HESC expressing the SV40 large T antigen alone, Fn was localized to the perinuclear region and also occurred as parallel strands between cells. When these cells were treated with DiC8, Fn localization changed to intense punctate regions at the cell periphery or to matrix-like patterns between cells. Also, in these cells, DiC8 induced greater detachment of cells from the substrate than from other cells, resulting in an apparent piling up of cells. Control and treated SV40/EJ ras cells and uterine sarcoma cells expressed Fn in a matrix-like pattern between cells. The rounded cellular morphology of treated HESC and treated cells expressing SV40 resembled the morphology of control or treated SV40/EJ ras cells and uterine sarcoma cells. Thus, treated cells expressing the SV40 large T antigen resembled the SV40/EJ ras cells and uterine sarcoma cells with respect to Fn localization and cellular morphology. DiC8 did not appear to further transform HESC expressing SV40 and EJ ras. However, with regard to cell shape and Fn localization, our results suggest that DiC8 may function as a second oncogene in the signal transduction pathway, in cells expressing SV40 alone. It appears that, with regard to Fn localization, DiC8 may alter signal transduction analogously to that caused by the activated Ha-ras oncogene in HESC expressing the SV40 large T antigen.     

Selection of a stable clone of the MVPK-1 fetal porcine kidney cell for assays of foot-and-month disease virus.

The MVPK-1 cell line, derived from fetal porcine kidney cells, supports the replication of foot-and-mouth disease (FMD) virus. The cell line was adapted to grow in medium containing 5% bovine serum. The susceptibility of the adapted cells decreased as they aged at 37 degrees C. Various clones were isolated from the adapted cells and their growth characteristics and sustained susceptibility to FMD virus were compared. Clone 7 maintained uniform susceptibility to FMD virus over a 3-day period at 37 degrees C and proved superior to other clones in the characteristics studied. The clone has maintained satisfactory susceptibility to FMD virus through 40 subcultures. Clone 7 can replace primary bovine kidney cells for routine viral assays, but cannot detect as much FMD virus in animal specimens as primary bovine kidney, bovine thyroid, or swine kidney cells.     

Alternative excision products originating from a single integration of polyomavirus DNA.

The Cyp cell line consists of mouse cells transformed by a thermosensitive polyomavirus (Py) genome and routinely propagated at 39 degrees C. Cyp cells are readily induced to synthesize free Py DNA by being transferred to 33 degrees C. In one subclone (C12/a1/S48, or S48) of this line, such induction resulted in the intracellular accumulation of three discrete species of cyclic DNA, i.e., genomic Py DNA, RmI, and RmII. RmI and RmII are Py-mouse chimeras, each of which contains a distinct set of sequences originating from the site of integration. Conceivably, genomic Py DNA, RmI, and RmII could persist at 39 degrees C as free replicating plasmids or originate from distinct populations of cells in S48 cultures. The data indicated that all three species arise at 33 degrees C from a genetically homogeneous cell population in which neither RmI nor RmII replicates at 39 degrees C. Examination of the sequence at the viral-cellular junction unique to RmII indicated that this chimera is excised from the host chromosome through a recombination event involving a complex viral sequence and a simple cellular sequence. Therefore, RmII provides another example of precise recombination occurring between nonhomologous sequences in a mammalian cell, as already observed for RmI (B. S. Sylla, D. Huberdeau, D. Bourgaux-Ramoisy, and P. Bourgaux, Cell 37:661-667, 1984).     

Temperature-dependent transmembrane potential changes in cells infected with a temperature-sensitive moloney sarcoma virus

Normal rat kidney cells (NRK) infected with the temperature-sensitive (ts) transformation mutant of Moloney murine sarcoma virus yielded a clone of cells, 6m2, that exhibited a transformed morphology at 33°C and a normal morphology at 39°C. Transmembrane potential (Em) was measured fluorometrically using a cyanine dye diS-C₃-(5). Fluorescence was inversely correlated with Em. Cells at 33°C had lower Em. Em changes were recorded within 15 minutes of temperature shift from 33°C to 39°C in both directions, increasing in the 33°C to 39°C direction and decreasing in the 39°C to 33°C direction. Uninfected NRK cells when shifted under the same condition exhibited small fluorescence changes in the 33°C to 39°C direction. Shifting from 39°C to 33°C resulted in Em changes similar to those in 6m2 cells. Also studied was a cell line infected with a spontaneous revertant of the ts mutant, designated 54-5A4; it was transformed at both temperatures. Shifting from 33°C to 39°C in both directions yielded small changes. Transmembrane potential changes in 6m2 cells precede other transformation-specific changes that occur after a temperature shift.     

Simian virus 40 gene A regulation of cellular DNA synthesis. I. In permissive cells.

The kinetics of host cellular DNA stimulation by simian virus 40 (SV40) tsA58 infection was studied by flow microfluorometry and autoradiography in two types of productively infected monkey kidney cells (AGMK, secondary passage, and the TC-7 cell line). Prior to infection, the cell populations were maintained predominantly in G0-G1 hase of the cell cycle by low (0.25%) serum concentration. Infection of TC-7 or AGMK cells by wild-type SV40, viable deletion mutant dl890, or by SV40 tsA58 at 33 degrees C induced cells through S phase after which they were blocked with a 4N DNA content in the G2 phase. The infection of TC-7 cells by tsA58 at 41 degrees C, which was a nonpermissive temperature for viral DNA replication, induced a round of cell DNA synthesis in approximately 30% of the cell population. These cells proceeded through S phase but then re-entered the G1 resting state. In contrast, infection of AGMK cells by tsA58 at 41 degrees C induced DNA synthesis in approximately 50% of the cells, but this population remained blocked in the G2 phase. These results indicate that the mitogenic effect of the A gene product upon cellular DNA is more heat resistant than its regulating activity on viral DNA synthesis and that the extent of induction of cell DNA synthesis by the A gene product may be influenced by the host cell.     

Epstein-Barr virus DNA is amplified in transformed lymphocytes.

Leukocytes isolated from two adult donors who lacked detectable antibodies to antigens associated with Epstein-Barr virus were exposed to an average of 0.02 to 0.1 DNA-containing particles of Epstein-Barr virus per cell and immediately clones in agarose. Within about 30 generations all transformed cell clones contained between 5 and 800 copies of viral DNA per cell. Only 1 in 10(4) to less than 1 in 10(5) of the cells of each clone release virus, and the frequency of release did not correlate with the average number of copies of viral DNA in the cells of each clone. One clone that had an average of five copies of viral DNA per cell was recloned, and the average number of copies in four of six subclones increased 15-to 50-fold while the subclones were being propagated sufficiently to study them. These results indicate that Epstein-Barr virus DNA can undergo amplification relative to cell DNA at different times after it transforms cells.     

Biological and biochemical characterization of an SV40-transformed xeroderma pigmentosum cell line

We have isolated a subclone of the SV40-transformed xeroderma pigmentosum (XP) cell line SV40XP12RO. The cell line, designated M1, is highly sensitive to ultraviolet light and is deficient in unscheduled DNA synthesis. The isoenzyme, HLA profile and karyotype of the cell line is presented. The structure and function of the resident SV40 genome is analysed. The M1 clone contains a complete copy of the SV40 genome flanked by partial SV40-DNA copies in a head-to-tail arrangement. The large T-antigen is defective in the ability to induce SV40-DNA replication. The M1 subclone is an efficient recipient of DNA in transfection experiments. Transfection of these cells with the pSV₂gpt plasmid shows that the M1 subclone is as efficient as the NIH 3T3 cell line in uptake and expression of foreign DNA. This cell line should be suitable for genetic analysis of the xeroderma pigmentosum defect. It should also be useful for the study of gene expression in human cells.     

NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity.

A 78 residue antimicrobial, basic peptide, NK-lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK-lysin. The clone was used to show that stimulation with human interleukin-2 induced synthesis of NK-lysin-specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin-2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK-lysin was present in cells positive for CD8, CD2 and CD4. NK-lysin showed high anti-bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK-sensitive mouse tumour cell line, YAC-1, but it did not lyse red blood cells. The amino acid sequence of NK-lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK-lysin is a new effector molecule of cytotoxic T and NK cells.     

Immunologic selection of simian virus 40 (SV40) T-antigen-negative tumor cells which arise by excision of early SV40 DNA.

A clonal line of highly oncogenic spontaneously transformed mouse cells (104C) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned (106CSC). This 106CSC cell line expressed T antigen and transplantation antigen but was about 100 times less tumorigenic than the 104C parent. When 10(5) 106CSC cells were injected into immunocompetent syngeneic mice, tumors were produced. From such tumors, cell lines were established in culture, all of which were consistently negative for T antigen. We found previously by solution DNA hybridization methods that the tumor cells were depleted in the early region of SV40 DNA which codes for the T antigen. We postulated that this loss occurs through a DNA rearrangement of unknown mechanism in one or a few 106CSC cells and that the tumors are then produced from such a cell or cells, whereas all the T-antigen-positive 106CSC cells are rejected by immunologic means. In this investigation we showed by the DNA transfer method using appropriately selected SV40 DNA probes that indeed the tumor cell clone (130CSCT) we selected to investigate came from one 106CSC cell in which the T-antigen-coding SV40 DNA sequences (but not all the early SV40 DNA sequences) were lost by an excision and recombination mechanism. We also showed that the 130CSCT cells, which are highly tumorigenic, could again be transformed by SV40 and that the resulting T-antigen-positive cloned derivative cells became much less tumorigenic (approximately 10(5)-fold), apparently again because of immunologic recognition and rejection. Indeed, when 10(7) T-antigen-positive cloned cells were injected, all the T-antigen-positive cells were rejected and the tumor was produced again from one or more T-antigen-negative cells. Thus, a one-step in vivo transplantation experiment allowed a selection (for tumorigenicity and against the SV40 T antigen) of a mutant mammalian cell with a DNA deletion at a definable site.     

RNA metabolism of murine leukemia virus. III. Identification and quantitation of endogenous virus-specific mRNA in the uninfected BALB/c cell line JLS-V9.

mRNA containing type C endogenous virus-specific sequences was indentified in JLS-V9 cells (an uninfected BALB/c-derived cell line) by annealing extracted RNA with 3H-labeled virus-specific DNA. The criterion for virus-specific RNA being mRNA was that it co-sedimented with polyribosomes in a sucrose gradient and that it changed to lower sedimentation value if polyribosomes were disagregated prior to centrifugation. It was not possible to identify virus-specific mRNA in unfractionated cytoplasm from JLS-V9 cells since large amounts of virus-specific ribonucleoprotein which was not mRNA had sedimentation values similar to polyribosomes and obscured the analysis. Virus-specific mRNA could be readily identified in polyribosomes which had been purified through a step gradient of 1 and 2 M sucrose, and consisted of two species with sedimentation values of 38S and 27S. The amount of virus-specific RNA in different JLS-V9 cell fractions was quantitated in comparison to cell fractions obtained from M-MuLV clone no. 1 cells (a line of NIH 3T3 cells producing Moloney murine leukemia virus). Approximately 40% of the total virus-specific mRNA was recovered in the purified polyribosomes in M-MuLV no. 1 cells. The amount of virus-specific RNA on polyribosomes appeared to be quite similar for JLS-V9 cells and M-MuLV clone no.1 cells .In contrast, the level of virus-specific protein in JLS-V9 cells (as monitored by radioimmunoassay of the internal structural protein p30) was less than 2% the level in the M-MuLV clone no. 1 cells.     

SV40 T基因转化的山羊乳腺上皮细胞系及其生物学特性

目的建立能用于乳腺特异表达基因构件质量检验的山羊乳腺上皮细胞系.方法根据已发表的SV40病毒T基因序列设计引物,以整合有SV40 DNA早期基因区的COS-1细胞基因组DNA为模板,用高保真PCR扩增SV40 T基因.将获得的SV40 T基因克隆入真核表达载体,并用获得的重组表达质粒转染山羊原代乳腺上皮细胞.经有限稀释和反复传代后获得转化细胞克隆,对其生物学特性进行研究.结果扩增出序列正确的SV40T基因,重组质粒转染获得的转化细胞的对数生长期为接种后第4天,细胞群体倍增时间为23.5*!h,克隆形成率为26.7%.DNA斑点杂交试验证明转化细胞的基因组中整合有SV40 T基因,染色体核型分析试验表明转化细胞的核型无明显异常,裸鼠接种试验证明转化细胞不能形成肿瘤,软琼脂集落形成试验表明转化细胞在软琼脂中不能生长.部分细胞克隆已在体外传30代以上,保持正常乳腺上皮细胞的形态特征,在胶原基质上能形成腺泡样结构.结论本研究获得的SV40 T基因转化的山羊乳腺上皮细胞具有转化细胞系的生物学特性.     

A novel Vero cell line for use as a mammalian host-vector system in serum-free medium

We have established a novel cell line from a Vero cell derivative that is useful for expression of exogenous genes and protein production. Parental Vero-317 cells can grow in biotin-containing Eagle's MEM without supplements. By transforming this cell line with replication origin-defective SV40 DNA, which contains a temperature-sensitive tsA58 large T antigen gene, we established the Verots S3 cell line that amplified a SV40-origin containing plasmid. The cell line expressed a human growth hormone (hGH) gene insert with higher efficiency than COS-7 cells in 5% serum-containing MEM and could grow and continue hGH expression in protein-free MEM. However, temperature-sensitive shut down of hGH production was observed not immediately but 3 days after the temperature shift from 33°C to 39.5°C.     

Characterization of simian virus 40 transformed African green monkey cells (CV-1). I. Defective virion and viral genome.

Several clones of SV40 transformed CV-1 cells have been characterized for the production of T- and V-antigens and for the state of viral genome. The transformed CV-1 cells failed to produce infectious virions as assayed after sonication or cocultivation and fusion with normal CV-1 cells, and were resistant to super-infection by SV40. Some clones of the transformed cells contained V-antigens. The population of V-antigen positive cells varied from 0 to 100% depending on the passage number while the T-antigen positive cells were always 100%. The virions isolated from the transformed cells were similar in morphology to complete SV40, but lighter in density than complete SV40. In one clone, a small amount of SV40 DNA was detectable in a free state while a large proportion of the DNA hybridizable with SV40 3H cRNA was linearly integrated into the cell DNA. The free SV40 DNA was noninfectious, closed circular DNA with a size smaller than infectious SV40 DNA component I. Since the cell extracts of the transformed cells contained an agent(s) which induced T- and V-antigens in normal CV-1 cells, it was suggested that the SV40 transformed CV-1 cells contained free as well as integrated defective SV40 genomes responsible for the synthesis of T- and V-antigens.