A new cell line from larval fat bodies of the bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Summary A new cell line, designated IOZCAS-Ha-I, was initiated from the fat body of larvae of Helicoverpa armigera (Lepidoptera: Noctuidae) in TNM-FH medium containing 10% fetal bovine serum. Spherical cells were predominant among the various cell types. The cell line showed a typical lepidopteran chromosome pattern ranging from 58 to 239 chromosomes in the majority of the cells, it was confirmed to have originated from the H. armigera by the DNA amplification-fingerprinting polymerase chain reaction (DAF-PCR) technique. The new cell line was only slightly susceptible to the multiple nucleocapsid nuclear polyhedrosis viruses (NPV) from H. armigera.     

Multiplication of a granulosis virus isolated from the potato tuber moth in a new established cell line of Phthorimaea operculella

Summary A newly established cell line was obtained from the culture of embryonic cells of the potato tuber moth Phthorimaea operculella in low temperature conditions (19° C) using modified Grace’s medium supplemented with 10% fetal bovine serum. The population doubling time was about 80 h when cells were cultivated at 19°C and 38 h at 27° C. The cell line had a relatively homogeneous population consisting of various sized spherical cells. The cells were cultivated for more than 25 passages. Their polypeptidic profile was different from profiles of other P. operculella cell lines we previously described and from other lepidopteran cells. The new cell line was designated ORS-Pop-95. The complete replication of the potato tuber moth granulosis virus (PTM GV) was obtained in vitro by both viral infection and DNA transfection. PTM GV multiplied at a significant level during several passages of the cell line that was maintained at 19° C. As long as the cells were maintained at 19° C, virus multiplication could also be obtained at the same rate at 27° C. To compare PTM GV multiplied both in vivo and in vitro, we used morphological identification, serological, DNA probe diagnosis and endonuclease digest profile analysis and confirmed the identity of the virus.     

Development and Characterization of a Nonmorphogenetic Cell Line of Wheat (Triticum aestivum)

This study was conducted to compare characteristics of a wheat (Triticum aestivum L.) cell line to those of the maize (Zea mays L.) black Mexican sweet (BMS) cell line and to compare protoplasts isolated from suspension cells of these cell lines. The wheat cell line was established from immature-embryo derived callus of the experimental line ‘ND7532’ and was conditioned for growth in suspension culture. For both cell lines, measurements of packed cell volume (PCV), fresh weight (FW), and dry weight (DW) were taken at 3 day intervals from suspension cultures. Measurements of FW of calluses cultured from suspension cells of both cell lines were taken at 6 day intervals. The morphogenetic potential of the wheat ND7532 cell line was tested in both callus and suspension cultures using media promoting regeneration and/or organogenesis. Growth rates of ND7532 cells in suspension culture were comparable to those of BMS cells. However, relative growth rates of calluses recovered from ND7532 suspension cells were slower than those of calluses recovered from BMS suspension cells. The ND7532 cell line has very limited morphogenetic potential and has been maintained as rapidly growing callus tissue for 11 years. Yields of protoplasts from suspension cells of the two cell lines were comparable, though ND7532 protoplasts were typically smaller. The wheat cell line has is now designated ND7532-NM (nonmorphogenetic) and is available for cellular and molecular biology research.     

A new cell line from the wax moth Galleria mellonella Linne (lepidoptera: Pyralididae)

Summary A cell line derived from the larval-fat body tissues of the wax moth, Galleria mellonella Linne, was established in MGM-450 medium. The cells grew in suspension and were mainly spherical in shape. Population doubling time was between 1.4 and 1.7 d over a range of 15 to 35°C, and the maximum growth rate was at 25°C. The chromosome number ranged from 70–239, with a mode of 170. The cells were sensitive to 20-hydroxyecdysone, which stimulated their growth and induced morphological changes. The cell line was designated GaMe-LF1.     

Molecular cytogenetic analysis of repeated sequences in a long term wheat suspension culture

A rapidly growingTriticum aestivum L. (wheat) derived long term suspension culture (named TaKB1), that is probably not regenerable, was analysed for karyotype rearrangements, stability and changes in repetitive DNA. The cell line has an average chromosome number of 21 and the DNA amount of unreplicated cells of TaKB1 measured by flow cytometry is about 30% lower than an unreplicated (1C) bread wheat genome.In situ hybridization of a repetitive DNA sequence (pSc119.2), which occurs as tandemly repeated blocks (heterochromatin) in wheat, shows that chromosomes from the TakB1 line have fewer and weaker subtelomeric locations of the sequence than wheat, suggesting deletions of distal chromosome segments and a reduction in the sites and copy number of the sequence. Thein situ hybridization pattern and chromosome morphology allowed 27 chromosome types to be identified in the cell line. No two analysed cells contained the same chromosome complement, although some chromosome types were present in every cell. Using Southern hybridization the structure and copy number of a retroelement (Wis-2) and its flanking sequence was shown to be the same in the TaKB1 cell line and wheat. Anin situ analysis of rDNA in the TaKB1 cell line (using the probe pTa71) showed a reduction in number of sites and rRNA genes in each cell from that in wheat. Interphase cells of the cell line showed dispersed signal throughout the nucleolus with no evidence for clusters of condensed and inactive rRNA genes.     

A genetic system to detect mitotic recombination between repeated chromosomal sequences in Drosophila Schneider line 2 cells

In order to study mitotic homologous recombination in somatic Drosophila melanogaster cells in vitro and to learn more on the question how recombination is influenced by mutagens, a genetic system was developed where spontaneous and drug-induced recombination could be monitored. Two recombination reporter substrates were stably introduced in multiple copies into the genome of established D. melanogaster Schneider line 2 cells: one plasmid (pSB310) contained the 5′ and 3′ deleted neomycin phosphoribosyltransferase alleles neoL and neoR as direct repeats; the other (pSB485) contained similar deletions (lacZL and lacZR) of the β-galactosidase gene (lacZ). Restoration of a functional neo gene upon mitotic recombination between homologous sequences allowed direct selection for the event, whereas recombination in single cells harbouring the integrated lacZ-based reporter plasmid was detected by histochemical staining or flow cytometric analysis (FACS). The neo-based construct in the clonal transgenic cell line 44CD4 showed a spontaneous recombination frequency of 2.9×10⁻⁴, whereas the 485AD1 cell line harbouring the lacZ-based construct exhibited a frequency of 2.8×10⁻⁴. The alkylating agents EMS and MMS and the clastogen mitomycin C were able to induce recombination in the 485AD1 cell line in a dose-dependent manner. The results obtained from these studies suggest that the transgenic cell lines are potentially useful tools for identifying agents which stimulate direct repeat recombination in somatic Drosophila cells.     

Growth, metabolism and baculovirus production in suspension cultures of an Anticarsia gemmatalis cell line

The UFL-AG-286 cell line, established from embryonic tissue of the lepidopteran insect Anticarsia gemmatalis, has been identified as a good candidate to be used as a cellular substrate in the development of a process for in vitro production of the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, a baculovirus widely used as bioinsecticide. In order to characterize the technological properties of this cell line and evaluate its feasibility to use it for the large-scale production of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, UFL-AG-286 cells were adapted to grow as agitated suspension cultures in spinner-flasks. Batch suspension cultures of adapted cells in serum-supplemented TC-100 medium grew with a doubling time of about 29 h and reached a maximum cell density higher than 3.5 × 10⁶ viable cells ml⁻¹. At the end of the growth period glucose was completely depleted from the culture medium, but l-lactate was not produced. Amino acids, with the exception of glutamine, were only negligibly consumed or produced. In contrast to other insect cell lines, UFL-AG-286 cells appeared to be unable to synthesize alanine as a metabolic way to dispose the by-product ammonia. The synchronous infection of suspension cultures with Anticarsia gemmatalis multicapsid nucleopolyhedrovirus in the early to medium exponential growth phase yielded high amounts of both viral progenies per cell and reduced the specific demands of UFL-AG-286 cells for the main nutrients.     

The development of a medium supplemented with egg extract for the maintenance ofDrosophila cell lines

Summary A saline extract was prepared fromDrosophila eggs. When diluted to a concentration of 1% withDrosophila tissue culture medium, it did not support growth of cells from theDrosophila line D1 during the first few days of subculture as well as medium containing serum. When cells reached a stationary phase, however, the cell density in medium containing extract was greater than in medium containing serum. By altering the concentrations of the extract, and by adding bovine albumin, a medium was obtained in which D1 cells survived initial culturing, and which supported cell growth by day 4 as well as medium plus serum. The initial retardation of growth in medium containing egg extract might be due to the need of the cells to adapt to the new medium. At the present time fourDrosophila cell lines have been maintained in this medium for more than 16 passages. Preliminary experiments with primary embryonicDrosophila cells indicate that medium containing 2% extract and bovine albumin retards the differentiation of these cells. This work was supported by a grant from the Science Research Council of Great Britain.     

An established cell line from the beetle, Xylotrechus pyrrhoderus (coleoptera: Cerambycidae)

Summary A continuous cell line has been established from larval fat body tissues of the cerambycid beetle Xylotrechus pyrrhoderus Bates. These cells were cultured in MGM-450 medium. The cell line, designated as XP-1, showed a heterogeneous population consisting of spherical and spindle-shaped cells with some capacity to adhere and a doubling time of 5 d. The chromosome number of the cell line ranged from 18 to 42 with a mode of 20. Isozyme analysis showed that the cells had patterns distinctive from those of other insect cell lines. The cells were sensitive to insect hormones, and when continuously treated with 20-hydroxyecdysone and juvenile hormone, they assumed a floating elongated-spindle shape and became strongly adherent, respectively.     

CDK inhibitors suppress apoptosis induced by chemicals and by excessive expression of a cell death gene, reaper, in Drosophila cells

The present study was aimed to investigate whether or not cyclin-dependent kinases (CDKs) participate in different cascades leading to apoptosis. We examined the effects of two CDK inhibitors, olomoucine (OLM) and buty-rolactone-I (BL-I), on apoptosis induced in two kinds of Drosophila cell lines. Increases of caspase activity induced by actinomycin D, cycloheximide, H-7 or A23187 in a Drosophila neuronal cell line, ML-DmBG2-c2, and induced by excessive expression of a Drosophila cell death gene, reaper, in Drosophila S2 cells were suppressed by 24-h pretreatment of each CDK inhibitor. Concomitant with the suppression of the caspase activity, fragmentations of cells and DNA, representatives of apoptosis, were also inhibited. These results suggest that CDK(s) participates in progression of apoptosis. However, these effects of the CDK inhibitors were also observed even at lower doses which did not affect cell proliferation. Therefore, it was shown that apoptosis is not always related to cell cycle in Drosophila cells. It was also suggested that the target(s) of the CDK inhibitors locates upstream of caspase in the cascade(s) of apoptosis.     

Development and characterization of a continuous cell line,AFKM-On-H,from hemocytes of the European corn borer <Emphasis Type="BoldItalic">Ostrinia nubilalis</Emphasis> (Hübner) (Lepidoptera,Pyralidae)

The corn borer, Ostrinia nubilalis, is a very important pest in different countries, and the in vitro system of the insect could be a useful tool for isolation and characterization of the pathogens and physiological responses of the insect. In this context, a cell line was derived from the hemocytes of the European corn borer and was named AFKM-On-H for, respectively, O. nubilalis, Armand Frappier, King Mongkut Institutes, and Hemocytes. This cell line was initiated and maintained in Ex-Cell 400 medium supplemented with 10% heat-inactivated fetal bovine serum. The cells, mostly spherical in shape, not firmly attached to the plastic culture flasks, were passaged up to 200 times by repeated gentle pipetting of the cells. The doubling times at the 80th and 125th passages at 28°C and at the 122th and 169th passages at 25°C were 40, 29, 35, and 34 h, respectively. The AFKM-On-H cell line was further characterized by the morphology, karyotype, random amplified polymorphic DNA analysis, and isozyme profiles. Susceptibility of the cell line to cytoplasmic polyhedrosis viruses (CPV) Euxoa scandens (EsCPV), Dendrolimus punctatus (DpCPV), and Choristoneura fumiferana (CfCPV); nuclear polyhedrosis viruses [Autographa californica (AcMNPV) wild type and recombinant, Antherea yammamai (AnyaNPV)]; and Chilo iridescent virus was demonstrated. Relative sensitivities of the cell line to Bacillus thuringiensis and Metarhizium anisopliae toxins and effects of the molting hormone 20-hydroxyecdysone on this new hemocyte cell line were characterized.     

Establishment of a cell line (SPC G1 13) from dorsal vessels ofGromphadorhina laevigata,saussure and zentner (dictyoptera: Blattidae)

Summary A new cell line, designated SPC G1 13, derived from dorsal vessels of larvae ofGromphadorhina laevigata has been established. The medium used is D. 73. The cells are fibroblastlike cells. Some of them are fixed; others, spindle-shaped or round, are in suspension. Most of the cells have the diploid chromosome number. The SPC G1 13 cell line is permissive to the chlamydiaRickettsiella grylli Research was supported, in part, by “A.T.P. Microbiology” INRA-CNRS.     

Establishment,Characterization, and Virus Susceptibility of a New Marine Cell Line from Red Spotted Grouper (<Emphasis Type="Italic">Epinephelus akaara</Emphasis>)

A marine fish cell line from the snout of red spotted grouper Epinephelus akaara, a protogynous hermaphrodite, was established, characterized, and subcultured with more than 60 passages. The grouper snout cell line (GSC) cells multiplied well in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum. The optimal growth temperature was 25°C, and morphologically the cells were fibroblastic. Chromosome analysis revealed that the GSC cell line has a normal diploid karyotype with . A virus titration study indicated that the cells were susceptible to turbot Scophthalmus Maximus rhabdovirus (SMRV) (10^8.5 TCID₅₀ ml⁻¹), while the viral titer of frog Rana grylio virus 9807 (RGV₉₈₀₇) reached 10^3.5 TCID₅₀ ml⁻¹. The infection was confirmed by cytopathic effect (CPE), immunofluorescence, and electron microscopy experiments, which detected the viral particles in the cytoplasm of virus-infected cells, respectively. Further, significant fluorescent signals were observed when the GSC cells were transfected with pEGFP vector DNA, indicating their potential utility for transgenic and genetic manipulation studies.     

Follicle cells and germ line cells both affect polarity in dicephalic chimeric follicles of Drosophila

Summary In aberrant egg follicles of the pattern mutant dicephalic (dic) the oocyte is wedged in between two groups of nurse cells, and this condition may give rise to embryos which express anterior traits at both ends. We have analysed the role of the dic genotype of the germ line cells and the surrounding somatic follicle cells in the formation of the dic follicular phenotype. By means of pole cell transplantations into Fs (1) K 1237 hosts (this cell-autonomous mutation causes degeneration of the host's germ line cells early in oogenesis), we constructed chimeras in which either the follicle cells, the germ line cells, or both were homozygous for the dic mutation. In all three combinations the dic phenotype was expressed but not in controls with dic ⁺ in both germ line cells and follicular epithelium. Since follicles with the dic phenotype may be produced if either the germ line cells or the follicle cells lack dic ⁺ gene activity we suggest that cellular interactions between both cell types are required for the correct positioning of the oocyte at the follicle's posterior pole.     

Highly passage of <Emphasis Type="Italic">Spodoptera litura</Emphasis> cell line causes its permissiveness to baculovirus infection

It is well known that the characteristics of cell lines possibly alter when cell lines are at high-passage number because of the environmental selection. We do not know whether non-permissive or low-permissive cell lines could become permissive or more permissive to virus infection after over-high passage. In the present studies, the alteration of the permissiveness of Spodoptera litura cell line Sl-zsu-1 to three baculovirus infection was investigated after over-high passage, and the possible mechanisms are also investigated. Vigorous apoptosis in Sl-zsu-1 cells was induced by both the recombinant Autographa californica multiple nucleopolyhedrovirus AcMNPV-GFP-actin and the celery looper Anagrapha falcifera multiple nucleopolyhedrovirus AfMNPV, suggesting the replication of the two viruses was blocked by apoptosis. However, the cells infected by S. litura multicapsid nucleopolyhedrovirus SpltMNPV did not undergo apoptosis, but the SpltMNPV titre of the supernatant was not detectable, suggesting this cell line was low-permissive for this virus infection and other factor(s) involved in blockage of the virus replication except apoptosis. However, when Sl-zsu-1 cells had been subcultured continuously for more than 4 years (high-passage cell), which was named as Sl-HP cell line afterwards, no significant apoptosis was induced by the three baculovirus in Sl-HP cells, and many replicated virions or nucleocapsids were observed in the cells. But the permissiveness of Sl-HP cells to the three viruses was very different according to the titre of viruses in the cell cultures. Interestingly, the DNA extracted from SpltMNPV could induce vigorous apoptosis of Sl-HP cells. Altogether, Sl-zsu-1 cell line becomes more permissive to baculovirus infection after over-high passage and multiple paths can block the baculovirus infectivity.     

Fish cell line (ULF-23HU) derived from the fin of the central mudminnow (Umbra limi): Suitable characteristics for clastogenicity assay

Summary A cell line (ULF-23HU) from the fin of the central mudminnow (Umbra limi) was characterized and tested for its suitability to assess cytogenetic damages induced by chemicals in fish. Cells of this line exhibit a fibroblastlike appearance and grew optimal at 25°C in, TC-199 medium containing 10% fetal bovine serum, but slower growth continued down to 4°C, where they could be stored for prolonged periods. Seeding efficiency of ULF-23HU cells on the plastic substratum was approximately 85% in the above culture medium at 25°C. They had a 32-h cell cycle time taken up by a 20-h S period as determined by the autoradiographic analysis of the fraction of labeled mitosis. Cultures showed relatively high mitotic index (0.84 to 2.35%) during exponential growth phase lasting about 7 d. Karyological analysis of the cells at the different subculture passages revealed constant chromosome modal number of 23 consisting of metacentric or submetacentric chromosomes, which were primarily similar to those of in vivo cells, with one additional chromosome. The spontaneous sister chromatid exchange rate was 5.3 per metaphse. When ULF-23HU cells were exposed toN-nitroso-N-methylurea, a clastogen in the mammalian cells, dose-dependent increases both in sister chromatid exchanges and chromosome aberrations were clearly detected. These results on the growth kinetics and cytogenetic characteristics offered the high possibility of the use of this cell line as a suitable in vitro model for clastogenicity studies in fish. This work was supported by grants from Korea Sciences and Engineering Foundation and Japanese Government Research Awards for Foreign Specialists to E.-H. Park.     

The isolation of functional pole cells from theDrosophila melanogaster maternal effect mutantmat(3)1

Summary A procedure for pole cell isolation has been developed that takes advantage of theDrosophila melanogaster maternal effect mutantmat(3) 1. Embryos derived from homozygousmat(3)1 mothers form exclusively pole cells. By outcrossing we could substantially increase the expressivity of the original mutant stock. We further introduced theTM8 balancer chromosome, which carries the dominant temperature sensitive mutationDTS-4. This allows the accumulation of large homozygousmat(3) 1 fly populations by eliminating the heterozygous flies at the restrictive temperature.Early embryos were mechanically fragmented and the cells were isolated by means of metrizamide step gradients. The isolated cells were demonstrated to exhibit the various ultrastructural and histochemical characteristics of pole cells. The isolated cells were transplanted into genetically marked host embryos. The germ line mosaics that were obtained indicate that the isolated cells represent functional pole cells.Proteins synthesized by the isolated pole cells during short term in vitro labelling with³⁵S-methionine were compared to the proteins synthesized by blastoderm cells fromOregon-R embryos. At least one protein could be demonstrated in the pole cell samples that is not synthesized byOregon-R blastoderm cells.The method allows a fast and gentle isolation of highly enriched pole cell populations which are a prerequisite for the biochemical analysis of germ cell determination and differentiation.     

Development of a continuous cell line from the insect egg parasitoid,Trichogramma pretiosum (Hymenoptera; Trichogrammatidae)

Summary A cell line (IPLB-TpE1) was established from embryos of the hymenopteran parasitoid,Trichogramma pretiosum Riley. Cultures contain a mixture of attached, elongate spindle-shaped cells and large aggregates of suspended cells. Chromosomes of the cells were typical ofTrichogramma species and isozyme characterization showed patterns similar toT. pretiosum adults, but distinctly different fromHeliothis zea, the lepidopteran host from which parasite eggs were obtained. The cells are capable of growth over a wide range of osmotic pressures with equal growth between 350 and 600 mOsm/kg. Optimal growth was obtained with a pH of 6.5. Doubling time at the 40th passage was 72 h and cultures are currently subcultured at weekly intervals. The mention of a commercial product does not constitute an endorsement by the U. S. Department of Agriculture.     

Characterization of cultured insect cells selected by <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> crystal toxin

Summary Selection for resistance against Bacillus thuringiensis (Bt) Cry1Ac10 in the Trichoplusia ni (Hübner) cell line BTI-TN-5B1-4 (TnH5) was tested, and the development of resistance in the selected cells was like a S-form curve. Monitoring at the Cry1Ac10 50th challenge, the resistance ratio was 1, 294-fold as many as that of initial cells. But the resistance to Cry1Ac10 declined gradually when the selection was relaxed. The resistance declined rapidly at the low level of resistance and slowly at the high level of resistance. This resistant cell had high resistance to all the tested solubilized trypsin-treated mixture of crystal multitoxins of B. thuringiensis subsp. aizawai GC-91, an engineering bacterium of Bt, B. thuringiensis subsp. aizawai HD-133 and B. thuringiensis subsp. kurstaki HD-1, and low cross-resistance (19.7-fold) to activated Cry1C. Both N-acetyl-d-galactosamine (GalNAc) and tunicamycin did not inhibit the toxicity of Cry1Ac10 to the susceptible TnH5 cells. Comparison of the total proteins of the selected resistant cells with that of the nonselected susceptible cells by two-dimensional electrophoresis analysis showed that were obvious differences among the 11 protein expression. These results strongly suggest that there exists an unknown mechanism of resistance in the cell line that was different from the reported mechanisms in insects.     

Establishment of three human breast epithelial cell lines derived from carriers of the 999del5 <Emphasis Type="Italic">BRCA2</Emphasis> icelandic founder mutation

Summary Germ line mutations in BRCA1 and BRCA2 account for a large proportion of inherited breast and ovarian cancer. Both genes are involved in DNA repair by homologous recombination and are thought to play a vital role in maintaining genomic stability. A major drawback for long-term functional studies of BRCA in general and BRCA2 in particular has been a lack of representative human breast epithelial cell lines. In the present study, we have established three cell lines from two patients harboring the 999del5 germ line founder mutation in the BRCA2 gene. Primary cultures were established from cellular outgrowth of explanted tissue and subsequently transfected with a retroviral construct containing the HPV-16 E6 and E7 oncogenes. Paired cancer-derived and normal-derived cell lines were established from one patient referred to as BRCA2-999del5-2T and BRCA2-999del5-2N, respectively. In addition, one cell line was derived from cancer-associated normal tissue from another patient referred to as BRCA2-999del5-1N. All three cell lines showed characteristics of breast epithelial cells as evidenced by expression of breast epithelial specific cytokeratins. Cytogenetic analysis showed marked chromosomal instability with tetraploidy and frequent telomeric association. In conclusion, we have established three breast cpithelial cell lines from two patients carrying the BRCA2 Icelandic 999del5 founder mutation. These cell lines from the basis for further studies on carcinogenesis and malignant progression of breast cancer on a defined genetic background. Agla J. Rubner Fridriksdottir and Thorarinn Gudjonsson contributed equally to this study.