Effects of simian virus 40 T-antigens on normal human mammary epithelial cells reveal evidence for spontaneous alterations in addition to loss of p16(INK4a) expression

Under standard culture conditions, normal human mammary epithelial cells (HMECs) divide a limited number of times before proliferation ceases in a growth-arrested state referred to as selection. Cells that have undergone spontaneous loss of p16(INK4a) expression due to hypermethylation of the p16(INK4a) CpG island emerge from selection and proliferate for an extended, but limited, period before senescence. Here we show, as expected, that selection was bypassed by expression of SV40 large T-antigen proteins containing an intact pRb-binding domain in preselection cells. These cells were immortalized with high efficiency (seven of nine separate cultures). Also as expected, postselection cells were immortalized by expression of the human papillomavirus-16 E6 oncoprotein (four of four cultures), which inactivates p53 protein. In contrast, we found that expression of SV40 large T-antigen protein, which also inactivates p53, was poorly maintained in postselection cultures due to its growth-suppressive effects; consequently, these cells became immortalized at low efficiency (one of 11 cultures). Reexpression of p16(INK4a) in postselection HMECs by the demethylating agent, 5-azacytidine, or transfection of a p16(INK4a) expression plasmid did not restore the ability of these cells to undergo SV40-induced transformation. Postselection HMECs are a widely used in vitro model system, but these observations indicate they have undergone changes in gene expression in addition to loss of p16(INK4a) expression.     

Cytogenetic characterization of embryogenic callus and regenerated plants of Pennisetum americanum (L.) K. Schum

Summary Embryogenic calli were derived from cultured segments of immature inflorescences of Pennisetum americanum (pearl millet). The original explants as well as the embryogenic calli and the plants regenerated via somatic embryogenesis were examined cytogenetically. Embryogenic calli were predominantly diploid (2n=14) after one month and six months in culture (92% and 76%, respectively). Tetraploid and aneuploid cells were observed in the original explant (2.5% and 1.2%) as well as in one (4.0% and 4.0%) and six-month-old calli (10.0% and 14.0%). Plants were regenerated from calli that had been in continuous culture for two, four and six months. Of the 101 regenerants, 100 were diploid and 1 was tetraploid. The tetraploid was an albino as were three of the diploid regenerants. Examination of 30 of the regenerants in meiotic diakinesis, anaphase I, anaphase II and quartet stages revealed no cytogenetic differences between control and regenerated plants. Gel electrophoresis for total protein content and alcohol dehydrogenase and malate dehydrogenase activity also did not reveal any differences between the controls and regenerants. The results of this study show that a slight shift toward aneuploidy and polyploidy may occur in embryogenic cultures, but there also is a strong selection in favor of plant regeneration from cytogenetically normal cells.     

Accelerated malignant conversion of human HBL-100 cells by the v-Ki-ras oncogene

The human epithelial HBL-100 cell line harbors SV40 genetic information and has an unlimited growth potential. Despite displaying properties characteristic of transformation since its early in vitro passages, it is capable of producing progressively growing tumors in nude mice only after long-term culture. This is a reproducible phenomenon and apparently not the consequence of a selection of preexisting malignant cells. Superinfection of early passage nontumorigenic HBL-100 cells with Kirsten murine sarcoma virus, which contains a Ki-ras oncogene having undergone multiple activating events, induces morphologic alterations and rapidly converts the cells to neoplastic cells, further supporting the hypothesis of multistep carcinogenesis. The HBL-100 cell line might be useful in defining the oncogenes representative of different families, which are able to complement SV40 in this system.     

Response of carrot protoplasts and protoplast-derived aggregates to selection using a fungal culture filtrate of Alternaria radicina

Protoplasts isolated from three accessions of cultivated carrot and 5-day-old protoplast-derived aggregates were subjected to selection to identify somaclonal variants with enhanced tolerance to the fungal disease black rot incited by Alternaria radicina. Different concentrations [1, 2, 3.5, 5, 10, 20, 35 and 50 % (v/v)] of a fungal culture filtrate (FCF) from 2-week-old liquid cultures of A. radicina were used. Protoplasts and aggregates were subjected to short-term selection for a period of 10 days. All FCF concentrations added to the cultures on the day of isolation decreased protoplast survival frequency and plating efficiency, while FCF applied 5 days later inhibited cell divisions in 5–50 % concentrations. The responses of protoplasts to the treatment were genotype dependent. Most R0 plants were regenerated in all accessions from cell lines grown with 1 % FCF, while only a few plants were produced from 2 to 3.5 % FCF-treated cultures of ‘Dolanka’ and the breeding line ‘9304B’, respectively. Nineteen-percent of putative stress-tolerant regenerants were tetraploids, while only 5 % tetraploids were observed in the control. The incidence of unique random amplified polymorphic DNA fragments indicating possible chromosomal rearrangements was low and did not differ among regenerants after selection and those derived from the control. Mobilization of miniature inverted repeat transposable elements was not observed. Some R0 individuals regenerated both from FCF-treated and untreated cultures showed lower susceptibility to A. radicina in a laboratory assay in comparison to control plants grown from seed. Regenerants from FCF-treated cultures showed lower frequency of flowering plants and a higher rate of male sterility. Pollen viability of the putative stress-tolerant regenerants varied over a wide range (6–98 %), independently of in vitro selection conditions. Our data suggest that A. radicina FCF may be feasible for the in vitro selection to generate plants with superior phenotypic performance against A. radicina.     

Cell surface changes correlated with density-dependent growth inhibition. Glycosaminoglycan metabolism in 3T3, SV3T3, and con A selected revertant cells.

A 35SO4-labeling/chromatography technique has been developed which facilitates quantitation of sulfated glycosaminoglycan (GAG) synthesis in mammalian cell cultures. The technique has been used to compare sulfated GAG biosynthesis, degradation, and turnover in three related cell lines with differing degrees of density-dependent inhibition of growth in vitro (Balb/c 3T3, SV3T3, and SV3T3 revertant cells). Viral transformation of Balb 3T3 cells is accompanied by a 2-5-fold decrease in cell associated sulfated GAG. SV3T3 revertant cells, which show partial reversion to low saturation density in vitro, show a 2.5-8-fold increase in cell-associated sulfated GAG compared to the parental SV3T3 cells from which they were selected. In addition, the distribution of 35SO4 and [3H]glucosamine among the different GAG species produced by SV3T3 revertant cells reverts so that it is similar to the distribution characteristic of untransformed 3T3 cells rather than SV3T3 cells. Mild trypsin treatment of 35SO4-labeled cells removed 68-84% of the cellular sulfated GAG, suggesting that at least this proportion of the total cellular sulfated GAG was located at the cell periphery. Removal of 35SO4-labeled cells from the Petri dish with a Ca2+ selective chelating agent revealed a fraction of the sulfated GAG that remained tightly bound to the Petri dish. A higher proportion of the total cell-associated sulfated GAG remained attached to the Petri dish in cultures of untransformed and revertant cells compared to that present in cultures of transformed cells. A role for sulfated GAG in density-dependent growth inhibition of fibroblast cultures is proposed and discussed in the light of the data obtained.     

Anchorage-independent colony formation of SV40 transformed BALB/c-3T3 cells in serum-free medium: role of cell- and serum-derived factors

The colony-forming response of SV40 transformed BALB/c-3T3 cells in agarose suspension culture was studied in a serum-free medium (with insulin, transferrin and serum albumin as the only macromolecular supplements) that was optimized for colony formation of fibronectin-attached monolayer cultures. In this serum-free medium, the SV3T3 cells fail to form colonies in agarose suspension. However, they can be induced to anchorage-independent colony formation by the growth factors that are additionally required by their untransformed counterparts for proliferation in monolayer culture. The SV3T3 cells are also rendered anchorage-independent for colony formation in serum-free medium by conditioned medium from dense monolayer serum-free SV3T3 cultures. These experiments suggest that it is the cell-substrate interaction that is responsible for the growth factor autonomy of fibronectin-attached transformed cells.     

Inhibition of DNA synthesis in SV3T3 cultures by isolated 3T3 plasma membranes

3T3 plasma membranes were added to subconfluent cultures of SV3T3 cells in the presence of fusogens. If this protocol results in the introduction into the SV3T3 cell membrane of 3T3 plasma membrane components responsible for density-dependent inhibition of growth, then the SV3T3 cell cultures would be expected to show decreased rates of DNA synthesis as they approach confluence. Results of these experiments indicate that rates of DNA synthesis in SV3T3 cultures so treated were as much as 63% less than in untreated controls. This effect could not be attributed to the fusogens or to the 3T3 plasma membranes alone. This growth-inhibitory effect is specific for 3T3 membranes and is not observed when SV3T3 plasma membranes are fused with SV3T3 cell cultures. These data support the hypothesis that one aspect of the loss of density-dependent inhibition of growth in SV3T3 cells is a deletion or alteration in plasma membrane components and, further, that density- dependent inhibition of growth can be in part restored to SV3T3 cell cultures by fusing the cells with 3T3 plasma membranes.     

Efficient immortalization and morphological transformation of human fibroblasts by transfection with SV40 DNA linked to a dominant marker

Immortal cell lines are essential for genetic and biochemical studies. Unlike rodent cells, which will form continuously growing cultures either spontaneously or after infection with an oncogenic virus (e.g., Simian Virus 40 (SV40)), human cells fail to form continuous cell lines spontaneously and in only rare cases from cell lines after oncogenic virus infection. We have used a plasmid (pSV3gpt) containing both the SV40 early region encoding T antigen and the bacterial gene xanthine-guanine phosphoribosyl transferase (gpt) to achieve high efficiency morphological transformation and immortalization of primary human skin fibroblasts. Transfection of this plasmid into primary human skin fibroblasts derived from a normal individual, two Cockayne's syndrome patients, and an immuno-deficient patient and selection for the gpt gene resulted in an altered cell morphology and growth properties characteristic of previously described SV40-transformed cells. Transfected cultures subsequently senesced, entered crisis and in each case formed a rapidly growing culture. The high efficiency of immunortalization described here (four out of four cell strains) is in contrast to previously described procedures utilizing focal overgrowth. We suggest that the use of a dominant selectable marker linked to the SV40 early region increases the probability of establishing an immortal human cell line.     

In Vitro Selection Against Ascochyta Blight of Chickpea (Cicer arietinum L)

Callus cultures established on MS medium containing 2.0 mg l^-1 2, 4-D were inoculated on the regeneration medium supplemented with different concentrations (0.5, 1.0, 1.5, 2.0, 2.5 and 3%, v/v) of culture filtrate (CF) of Ascochyta rabiei infesting chickpea. Out of 486 callus pieces and 270 regenerants obtained from immature embryo derived callus screened, 50 callus lines and 74 regenerants were found resistant. Further, these resistant callus lines and regenerants were subjected to stability test by growing them on a medium containing 3% CF. Seventeen callus lines and 28 regenerants of the selected lines showed normal growth on the selection medium. The regenerated plants were tested in pots under artificial epiphytotic conditions where they showed normal growth behaviour and high degree of resistance.     

In vitro selection for salt tolerance in crop plants: Theoretical and practical considerations

Summary In recent years attempts have been made to supplement traditional breeding for the production of salt-tolerant plants with variability existing in cell culture. The potential causes suggested as an explanation for the limited success of the in vitro approach include: a) lack, or loss during selection, of regeneration capability; b) the development of epigenetically adapted cells; c) lack of correlation between the mechanisms of tolerance operating in cultured cells and mechanisms that operate in cells in the intact plant; and d) multigenicity of salt tolerance. The recent successful production of healthy, fertile, and genetically stable salt-tolerant regenerants from cells obtained from highly morphogenic explants which are selected early in culture (using one-step or short-term strategies) for salt tolerance, together with the demonstration that salt-sensitive plants can become tolerant by mutations in one or few genes, suggest that some of the potential limitations can be overcome and that some of them may not exist at all.     

In vitro selection for methomyl resistance in CMS-T maize

Summary Many plants resistant to methomyl (Lannate), an insecticide which selectively damages maize with the Texas (T) type of cytoplasmic male sterility (CMS-T), were obtained by in vitro selection and also without selection. The selection procedure used 0.6–0.7mM methomyl and callus from CMS-T versions of several field and sweet corn genotypes (W182BN, Wf9, P39, MDM1, SW1 and hybrids of SW1, IL766A1, IL766A2, and 442 with W182BN-N). Addition of 1 mM methomyl to the regeneration medium greatly reduced recovery of methomyl-sensitive escapes. Resistance was linked with reversion to male fertility and maternally inherited. Most progeny of resistant plants exhibited stable maternally inherited resistance for two generations in field tests. First-generation progeny of seven culture-derived plants segregated for resistance and sensitivity; this suggests that ears of these seven regenerants were cytoplasmically chimeral. Resistance to methomyl was associated with resistance to T toxin from Helminthosporium maydis race T and with changes in mitochondrial physiology. Prolonged culture (14–16 months versus 6–8 months) increased the frequency of resistance among both selected and non-selected regenerants. Little or no resistance was found among regenerants from certain genotypes. Selection with methomyl may be useful for production of improved sweet corn lines and as a source of mitochondrial mutants. This system is also convenient for studies of the effects of nuclear background and of culture and selection systems on the generation of cytoplasmic mutants.     

Karyological characterization of sugar beet gynogenetic lines cultured in vitro

Flow cytometry was used to screen ploidy levels in 47 cultured in vitro sugar beet gynogenetic lines of various origin and age, obtained after plant regeneration from unfertilized ovules. When donor plants were diploid, the majority of regenerants were found to have cells with 1C, 2C and 4C relative DNA content (mainly haploid and diploid) and there were large differences in the rate of spontaneous in vitro chromosome doubling between individual homozygous lines. Six ovule-derived lines regenerated from fertile and sterile diploid donors of forty-five lines were solid diploids from the very early stages of their in vitro cultivation, and thus could not be classified as doubled haploids. In the case of tetraploid donor plants, the gynogenetic regenerants demonstrated 2x-ploidy level. The results obtained in chimeric plants with both haploid and diploid cells indicated the possibility to overcome mixoploidy by their re-cultivation through generative shoot tip culture. The flow cytometry method confirmed data obtained by conventional microscopic chromosome counting in dividing leaf cells and was found very useful for screening of a large number of regenerants and for characterizing the process of in vitro gynogenetic lines formation in sugar beet.     

Development of a highly-efficient CHO cell line generation system with engineered SV40E promoter

Chinese hamster ovary (CHO) cells have been one of the most widely used host cells for the manufacture of therapeutic recombinant proteins. An effective and efficient clinical cell line development process, which could quickly identify those rare, high-producing cell lines among a large population of low and non-productive cells, is of considerable interest to speed up biological drug development. In the glutamine synthetase (GS)-CHO expression system, selection of top-producing cell lines is based on controlling the balance between the expression level of GS and the concentration of its specific inhibitor, l-methionine sulfoximine (MSX). The combined amount of GS expressed from plasmids that have been introduced through transfection and the endogenous CHO GS gene determine the stringency and efficiency of selection. Previous studies have shown significant improvement in selection stringency by using GS-knockout CHO cells, which eliminate background GS expression from the endogenous GS gene in CHOK1SV cells. To further improve selection stringency, a series of weakened SV40E promoters have been generated and used to modulate plasmid-based GS expression with the intent of manipulating GS-CHO selection, finely adjusting the balance between GS expression and GS inhibitor (MSX) levels. The reduction of SV40E promoter activities have been confirmed by TaqMan RT-PCR and GFP expression profiling. Significant productivity improvements in both bulk culture and individual clonal cell line have been achieved with the combined use of GS-knockout CHOK1SV cells and weakened SV40E promoters driving GS expression in the current cell line generation process. The selection stringency was significantly increased, as indicated by the shift towards higher distribution of producing-cell populations, even with no MSX added into cell culture medium. The potential applications of weakened SV40E promoter and GS-knockout cells in development of targeted integration and transient CHO expression systems are also discussed.     

Development of a non-lethal selection system by using the aadA marker gene for efficient recovery of transgenic rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The application of aminoglycoside-3-adenyltransferase (aadA) gene-mediated streptomycin resistance for non-lethal selection of transgenic rice resulted in plant regeneration frequencies under selection pressure as high as those in non-transformed controls without selection. Since streptomycin does not kill non-transgenic cells, and allows plant regeneration from them, a selection procedure was developed that made the visual identification of transgenic calli and regenerants possible. For callus-level selection, a vital pH indicator—Chlorophenol Red—was applied together with streptomycin, making use of the phenomenon that fast-growing cell lines lower the pH in the culture medium. Transgenic plants were selected according to their main distinctive features; their green colour (photomixotrophic assimilation), and more intense growth. At the same time, non-transgenic regenerants were bleached (heterotrophic assimilation), and growth was retarded in the presence of streptomycin and sucrose. The final efficiency of genetic transformation based on streptomycin resistance was found to be double that of transformations where the selective agent was l-phosphinothricin, and nearly three times more compared to transformations resulting in hygromycin-resistant regenerants. To the best of our knowledge, this is the first report on producing nuclear transformed rice plants by using a non-lethal selection strategy based on the chimaeric aadA gene.Communicated by D. Dudits     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells IV. Two Different Requirements for Protein Synthesis During Simian Virus 40 Deoxyribonucleic Acid Replication

The replication of simian virus 40 (SV40) deoxyribonucleic acid (DNA) was inhibited by 99% 2 hr after the addition of cycloheximide to SV40-infected primary African green monkey kidney cells. The levels of 25S (replicating) and 21S (mature) SV40 DNA synthesized after cycloheximide treatment were always lower than those observed in an infected untreated control culture. This is consistent with a requirement for a protein(s) or for protein synthesis at the initiation step in SV40 DNA replication. The relative proportion of 25S DNA as compared with 21S viral DNA increased with increasing time after cycloheximide treatment. Removal of cycloheximide from inhibited cultures allowed the recovery of viral DNA synthesis to normal levels within 3 hr. During the recovery period, the ratio of 25S DNA to 21S DNA was 10 times higher than that observed after a 30-min pulse with (3)H-thymidine with an infected untreated control culture. The accumulation of 25S replicating SV40 DNA during cycloheximide inhibition or shortly after its removal is interpreted to mean that a protein(s) or protein synthesis is required to convert the 25S replicating DNA to 21S mature viral DNA. Further evidence of a requirement for protein synthesis in the 25S to 21S conversion was obtained by comparing the rate of this conversion in growing and resting cells. The conversion of 25S DNA to 21S DNA took place at a faster rate in infected growing cells than in infected confluent monolayer cultures. A temperature-sensitive SV40 coat protein mutation (large-plaque SV40) had no effect on the replication of SV40 DNA at the nonpermissive temperature.     

Long-term cultures of chick embryo fibroblasts transformed by the Schmidt-Ruppin strain (D) of Rous sarcoma virus.

Attempts have been made to keep in vitro, for extended periods of time, cultures of chick embryo fibroblasts transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D. Roller cultures of transformed chick cells kept in serum-deficient medium can be maintained without subcultivation for up to 6 months. The confluent cultures continuously release viruses and viable tumor cells into the medium. The released cells can be plated and have characteristics of growth and morphology which are relatively stable with time until the culture degenerates. Cells released at later stages of the culture produced substantially more viruses than those released earlier, suggesting that cell selection or differentiation occurs during long-term cultivation in low serum concentration. Long-term cultures of untransformed chick embryo fibroblasts can also be maintained in the same way. The release of viable cells by these confluent cultures, however, is negligible.     

Application of the salt stress to the protoplast cultures of the carrot (Daucus carota L.) and evaluation of the response of regenerants to soil salinity

This is the first study to generate carrot plants for enhanced salinity tolerance using a single-cell in vitro system. Protoplasts of three carrot accessions were exposed to treatment by seven different concentrations of NaCl (10–400 mM). Salt concentrations higher than 50 mM decreased plating efficiency and those of 200–400 mM of NaCl completely arrested mitotic divisions of cultured cells. The protoplast-derived plants from the control and 50–100 mM NaCl treatment were subjected to an 8-week salt stress in greenhouse conditions induced by salinized soil (EC 3 and 6 mS cm^?1). 50 mM NaCl stress applied in vitro induced polyploidy among regenerated plants. The regenerants obtained from the 50 and 100 mM NaCl-treated protoplast cultures grown in saline soil had a higher survival rate compared to the regenerants from the control cultures. The salt-stressed plants accumulated anthocyanins in petioles and produced denser hairs on leaves and petioles in comparison to the control plants. Salt stress influenced pollen viability and seed setting of obtained regenerants. The results suggest that salt stress applied in vitro in protoplast cultures creates variation which allows alleviating the negative effects of salt stress on the development and reproduction of the carrot.

     

The detection of genome polymorphism in Stachys species using RAPD

Molecular genome analysis was for the first time carried out in the genus Stachys. RAPD analysis proved to be suitable for identifying the species-specific markers, studying the interspecific DNA polymorphism, and detecting the genetic changes that arise during in vitro culturing of Stachys sieboldii. In addition, RAPD was used for screening genetic variation in S. sieboldii regenerants obtained at various phytohormone concentrations. High cytokinin concentrations and multiple regeneration were shown to induce genetic changes detectable with RAPD patterns. High DNA polymorphism was demonstrated for two types of S. sieboldii callus cultures and for plants regenerated from a callus culture.     

The morphology of mesangial cells cultured at high density and in collagen gels

Primary mesangial cells (rat) from monolayer cultures of the 6th to 12th passage and permanent SV40 Mes13 cells were grown at high density in organoid culture at the medium/air interphase. After adaptation to the in vitro conditions, both mesangial cell types developed after 7 days a synthesis apparatus (endoplasmic reticulum, Golgi apparatus) and produced matrix which consisted of Lamina densa-like material, collagenous fibrils and filaments. Unspecific contacts, gap junctions and adhesion belts could be demonstrated in the contact areas. Additionally, some cells exhibited thick bundles of actin filaments. A close resemblance of the mesangial cells in high density culture to those in vivo can, therefore, be stated. Hence, they differentiated with regard to their matrix formation, contraction and contact behaviour and can therefore be used for experimental studies within a short culture period of 7 days. Cell aggregates in monolayer culture and in cultures in collagen gels had not differentiated at this stage.     

Function of small hydrophobic proteins of paramyxovirus

Mumps virus (MuV), a rubulavirus of the paramyxovirus family, causes acute infections in humans. MuV has seven genes including a small hydrophobic (SH) gene, which encodes a type I membrane protein of 57 amino acid residues. The function of the SH protein is not clear, although its expression is not necessary for growth of MuV in tissue culture cells. It is speculated that MuV SH plays a role in viral pathogenesis. Simian virus 5 (SV5), a closely related rubulavirus, encodes a 44-amino-acid-residue SH protein. Recombinant SV5 lacking the SH gene (rSV5DeltaSH) is viable and has no growth defect in tissue culture cells. However, rSV5DeltaSH induces apoptosis in tissue culture cells and is attenuated in vivo. Neutralizing antibodies against tumor necrosis factor alpha (TNF-alpha) and TNF-alpha receptor 1 block rSV5DeltaSH-induced apoptosis, suggesting that SV5 SH plays an essential role in blocking the TNF-alpha-mediated apoptosis pathway. Because MuV is closely related to SV5, we hypothesize that the SH protein of MuV has a function similar to that of SV5, even though there is no sequence homology between them. To test this hypothesis and to study the function of MuV SH, we have replaced the open reading frame (ORF) of SV5 SH with the ORF of MuV SH in a SV5 genome background. The recombinant SV5 (rSV5DeltaSH+MuV-SH) was analyzed in comparison with SV5. It was found that rSV5DeltaSH+MuV-SH was viable and behaved like wild-type SV5, suggesting that MuV SH has a function similar to that of SV5 SH. Furthermore, both ectopically expressed SV5 SH and MuV SH blocked activation of NF-kappaB by TNF-alpha in a reporter gene assay, suggesting that both SH proteins can inhibit TNF-alpha signaling.