Ultrastructural and cytochemical features of SV 40 transformed hypothalamic cell lines

Summary A continuous cell line was previously obtained by Simian Virus (SV) 40 transformation of primary cultures of dissociated mouse fetal hypothalami. One clone from this cell line has been previously shown to possess some of the ultrastructural features, immunological properties and synthesizing capacities of magnocellular hypothalamic neurons which secrete vasopressin and neurophysins. The present paper reports on the morphological characterization of 14 other clones or subclones of the original cell line, using the following criteria: phase contrast microscopy, electron microscopy, Gomori's aldehyde fuchsin staining, cytochemical detection of -glucuronidase, immunochemical staining with antisera against bovine neurophysin I, bovine neurophysin II, lys-vasopressin, oxytocin, LH-RH and TRH.The results allowed the conclusion that the clones as well as the subclones can be distributed into two groups: 1) neurosecretory neurons which all possess several of the ultrastructural and cytochemical features of the neurophysin-vasopressin synthesizing clone, and 2) primitive nerve cells which are devoid of such features but display numerous bundles of filaments. In addition some clones were found to display intermediate features between groups 1 and 2. A similar diversity was observed within clones of the original strain and subclones of a neurosecretory clone. It is suggested that the primitive clones could represent precursors of the neurosecretory clones.This work is dedicated to Professor W. Bargmann in honour of his 70th birthday     

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.     

Properties of a macrophage cell line transformed by simian virus 40. Morphological changes related to cell functions

A mouse macrophage clone (line nH-1) transformed by simian virus 40 (SV40) was examined by electron microscopy. In the growing phase of the cultures, NH-1 cells were non-phagocytic and SV40 T antigen-positive, and contained a large number of filament sheaths within their pseudopodia. In the late stationary phase, they became phagocytic, SV40 T antigen-negative and contained a filamentous network within their psudopodia. In addition, NH-1 cells in the late stationary phase were very similar to normal macrophages in other morphological properties.     

Characterization of a simian virus 40-transformed Fanconi anemia fibroblast cell line

We have characterized a SV40-transformed human fibroblast cell line (GM6914) derived from a patient with Fanconi anemia (FA) in order to establish its usefulness for biochemical and genetic experiments, including DNA-mediated gene transfer. GM6914 cells have a growth rate similar to that of SV40-transformed normal human fibroblasts and an indefinite lifespan in culture. As has been established for other FA cell types, GM6914 cells have an increased sensitivity to DNA-crosslinking agents such as mitomycin C (MMC). The D10 for GM6914 cells is 8 times lower than for equivalent controls. GM6914 cells also have an elevated frequency of spontaneous chromosome aberrations and this frequency can be increased by MMC concentrations which show no effect on control cells. Genetic complementation studies with lymphoblasts derived from two affected sibs of the donor of GM6914 cells show that GM6914 belongs to FA complementation group A. In DNA-transfection studies using plasmid pRSVneo, colonies of GM6914 cells resistant to the drug G-418 were observed at frequencies ranging from 1.7 to 16 X 10(-4), values similar to those observed with several other SV40-transformed human cell lines. GM6914 should be a useful recipient cell line in experiments using DNA-mediated gene transfer to clone the normal allele of the gene which is defective in FA complementation group A. GM6914 would also be an excellent cell line for studies on mutagenesis, recombination and repair using plasmid vectors.     

Establishment and characterization of a permanent pSV ori--transformed ataxia-telangiectasia cell line

A permanent ataxia-telangiectasia (A-T) cell line has been established from the fibroblast strain AT2SF after transfection with the bacterial plasmid pSV ori-, which contains replication origin-defective SV40 sequences. The original transfection frequency, as measured by transformed foci, was markedly reduced in two A-T strains when compared with either normal or xeroderma pigmentosum fibroblasts. As with SV40 virion-transformed fibroblasts, pSV ori--transformed cells entered a crisis phase, from which about one-fourth of the original clones from A-T and normal fibroblasts recovered. Both the pSV ori--transformed TAT2SF cell line and an SV40 virion-transformed AT5BI (GM5489) cell line retained their characteristic sensitivity to the lethal effects of ionizing radiation, as well as their X ray-resistant DNA synthesis. Southern blot analysis of cellular SV40 sequences demonstrated a single major integration site of pSV ori- in the AT2SF cells. In contrast, AT5BI cells transformed with SV40 virions demonstrated a high degree of heterogeneity of integrated viral sequences. Neither the TAT2SF nor the GM5489 transformed cell line contains any detectable freely replicating SV40 viral sequences, which are seen in many other semipermissive SV40-transformed cells.     

Spontaneous rearrangement of integrated simian virus 40 DNA in nine transformed rodent cell lines.

Frequencies of spontaneous DNA rearrangement within or near integrated simian virus 40 (SV40) DNA were measured in four transformed mouse and rat cell lines of independent origin and in five clones of the SV40-transformed mouse line SVT2. Rearrangements were detected as polymorphisms of restriction enzyme fragment length in subclones of the lines. At least 17% of the subclones of each line had detectable rearrangements. The rate of rearrangement was calculated to be at least 5 x 10(-3) events per cell per division. No rearrangements were detected in sequences of an immunoglobulin gene, part of the coding region of the mouse protein p53, and five proto-oncogenes. The possible role of recombination between duplicated segments of integrated SV40 DNA in generating rearrangements was studied in the five SVT2 clones, which differed in the number of duplications within a single SV40 DNA segment. The SVT2 clone that had no duplications, M3, became rearranged further at least as frequently as did closely related lines with one, two, or three duplications. Another line in this group that had one small duplication, X1, had a much higher frequency of rearrangement than did the others; integrated SV40 DNA of X1 became mostly rearranged within 100 cell divisions. The examples of M3 and X1 suggested that the high rate of rearrangement characteristic of integrated SV40 DNA was influenced more by the presence of particular sequences within or near integrated SV40 DNA than by the number or extent of duplicated sequences.     

Events preceding stable integration of SV40 genomes in a human cell line

We have examined the organization of integrated SV40 sequences in an uncloned population of a transformed human fibroblast cell line. Somatic cell hybrids between mouse B82 cells and human GM847 cells were examined for SV40 T-antigen expression and individual human chromosome presence. This analysis revealed that a functional SV40 genome is located on human chromosome 7. Restriction endonuclease digestion followed by blot hybridization of the parental human cell line revealed that it contains multiple normal and defective SV40 copies integrated into the host genome in tandem. A similar analysis of several T-ag+ hybrid cell lines indicated that the integrated viral sequences in different hybrid cell lines (thus in different cells of the original population) are very closely related but not always identical. Analysis of subclones of GM847 also revealed such differences. Based upon these results, we postulate that following the initial integration event, viral as well as the flanking host DNA sequences become unstable and are subject to deletions and rearrangements. This short-lived structural instability is followed by highly stable integration of SV40 which is maintained in these cells or their hybrid derivatives for at least hundreds of cell generations.     

Finite life span of hybrids formed by fusion of different simian virus 40-immortalized human cell lines.

Simian virus 40 (SV40) genes are able to induce immortalization of normal human cells after a culture crisis during which unknown cellular genetic changes presumably occur. To determine whether these genetic changes are always identical, we performed somatic cell hybridization analysis of an SV40-immortalized human bronchial epithelial cell line, BET-1A. Fusion of BET-1A with an SV40-immortalized fibroblast cell line resulted in hybrids that senesced, indicating that these cell lines are in different complementation groups for immortalization.     

Cell surface antigens coded for by the human chromosome 7

Human-mouse somatic cell hybrids, containing chromosome 7 from an SV40-transformed human cell line as the only human chromosome, were injected into the same inbred strain of mouse as the mouse parental cell, and the humoral immune response assayed. A cell-surface antigen(s) coded for by the chromosome 7 common to all human fibroblastic cell lines tested and also found on African green monkey kidney cell lines was demonstrated. No reactivity to SV40-induced TSTA was detected.Abbreviations used in this paper are as follows SV40 simian virus 40 - MEM minimal essential medium - FBS fetal bovine serum - FITC fluorescein isothiocyanate - C121 53-87 (1) clone 21 - Cl36 53-87-3 clone 36 - 52-62 52-62 (1) clone 5 subclone 9 - MSV murine sarcoma virus - T tumor - TSTA tumor-specific transplantation antigen - RIA radioimmunoassay - PBS phosphate-buffered saline - PBS⁺ PBS with sodium azide and FBS - IgG immunoglobulin - cpm counts per minute     

Karyology and tumorigenicity of a simian virus 40-transformed Chinese hamster cell clone.

A pseudodiploid clone of chinese hamster cells tranformed in vitro with Simian virus 40 (SV40) was isolated from soft agar and injected into nude mice through three successive passages with a short in vitro cultivation between each animal inoculation. the original clone and the three subsequent tumor populations were characterized in regard to SV40 T antigen staining, modal chromosome number, and Giemsa-banded karyotype. All tumor cell lines maintained the pseudodiploid mode, as well as the positive sv40 t antigen staining. Nonrandom chromosomal changes included loss of one of the X chromosomes, additions of abnormal variants of chromosomes No. 1 and No. 2, the appearance of unidentified marker chromosomes, and the loss of autosomes No. 5, No. 6, and No. 11. The deletion of one of the X chromosomes occurred with about the same frequency in all cell lines. Additions of abnormal chromosomes No. 1 and No. 2 tended to recur more often in the tumor cell lines than in the original clone. the appearance of marker chromosomes, as well as the loss of autosomes No. 5, No. 6, and No. 11 demonstrated a correlation with tumorigenicity. Yet, the three successive passages of the cells through the animal did not select for a tumor population with a single, homogeneous karyotype.     

Ultrastructural and biological characterization of human choroid cell cultures transformed by Simian Virus 40

A human diploid cell line of choroid origin was isolated from the retrouveal portion of an enucleated eye and designated HC. After 10 passages, when the proliferative capacity of HC cells decreased, they were infected and transformed by Simian Virus 40 (SV40). A proliferating long-term cultured cell line designated HC/SV40 was established and it has been maintained as monolayer for more than 100 passages so far. The two cell lines, HC and HC/SV40, were compared for growth characteristics, capacity to form colonies in soft agar, presence of nuclear T-antigen, and ultrastructure. Cytogenetic analysis was also performed to determine the presence of chromosomal aberrations due to the permanent viral transformation of the cell line. The results indicate that HC/SV40 should be considered the transformed counterparts of HC cells because they are morphologically similar to the latter but can grow in soft agar, possess T-antigen, and show a pattern of karyotypic changes similar to that induced by SV40 in human fibroblasts. The choroid origin of HC and HC/SV40 cell lines was confirmed by the presence, in their cytoplasm, of typical electron dense granules. Their neural origin will make these cell lines very useful for neuropharmacological and differentiation studies.     

Ultrastructural and biological characterization of human choroid cell cultures transformed by Simian Virus 40

Summary A human diploid cell line of choroid origin was isolated from the retrouveal portion of an enucleated eye and designated HC. After 10 passages, when the proliferative capacity of HC cells decreased, they were infected and transformed by Simian Virus 40 (SV40). A proliferating long-term cultured cell line designated HC/SV40 was established and it has been maintained as monolayer for more than 100 passages so far. The two cell lines, HC and HC/SV40, were compared for growth characteristics, capacity to form colonies in soft agar, presence of nuclear T-antigen, and ultrastructure. Cytogenetic analysis was also performed to determine the presence of chromosomal aberrations due to the permanent viral transformation of the cell line. The results indicate that HC/SV40 should be considered the transformed counterparts of HC cells because they are morphologically similar to the latter but can grow in soft agar, possess T-antigen, and show a pattern of karyotypic changes similar to that induced by SV40 in human fibroblasts. The choroid origin of HC and HC/SV40 cell lines was confirmed by the presence, in their cytoplasm, of typical electron dense granules. Their neural origin will make these cell lines very useful for neuropharmacological and differentiation studies. This work was supported by Grant 82.00346.96 from the C.N.R. finalized project “Control of Neoplastic Growth”.     

Genomic rearrangements in a mouse cell line containing integrated SV40 DNA

In the SV40-transformed mouse embryo fibroblast cell line SVT2/S, genomic rearrangements involving the SV40 DNA and flanking host sequences were identified by Southern blot hybridization using viral DNA as probe. No rearrangements of SV40 DNA integrated into nonpermissive mouse cells have been previously described. The standard arrangement found in the majority of subclones was mapped with 20 restriction enzymes, 10 of which cleave sites within the SV40 DNA. A single copy of a defective integrated viral genome is present, in which the late region is missing from about nucleotide 200 clockwise to about nucleotide 1750. The rest of the viral genome including the origin of replication and T antigen binding region is present and colinear with SV40 DNA, except for an internal repeat of about 1750 bp located between nucleotides 2750 and 4500. Rearrangements were found in 4 out of 20 random subclones of the parental SVT2/S cell line and 3 of the 4 continued to rearrange. The thioguanine-resistant cell line 281-1-4, derived from SVT2/S, remained stable on subculture but a chloramphenicol-resistant mutant, 107-6-4, derived from 281-1-4, was highly unstable. In 107-6-4, unique rearrangements were found in 6 of 31 subclones of a population that had undergone abut 25 doublings from a single-cell isolate. The high rate of rearrangement and the sporadic expression of rearrangement potential are characteristic of the transposable controlling elements discovered by McClintock.     

Establishment of a functional ovine fetoplacental artery endothelial cell line with a prolonged life span

To study mechanisms governing fetoplacental vascular function, we have established a primary ovine fetoplacental artery endothelial (OFPAE) cell line. These OFPAE cells produce nitric oxide (NO), proliferate, and migrate in response to fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF). To overcome the senescence crisis that this primary OFPAE cell line will eventually enter, we attempted to establish a functional OFPAE cell line with a prolonged life span by transfecting cells with plasmids containing a neomycin resistance gene and a simian virus 40 gene (SV40) expressing large T (T) and small t (t) antigens. The OFPAE cells at passage 8 were transfected. After neomycin selection, the surviving OFPAE (designated SV40 OFPAE) cells were expanded up to passage 80. Up to passage 30, these SV40 OFPAE cells maintained a morphology similar to untransfected OFPAE cells. Expression of T and t antigens in SV40 OFPAE cells was confirmed by immunocytochemistry. These SV40 OFPAE cells exhibited positive uptake of acetylated low-density lipoprotein (Ac-LDL) and positive staining for NO synthase 3 (NOS3) and formed capillary-like tube structures on Matrigel. Up to passages 20-23, these SV40 OFPAE cells proliferated (P < 0.05) and produced (P < 0.05) NO in response to both FGF2 and VEGF. Moreover, this cell proliferation stimulated by FGF2 and VEGF was dose-dependently inhibited (P < 0.05) by PD98059 (a selective mitogen-activated protein kinase 1 and 2 [MAP2K1/2, also termed MEK1/2] inhibitor) or by LY294002 (a selective phosphoinositide 3-kinase [PI3K] inhibitor). These data indicate that SV40 OFPAE cells, at least at passage 23, retain endothelial phenotypes and functions similar to their parental, untransfected OFPAE cells. Thus, a functional OFPAE cell line with an extended life span has been successfully established, potentially providing a valuable cell model for studying fetoplacental endothelial function.     

Isolation and characterization of a cloned growth factor dependent macrophage cell line, BAC1.2F5

The SV40 transformed murine macrophage cell line, BAC1, proliferates in response to the colony stimulating factor, CSF-1 (Schwarzbaum et al., J. Immunol., 132:1158, 1984). In order to obtain a cell line suitable for biochemical and genetic studies of CSF-1 signal transduction, clones of BAC1 were established. Clones ranged from being completely autonomous to being completely dependent on CSF-1 for growth. Cells of one clone (2F5), which proliferated in response to either CSF-1 or granulocyte-macrophage CSF (GM-CSF) were characterized in detail. The kinetics of receptor-mediated internalization and intracellular destruction of CSF-1 were comparable to the kinetics observed with peritoneal exudate macrophages. CSF-1 was shown to regulate cell spreading, cell survival, protein degradation, and the duration of the G1 and S phases of the cell cycle. The 2F5 clone therefore exhibits a number of CSF-1 stimulated responses and is being used for genetic and biochemical studies of CSF-1 action.     

Expression of transformation in cell hybrids. I. Isolation and application of density-inhibited Balb/3T3 cells deficient in hypoxanthine phosphoribosyltransferase and resistant to ouabain.

A cell line, THO2, was isolated from Balb/3T3 clone A31 after sequential nitrosoguanidine treatments and selection for resistance to 6-thioguanine and ouabain. THO2 retains the properties of density-dependent inhibition of growth and serum dependence of DNA synthesis characteristic of 3T3. The codominant expression of ouabain resistance and inability of THO2 to utilize exogenous hypoxanthine in the presence of aminopterin allows isolation of somatic cell hybrids involving THO2 and any ouabain-sensitive, hypoxanthine phosphoribosyltransferase-positive cell line. Hybrid clones derived from THO2 and SV40-transformed cells show dominant expression of the transformed phenotype with respect to multilayered arrangement of cells and ability to synthesize DNA in 1% calf-serum medium.     

Establishment of a SV40-transformed cell line from primary culture of rat dorsolateral prostatic epithelial cells

A permanent cell line of the rat dorsolateral prostate epithelium was established after transformation of primary cultured cells with simian virus 40 (SV40). The established cells had SV40 T-antigen in their nuclei but lacked such typical characteristics of transformed cells as piled-up growth. They grew in a monolayer with an epithelial morphology, were stained with antikeratin antisera, and also retained an ability to form dome-like structure at a confluent state.