Expression of two human growth hormone genes in monkey cells infected by simian virus 40 recombinants.

We have constructed simian virus 40 recombinants carrying two different human growth hormone (hGH) genes. Monkey kidney cells infected with these recombinants synthesize, process, and secrete hGH. The product of gene 1, which has coding sequences identical to those of a cloned hGH complementary DNA, is indistinguishable from pituitary hGH by several criteria. The product of gene 2, which is predicted to encode a variant protein, is less immunoreactive than pituitary hGH but binds efficiently to hGH cell surface receptors. These results show that gene 2 has the potential to be expressed into a previously unidentified form of hGH. They also demonstrate that it is possible to produce a mature hormone by gene transfer in eukaryotic cells and indicate the utility of the simian virus 40-monkey cell system for producing and characterizing secreted animal cell proteins.     

Reversible growth arrest in simian virus 40-transformed human fibroblasts.

A reversible growth arrest of simian virus 40-transformed human fibroblasts has been produced by replacement of methionine in the growth medium by its immediate metabolic precursor, homocysteine, Although these arrested cells exhibit a greatly reduced cloning efficiency when plated in methionine-supplemented medium, they resume rapid proliferation without a lag when subconfluent cells are refed with methionine-supplemented medium. This growth arrest is accompanied by a reduction in the percentage of mitotic cells in the cell population. Furthermore, data obtained using fluorescence-activated cell sorting techniques indicate that the cells are arrested i the S and G2 phases of the cell cycle. This is in contrast to a G1-phase accumulation of cells, which occurs only in methionine-supplemented medium at very high densities and which is similar to the G1 block seen in cultures of normal fibroblasts at high density. The apparent relationship between specific events in the DNA-synthetic and premitotic phase of the cell cycle and methionine dependence in these transformed cultures is discussed.     

Transformation of human skeletal muscle cells by simian virus 40.

Molecular studies of the biochemical alterations involved in human myopathies have been restricted because of the finite life-span and slow growth rate of cultures derived from primary tissue. Because the tumor virus simian virus 40 (SV40) can alter both the growth properties and longevity of human cells, we have infected skeletal muscle cultures derived from four biopsies with a small-plaque variant of SV40 and analyzed the biological and biochemical properties of cloned myoblast derivatives. At early times after infection, myoblasts fused normally into multinucleated myotubes, and both unfused and fused cells contained SV40 tumor antigen (T antigen). After six to eight subcultures after infection, the ability of myoblasts to fuse diminished, and clonal cell lines were generated with increased growth rates and saturation densities. Transformed cultures also lost contact inhibition of growth and became anchorage independent. Unlike untransformed myoblasts, SV40-transformed clones did not undergo an increase in creatine kinase activity or a transition of creatine kinase isoenzymes from the BB form to the muscle-specific MM form. Analysis of the pattern of SV40 DNA integration by Southern blotting hybridization analysis in two cloned SV40-transformed myoblast cell lines (KJ-SV40 and PK-SV40) indicated that KJ-SV40 contained at least one site of SV40 DNA integration into chromosomal DNA and PK-SV40 contained at least three sites of SV40 DNA covalently linked to cellular DNA. Cell lysates and growth medium from PK-SV40 transformants contained infectious small-plaque variant SV40, whereas KJ-SV40 did not contain or produce detectable virus. These studies demonstrate that human myoblasts can be immortalized by SV40. This procedure may prove useful for generating large quantities of genetically deficient human cells for biochemical and molecular analysis.     

Purification of simian virus 40 tumor antigen from a line of simian virus 40-transformed human cells.

Simian virus 40 tumor antigen can be isolated in a highly purified state from the nuclei ofSV80 cells, a continuous line of simian virus 40-transformed human fibroblasts. A five-step purification method was used. Its apparent molecular weight (in sodium dodecyl sulfate/polyacrylamide gels) is approximately 90,000-94,000. It contains a detectable amino-terminal residue.     

Fate of viral DNA in nonpermissive cells infected with simian virus 40.

Mouse cells are nonpermissive for simian virus 40 (SV40); replication of viral DNA is undetectable and progeny virions are not produced. Infection leads instead to the establishment of stably transformed cell lines in which viral DNA is covalently integrated into cellular DNA. We have followed the fate of SV40 DNA in infected mouse cells to define steps in viral DNA metabolism that precede integration. A novel high molecular weight form of SV40 DNA is synthesized shortly after infection by a process sensitive to the inhibition of DNA replication. This DNA represents polymers in which viral genomes are organized as tandem "head-to-tail" arrays. Recombination can be demonstrated with mutant viruses, but the recombination frequency is not high enough to account for the synthesis of polymers by recombination between infecting genomes. We conclude that polymers are synthesized by DNA replication and that they then recombine with one another. We believe that the polymers also recombine with cellular DNA and are thus the precursor to integrated viral DNA. Such a model accounts directly for the high frequency of tandemly duplicated viral insertions in transformed cells and also leads to experimentally testable predictions.     

Episomal simian virus 40 genomes in human brain tumors.

Eight out of 35 human intracranial tumors were shown by restriction enzyme analysis to contain unintegrated simian virus 40 (SV40) DNA molecules. The relative amount of viral DNA was estimated to be the equivalent of one viral genome within every 10th to 20th cell. No infectious virus was detected in tissue cultures established from the tumors. From only one tumor was it possible to rescue, by cell fusion, infectious SV40 displaying wild-type properties. In those cases that permitted a more detailed analysis, the restriction enzyme cleavage patterns appeared to correspond to the wild-type patterns with one exception, in which the SV40 episomes displayed a deletion of approximately 70 base pairs close to the origin of DNA replication. From one tumor, the SV40 genomes were transferred into permissive CV-1 monkey cells by transfection with the total tumor DNA. Despite their persistence as episomes no infectious virus was produced. Furthermore, no viral antigens were detectable, although the SV40 messengers for the small and the large tumor antigens were present. These cells had, however, acquired the ability to form colonies in low concentrations of serum. Thus this report provides, by restriction enzyme analysis, direct evidence for the presence of SV40 DNA in human tumors.     

Expression of the transformation-sensitive protein "cyclin" in normal human epidermal basal cells and simian virus 40-transformed keratinocytes.

A cell population highly enriched in human epidermal basal cells has been obtained and characterized by using antibodies specific for various cell types in the epidermis. Quantitative two-dimensional gel electrophoretic analysis (isoelectric focusing) of [35S]methionine-labeled polypeptides from basal cells and simian virus 40-transformed keratinocytes showed that the basal cells synthesize very low amounts (less than 0.02% of the total protein) of the nuclear, transformation-sensitive protein cyclin as compared to the transformed cells, which synthesize this protein constitutively (0.15% of the total protein). Very low levels of cyclin were observed in total human epidermis, and preliminary studies of two basaliomas have shown a significant synthesis of this protein in these tumors. Immunofluorescence studies using antibodies to proliferating cell nuclear antigen that immunoprecipitate cyclin confirmed the above observations at least in the case of the cultured cells. Taken together, these results support the notion that cyclin may be a central component of the pathway(s) that controls cell proliferation.     

Isolation of renin-producing human cells by transfection with three simian virus 40 mutants.

A human juxtaglomerular cell (JGC) tumor was used for the immortalization of renin-secreting cells. The transfection of primary JGC with three different simian virus 40 (SV40) mutants resulted in the continuous production of renin-secreting cells. The most efficient renin-producing cells (producing about 400 pg of renin per 24 hr per ml of culture medium) were those transfected with the PAS SV40 mutant. The renin production was stable and the cell cultures have been maintained for greater than 1 year. Two types of cells were cultured together and could not be separated: round and birefringent cells, which exhibited features of mast cells, and elongated cells containing myofilaments and secretory granules. Immunocytochemical staining showed the presence of renin in this latter cell type. The renin produced by the transfected cells was not stored within the cells but was released rapidly into the medium. More than 95% of the renin produced was prorenin, which, after activation, had characteristics similar to those of pure human standard renin as to its enzymatic, immunologic, and biochemical properties, except that it was less glycosylated. These stable JGC tumoral cell lines provide a unique system for studying human renin biosynthesis and its regulation in vitro.     

Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40.

To facilitate understanding of the mechanisms underlying pulmonary diseases, including lung cancer and cystic fibrosis, we have transformed and characterized cultures of human tracheal epithelial cells. Cells were transfected by calcium phosphate precipitation with a plasmid containing a replication-defective simian virus 40 (SV40) genome. Colonies of cells with enhanced growth potential were isolated and analyzed for transformation- and epithelial-specific characteristics. Precrisis cells were observed to express the SV40 large tumor antigen, produce cytokeratins, have microvilli, and form tight junctions. After crisis, cells continued to express the SV40 large tumor antigen as well as epithelial-specific cytokeratins and to display the apical membrane microvilli. Apical membrane Cl channels were opened in postcrisis cells exposed to 50 microM forskolin. These channels showed electrical properties similar to those observed in primary cultures. The postcrisis cells have been in culture for greater than 250 generations and are potentially "immortal." In addition to providing a useful in vitro model for the study of ion transport by human airway epithelial cells, the cells can be used to examine stages of neoplastic progression.     

Assignment of the integration site for simian virus 40 to chromosome 17 in GM54VA, a human cell line transformed by simian virus 40.

GM54VA human cells transformed by simian virus 40 (SV40) were fused with peritoneal macrophages obtained from three different mouse strains. All 27 hybrid clones studied were positive for SV40 tumor antigen in 100% of their cells and contained human chromosome 17. Human chromosome 17 was the only human chromosome present in five of the hybrid clones. Fusion of GM54VA cells and either thymidine kinase (EC 2.7.1.75)-deficient mouse or Chinese hamster fibroblasts resulted in the growth in hypoxanthine-aminopterin-thymidine medium of hybrid clones positive and negative for SV40 tumor antigen. Counterselection of the hybrid clones positive for tumor antigen in medium containing 5-bromodeoxyuridine resulted in the growth of hybrid cells that were negative for tumor antigen. These experiments indicate that negative for tumor antigen. These experiments indicate that SV40 is integrated in only one of the two parental human chromosomes 17. Because the genome of SV40 has been assigned to human chromosome 7 in two other SV40-transformed human cell lines, at least two different integration sites for SV40 would seem to be present in human cells: one located in human chromosome 7 and the other located in human chromosome 17.     

Presence of simian virus 40 DNA sequences in human lymphomas

Simian virus 40 (SV40)--a potent oncogenic virus--has been associated previously with some types of human tumours, but not with lymphomas. We examined human tumours for the presence of specific SV40 DNA sequences by PCR and Southern blotting. Viral sequences were present in 29 (43%) of 68 non-Hodgkin lymphomas, and in three (9%) of 31 of Hodgkin's lymphomas. Viral sequences were detected at low frequencies (about 5%) in 235 epithelial tumours of adult and paediatric origin, and were absent in 40 control tissues. Our data suggest that SV40 might be a cofactor in the pathogenesis of non-Hodgkin lymphomas.     

Methylated simian virus 40-specific RNA from nuclei and cytoplasm of infected BSC-1 cells.

Host cell and virus-specific poly(A)-containing RNAs isolated from nuclei and cytoplasm of monkey kidney cells infected with simian virus 40 contain different methylated nucleotides. In the cytoplasmic simian virus 40-specific RNA, about 75% of the radioactivity derived from (methyl-3-H)methionine was in N-6-methyladenosine (N-6mA) after digestion with Penicillium nuclease and bacterial alkaline phosphatase. The remainder was in a negatively charge component with properties of 5'-terminal structures, i.e., digestion with nucleotide pyrophosphatase and bacterial alkaline phosphatase released 2'-O-methyladenosine (A-m), 2'-O-methylguanosine (G-m), and 7-methylguanosine (m-7-G), consistent with a 5'-terminal structure of the type, m7-GpppNm. The nuclear virus-specific RNA contained N6mA, GM, 2'-O-methyluridine (U-m), and a smaller proportion (10%) of nuclease-, phosphatase-resistant presumptive 5' termini that also yielded A-m, G-m, and m7-G upon further hydrolysis. The infected cell nuclear and cytoplasmic RNAs that did not hybridize to DNA of simian virus 40 contained all four 2'-O-methylnucleosides. The possible role of methylation in the processing and translation of simian virus 40-specific mRNA is discussed.     

Release of interleukin-6 by human thyroid epithelial cells immortalized by simian virus 40 DNA transfection.

Interleukin-6 (IL-6) has actions on a variety of endocrine tissues. The cytokine is secreted by cells of the anterior pituitary and endocrine pancreas and has recently been shown to be produced by cultures of thyroid epithelial cells. In this study we have examined some of the factors which regulate IL-6 release from an immortalized human thyroid line (HTori3). IL-6 release over 24 h was stimulated by TSH (5000 microU/ml), by forskolin (0.01 mmol/l), by fetal calf serum (1-20%) and by epidermal growth factor (20 ng/ml). Stimulation was also apparent with gamma-interferon and with tumour necrosis factor at concentrations known to enhance class II major histocompatibility antigen expression by thyroid epithelium. The most potent factor tested was interleukin-1 (IL-1), which controls IL-6 release from other cell types. Threefold stimulation was found with 1 U/ml rising to 350-fold with 1000 U/ml. The effect of IL-1 took 2 h to develop and was blocked by cycloheximide (100 mumol/l). Stimulation was not markedly inhibited by pertussis toxin. Many of the actions of IL-1 are mediated by prostaglandin E2 (PGE2). At concentrations as low as 30 nmol/l, PGE2 stimulated IL-6 release but the maximum stimulation obtained with PGE2 was only threefold. The effect of IL-1 was not inhibited by indomethacin. These data provide further evidence that IL-6 is produced by human thyrocytes. The effect of IL-1 has not been demonstrated previously. Stimulation of IL-6 release by IL-1 did not appear to be mediated by prostaglandin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficient introduction of plasmid DNA into human hemopoietic cells by encapsidation in simian virus 40 pseudovirions.

Introduction of DNA into human hemopoietic cells is required for the study of regulatory mechanisms operating in these cells, as well as for possible procedures of gene therapy. However, with hemopoietic cells the conventional technique of calcium phosphate precipitation is inefficient. The pathway of encapsidation of plasmid DNA as simian virus 40 (SV40) pseudovirions for the introduction of new genetic material was therefore investigated. Encapsidation was achieved in COS (monkey kidney) cells, which express SV40 large tumor (T) antigen constitutively. The vector, pSO, was introduced to the COS cells by DNA transfection. It carried the SV40 origin of replication (ori), to facilitate replication of the plasmid in the COS cells. The SV40 capsid proteins were supplied in trans by a helper SV40 virus. The bacterial chloramphenicol acetyltransferase gene cat was used as a model for gene transmission. After encapsidation, the pseudovirions were used in infection of the human erythroleukemic cell line K562 and of normal human bone marrow cells. The results demonstrate that the cat gene can be transmitted with high efficiency. Over 40% of the infected K562 cells and 30% of the infected bone marrow cells were observed to contain plasmid DNA 48 hr after infection. Moreover, the results suggest that the efficiency of gene transmission by this vector can be improved and so may approach the theoretical 100%.