Adenovirus types 2 and 5 functions elicit replication and late expression of adenovirus type 12 DNA in hamster cells.

A recombinant plasmid harboring both genomic termini of tupaia herpesvirus (THV) DNA was characterized by restriction enzyme analysis and by determination of the nucleotide sequence. A unique NotI cleavage site was found that is located approximately 19 base pairs upstream of the THV terminal junction. THV DNA fragments from virion DNA were analyzed by using the same restriction enzymes as for the recombinant plasmid. The comparative fine mapping of virion THV DNA revealed heterogeneous molecules of variable lengths with the NotI cleavage site conserved. A number of short direct and inverted repeats and palindromes were found surrounding the THV terminal joint. The THV repetitive sequences were compared with the repeats reported for the DNA termini of herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus and are discussed in respect to signals for a site-specific endonuclease required for packaging.     

Arrangement of integrated viral DNA sequences in cells transformed by adenovirus types 2 and 5.

The organization of the viral DNA sequences in 15 adenovirus-transformed cell lines was analyzed by the Southern blotting procedure. The site of adenovirus integration in the cellular genome was found not to be unique, and the viral DNA sequences involved in integration were not confined to a specific region of the adenovirus genome. Several cell lines showed simple integration patterns that demonstrated the presence of large continuous stretches of viral DNA. In four cell lines, containing sequences from both molecular ends of the viral genome, the left- and right-hand-terminal sequences appeared to be linked to each other.     

Stimulation of cellular DNA synthesis by human cytomegalovirus

Human cytomegalovirus (CMV) is able to induce cellular DNA synthesis in both permissive (human embryonic lung) and nonpermissive (Vero) cells. The induction of cell DNA synthesis was assayed by the incorporation of [methyl-³H]thymidine into macromolecules having the buoyant density characteristics of cell DNA. The DNA synthesis induced by CMV infection appears to represent normal semiconservative replication as opposed to repair synthesis. Both strains of CMV tested were capable of inducing cell DNA synthesis. Virus exposed to heat or UV light prior to infection lost the ability to induce DNA synthesis, indicating that a virus-coded function expressed after infection is responsible for stimulation of cell DNA synthesis.     

Complementation of human adenovirus type 5 ts mutants by human adenovirus type 12.

Temperature-sensitive mutants of type 5 adenovirus belonging to eight complementation groups were complemented in mixed infection by type 12 adenovirus, whereas mutants of 7 other groups were not enhanced. In some crosses, phenotypic mixing took place. No evidence of recombination between type 5 ts mutants and type 12 was found.     

Stimulation of DNA synthesis in cultured primary human mesothelial cells by specific growth factors

Monolayer cultures of human mesothelial cells made quiescent by serum deprivation are induced to undergo one round of DNA synthesis by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor type beta 1 (TGF-beta 1). This one-time stimulation is independent of other serum components. The kinetics for induction of DNA synthesis observed for PDGF, EGF, and TGF-beta 1 are all similar to one another, with a peak of DNA synthesis occurring 24-36 h after the addition of the growth factors. Repetitive rounds of DNA synthesis and cell division do not ensue after addition of PDGF, EGF, or TGF-beta 1 alone or in combination; however, in media supplemented with chemically denatured serum, each of these factors is capable of sustaining continuous replication of mesothelial cells. Stimulation of growth by PDGF and TGF-beta 1 is unusual for an epithelial cell type, and indicates that mesothelial cells have growth regulatory properties similar to connective tissue cells.     

Stimulation of DNA synthesis in human fibroblasts by thrombin.

Earlier work showed that thrombin stimulates proliferation of human fibroblasts in serumfree medium. This work demonstrates (1) that thrombin has to be prensent during most or all of the G1 period to ensure maximal DNA synthesis, (2) that DNA synthesis increases about three hours later after thrombin than after serum treatment, (3) that both thrombin and serum activate transport of uridine, D--2-deoxy-glucose and putrescine, (4) that thrombin is able to increase 3H-thymidine incorporation also in SV40 transformed human fibroblasts, in HeLa cells and in two continuous monkey cell lines.     

Construction of restriction enzyme fragment libraries containing DNA from human adenovirus types 2 and 5

Restriction-fragment libraries containing adenovirus type 2 (Ad2) DNA have been constructed, using the pBR322 plasmid (Bolivar et al., 1977) as a vector. Clones have been isolated which contain all the HindIII fragments of Ad2 DNA except the terminal G- and K-fragments inserted into the HindIII cleavage site of the vector. All the 13 SmaI-fragments of Ad2 DNA were separately inserted into the PstI site of the pBR322 vector after addition of homopolymeric poly(dG) tails to the fragments and poly(dC) tails to the linearized plasmid. Two large fragments of adenovirus type 5 (AD5) DNA, located between map positions 17.0 and 59.5 and between map positions 59.5 and 97.3, respectively, were cloned using bacteriophage lambda as a vector. All clones, which are described in the present report, are available upon request.     

Recombination in hamster cell nuclear extracts between adenovirus type 12 DNA and two hamster preinsertion sequences.

A cell-free system of nuclear extracts from BHK21 cells has been developed to catalyse recombination in vitro between the DNA of adenovirus type 12 (Ad12) and two different hamster preinsertion sequences. The pBR322 cloned 1768 bp fragment p7 and the 3.1 kbp fragment p16 from BHK21 hamster DNA had previously been identified as the preinsertion sites corresponding to the junctions between Ad12 DNA and hamster DNA in cell line CLAC1 and in the Ad12-induced tumour T1111(2), respectively. Preinsertion sequences, which had recombined previously with foreign (Ad12) DNA, might again be recognized by the recombination system even in a cell-free system. PstI cleaved Ad12 DNA and the circular or the EcoRI linearized p7 or p16 preinsertion sequences were incubated with nuclear extracts. Recombinants were isolated by transfecting the DNA into recA- Escherichia coli strains and by screening for Ad12 DNA-positive colonies. Without a selectable eukaryotic marker, all Ad12 DNA positive recombinants were registered. Out of a total of greater than 90 p7-Ad12 DNA recombinants, 21 were studied by restriction-hybridization, and four by partial nucleotide sequence analyses. Among the p16-Ad12 DNA recombinants, four were analysed. The sites of linkage between Ad12 DNA and p7 or p16 hamster DNA were all different and distinct from the original CLAC1 or T1111(2) junction site between Ad12 and hamster DNA. The in vitro recombinants were not generated by simple end-to-end joining of the DNA fragments used in the reaction but by genetic exchange. Thirteen of the 25 recombinants were derived from the 61-71 map unit fragment of Ad12 DNA. Recombination experiments between Ad12 DNA and four randomly selected unique or repetitive hamster DNA sequences of 1.5-6.2 kbp in length did not yield recombinants. Apparently, the p7 and p16 hamster preinsertion sequences recombined with Ad12 DNA with a certain preference.     

Cloned fragments of human adenovirus type-12 DNA

The following restriction endonuclease fragments of human adenovirus type 12 (Ad12) DNA have been cloned in plasmid or bacteriophage lambda vectors using standard protocols: the EcoRI-A*, -B, -D, -E, and -F fragments, the BamHI-B, -C, -D, -F, -G, -H, and -I fragments, the HindIII-F and -I fragments, and the PstI-A, -D, -F, -G, and -H fragments. The EcoRI-A* fragment comprises the right terminal 5 kb of Ad12 DNA including the terminal 143 bp.     

Stimulation of DNA synthesis in senescent human cells following incubation with plasma membranes

DNA synthesis and mitosis were increased in mitogen-stimulated senescent WI-38 cells following incubation with plasma membranes prepared from young or senescent WI-38 cells, A431 cells, 3T3 cells, or NR6 cells. The percentage of [3H]thymidine-labeled nuclei in senescent cultures was two- to fivefold greater than that seen in controls in which cells were incubated in the absence of membranes or in the presence of boiled membranes. The effect was trypsin sensitive, suggesting that a protein moiety is necessary for stimulation of DNA synthesis. As the culture age increased, basal levels of DNA synthesis, as well as maximal stimulation of DNA synthesis following incubation with plasma membranes, decreased. These observations are consistent with the hypothesis that different subpopulations exist in senescing cultures and suggest a complex pattern of inhibitory and stimulatory regulation of cell proliferation.     

Stimulation of DNA synthesis and myo-inositol incorporation in mammalian cells

Phosphatidylinositol (PI) synthesis and its role in controlling the cell cycle has been investigated using fibroblasts and liver cells in culture. PI synthesis as measured by incorporation of [³H]-myo-inositol into trichloroacetic acid precipitable material during 0–60 min after serum or growth factor stimulation of serum-starved cells is increased in primary fetal rat liver cells, rat embryo fibroblasts, and 3T3 mouse cells. In contrast, growth stimulation of 3T3 cells and hepatocytes rendered quiescent in G₁ by amino acid starvation is not accompanied by increased incorporation of [³H]-myo-inositol into trichloroacetic acid precipitable material. This suggests that those cells might be arrested at a different point in G₁ than cells arrested by serum depletion. Inhibition of PI synthesis by δ-hexachlorocyclohexane (HCH), a steric analog of myo-inositol, during early times (e.g., 0–4 hr) after growth stimulation, reversibly blocks initiation of DNA synthesis in 3T3 cells. The results support the idea that increased PI synthesis in response to growth stimulation in the cell types studied here is a prerequisite for progression through G₁ and subsequent entry into S phase.     

Effects of bradykinin on DNA synthesis in resting NIL8 hamster cells and human fibroblasts

Bradykinin stimulates [3H]thymidine incorporation and DNA synthesis in resting, serum-deprived NIL8 hamster cells. The ED50 for this stimulation is 4.52 +/- 2.91 nM. Other kinin peptides including lys-bradykinin (kallidin) and met-lys-bradykinin also stimulate [3H]thymidine incorporation in the NIL8 cells, whereas desarg9-bradykinin is without effect, suggesting action of the kinin peptides through type B2 receptors. Bradykinin also stimulates DNA synthesis in IMR-90 human fibroblasts; however, this effect is observed only in the presence of indomethacin, which blocks prostaglandin synthesis. These results suggest that prostaglandins act as negative modulators of the growth-stimulatory effects of bradykinin in the fibroblasts. This conclusion is supported by the observation that exogenously added PGE1, PGE2, PGA1, PGA2, PGB1, and PGB2 strongly inhibit [3H]thymidine incorporation in the human fibroblasts. The direct effect of bradykinin observed in the NIL8 cells may be attributable to the relative resistance of these cells to growth inhibition by prostaglandins.     

Patterns of integration of viral DNA in adenovirus type 2-transformed hamster cells

The patterns of integration of viral DNA in five lines of adenovirus type 2-transformed hamster cells have been investigated. Cell lines HE1 to HE5 were obtained by in vitro transformation of hamster embryo cells by ultraviolet light-inactivated Ad2². In all lines, segments in the central parts of the viral genome are missing. The lines HE1, HE2, HE3, HE4 and HE5 contain 2 to 4, 2 to 4, 6 to 10, about 10, and 2 to 3 genome fragment equivalents per cell, respectively.The patterns of integration in lines HE2 and HE3 are identical; however, the viral genome has been amplified in these cell lines to different extents. This result provides evidence for the post-integrational amplification of inserted viral genomes. It is also conceivable that line HE2 may have undergone losses of integrated Ad2 genomes. The persisting Ad2 genomes in lines HE2 and HE3 have deletions in parts of the EcoRI F and D fragments. The remainders of these fragments are linked to cellular DNA. The termini of the segments of the viral genome have been inverted and linked to each other. This linkage could have occurred via a circular intermediate in integration or via tandemly integrated viral genomes with subsequent deletion events. The linkage of the termini of viral DNA might be mediated by short sequences of cellular DNA.In line HE5, approximately 40% of the Ad2 genome is deleted, and the truncated segments, again comprising the terminal Ad2 DNA fragments, have been fused. The termini of the viral DNA are linked to cellular DNA. In lines HE1 and HE4 complex deletion and fusion events have altered the inserted Ad2 genomes.     

Stimulation of DNA synthesis in human epidermis by UVB radiation and its inhibition by difluoromethylornithine

The purpose of this study was to determine whether the rate of DNA synthesis in human skin could be increased by UVB radiation and to determine the potential for reversing the stimulatory effects of UVB radiation by alpha-difluoromethylornithine (DFMO). Split-thickness facial skin was grafted onto athymic CD-1 Nu/Nu mice on the anterolateral dorsal surface. Following graft healing for 6 weeks, grafts were treated with 0%, 2%, or 5% DFMO (a potent inhibitor of polyamine biosynthesis) and subsequently irradiated with 0.15 J/cm2 of UVB light. Two days after UVB exposure, [3H]thymidine was injected and the grafts were dissected and counted. Ultraviolet radiation significantly increased thymidine incorporation, indicating increased DNA synthesis. The stimulatory effects of UV radiation were significantly reduced by topical application of 5% DFMO. Thus administration of DFMO most likely decreased the polyamine level and decreased the rate of DNA synthesis, which may have caused a decreased rate of epidermal proliferation. Thus the topical application of DFMO may prove beneficial for UVB exposure and other hyperproliferative states where a decrease in the rate of cell turnover might be desirable.     

Transformation of rat cells by cyt mutants of adenovirus type 12 and mutants of adenovirus type 5.

Several mutants with much reduced oncogenicity (spontaneous mutants H12 cyt 52 and H12 cyt 70 and UV-induced mutants H12 cyt 61, H12 cyt 62, and H12 cyt 68) of the highly oncogenic adenovirus type 12 (Ad12) were studied for their ability to transform primary baby rat kidney cells. Four of the mutants showed much reduced capacity to transform cells in vitro, while H12 cyt 61 transformed cells as efficiently as the wild-type virus. Viral gene expression in several cell lines established from cultures infected by cyt mutants was studied, and it was found that viral sequences belonging to the left 16% of Ad12 were always transcribed. These results suggest that the function of the transformed state is not defective in the cyt mutants studied. Heterotypic complementation studies showed that the defect(s) in a cyt mutant can be corrected by an Ad7 function. Ad5 dl 313, with a deletion between 3.5 and 10.5 map units, transformed rat cells only at high multiplicity. These results suggest that the region E1B of adenoviruses may be required for efficient transformation of rat cells.