Incorporation of glucosamine-3H into extracellular gamma-globulin by Ehrlich ascites tumor cells.

The incorporation of radioactivity from tritiated glucosamine into glycoproteins was studied in Strong A male mice, with and without Ehrlich ascites tumor. The proteins studied were mouse plasma, ascites fluid, and the extracellular fluid from in vitro incubations of Ehrlich ascites tumor cells. Of particular interest in the in vivo experiments were the relatively high specific radioactivity of the γ-globulin fraction of the ascites fluid as compared to mouse plasma and that the incorporation of glucosamine-³H into the γ-globulin fraction was greatest at 90 hr following inoculation of the tumor. At 66 hr of tumor growth, the radioactivity of the ascites γ-globulin fraction was slightly greater than that of the plasma γ fraction, but it increased to 15 times that of the plasma fraction at 90 hr. Although the specific activity of the ascites γ fraction had decreased markedly by 168 hr of tumor growth, it was still four times that of the plasma fraction. A similar pattern of radioactivity incorporation was observed when Ehrlich ascites tumor cells were incubated with tritiated glucosamine in vitro. The specific activity of the γ-globulin fraction of the extracellular fluid, following incubation with Ehrlich ascites tumor cells obtained from 90 hr of tumor growth, was more than twice that observed with cells from 66 or 168 hr of tumor growth.     

The effect of gossypol and Lonidamine on electron transport in Ehrlich ascites tumor mitochondria

The effect of the association of gossypol and Lonidamine on the electron transport in Ehrlich ascites tumor mitochondria has been investigated by addition of drugs to isolated mitochondria. The results may be summarized as follows. (1) Low concentrations of gossypol increase the rate of oxygen consumption at the level of three energy-conserving sites of the respiratory chain. Higher concentrations result in an inhibition of oxygen consumption at (or near) both energy-conserving sites 1 and 2, while energy-conserving site 3 is unaffected. (2) Gossypol, at concentrations at which it exerts its uncoupling effect, stimulates ATPase activity. Higher concentrations inhibit the enzyme activity. (3) The addition of gossypol to mitochondria respiring on pyruvate plus malate or succinate induces a more oxidized state of NAD+ and cytochrome b, respectively. (4) Gossypol enhances the effect of Lonidamine on oxygen consumption. Lonidamine does not affect state 4 respiration, but in the presence of gossypol, it determines a marked decrease in the rate of oxygen consumption. The inhibition of oxidation of NAD-linked substrates is greater than that of FAD-linked substrates. (5) It may be concluded that gossypol is very effective in potentiating the effect of Lonidamine. Moreover, it may be suggested that the antitumor activity of Lonidamine is enhanced if it is used in combination with other drugs and/or treatments, such as hyperthermia, which modify the energy status of mitochondria.     

Interferon action: transcriptional control of a gene specifying a 56,000-Da protein in Ehrlich ascites tumor cells

The exposure of cells to interferons enhances the accumulation of particular mRNAs and of the corresponding proteins. A cDNA clone (clone 202) complementary to an mRNA (202 mRNA) whose level is enhanced over 12-fold in mouse Ehrlich ascites tumor cells upon exposure to beta-interferon for 10 hr has previously been isolated. The level of this mRNA was also increased in other beta-interferon-responsive mouse cell lines (i.e., L929, L1210S) but not in a line (L1210R) which is not responsive to beta-interferon. The extent of induction in Ehrlich ascites tumor cells depended on the beta-interferon concentration and reached its maximal level between 300 and 1000 units of interferon/ml. Nuclei isolated from Ehrlich ascites tumor cells which had been exposed to beta-interferon produced in vitro more 202 specific RNA than nuclei from control Ehrlich ascites tumor cells: an increase in this production was detectable 2 hr after beginning the exposure of the cells to 1000 units/ml of beta-interferon and the increase reached its maximal level, around 18-fold, after 18 hr exposure. Much, if not all of this increase, appeared to be due to an increase in the rate of synthesis of the RNA and not to a decrease in its rate of turnover. The 202 mRNA was translated in a reticulocyte lysate into a 56,000-Da protein.     

State of viral DNA in BK virus-transformed rabbit cells

Semipermissive rabbit liver, brain, and kidney cells were transformed by BK virus (BKV). All cells of the three resulting cell lines produced BKV T antigen. Viral DNA was detected by DNA-DNA reassociation kinetics and by blot-transfer hybridization. Hybridization patterns were different for the three lines, indicating a different mode of integration for each line. In addition to integrated viral DNA, two of the lines contained also free viral DNA sequences, which in one case were defective viral genomes. Absence or splitting of particular regions of the viral genome was not a necessary condition for the maintenance of the transformed state. HindIII-generated segment A, which contains all the sequences coding for the late viral proteins, was absent (in an intact form) in the only line from which virus could not be rescued.     

Replication of vaccinia DNA in mouse L cells. IV. Protein synthesis and viral DNA replication

The requirement for protein synthesis during vaccinia DNA replication in mouse L cells was investigated. Within the first 30 min after reversal of a hydroxyurea (HU) block, viral DNA replication was not affected in cells treated with cycloheximide (100 μg/ml) to inhibit protein synthesis. During this period the intermediates in DNA replication detected, the rate of chain elongation, and the accumulation of crosslinked viral DNA molecules were all identical to those observed in vaccinia-infected cells not treated with cycloheximide. Thereafter, DNA replication, as measured by incorporation of [³H]thymidine, was inhibited in cycloheximide-treated infected cells (>90%, 2 hr post-HU reversal). Inhibition of viral DNA synthesis was further demonstrated by the sparse appearance and failure of cytoplasmic viral factories to increase in size after HU reversal, when protein synthesis was inhibitied. Density labeling of replicating viral DNA molecules with bromodeoxyuridine and analysis of equilibrium density centrifugation in CsCl showed that hybrid moelcules (hl, ? = 1.77 g/ml) accumulated in cycloheximide-treated cells. The hybrid molecules were not converted to “heavy” viral DNA (hh, ? = 1.825 g/ml), as was observed to occur during viral DNA replication in cells continuously synthesizing protein. The results of these experiments showed that after an initial round of viral DNA replication was completed, new protein synthesis was required to initiate additional rounds of viral DNA replication. The dissociation of viral DNA molecules, synthesized after HU reversal, from cytoplasmic DNA complexes was inhibited by cycloheximide but not rifampin. Continuous protein synthesis, apparently to permit expression of a “late” viral function, not related to viral assembly is required for release of the newly replicated viral genomes from complexes.     

The oncogenicity of avian adenoviruses. III. In situ DNA hybridization of tumor line cells localized a large number of a virocellular sequence in few chromosomes

The arrangement of the viral DNA in a CELO virus (an avian adenovirus)-induced rat tumor cell line was analyzed at the molecular and the cellular level. Blot hybridization of restriction endonuclease-digested tumor cell DNA and quantitative hybridization of nondigested tumor cell DNA with ³²P-labeled restriction endonuclease-generated fragments of the viral DNA indicate the following: (1) A contiguous portion of the viral DNA comprising 33% of the viral DNA molecules [i.e., 14.1 kilobases (kb)] is flanked at each terminus by a cellular DNA sequence. This viral DNA sequence, together with >6.1 kb of one of the flanking cellular DNA sequences and >6.8 kb of the other, forms repetition unit (total length >27kb). (2) During the evolution of the original tumor, this unit has attained as many as 160 copies per diploid amount of cellular DNA. In situ DNA hybridization of the cultured tumor cells with ³H-labeled viral DNA indicates that most of the repetition units are constituents of chromosomal DNA and their distribution is limited to only a few chromosomes per hypotetraploid tumor cell. The majority of chromosomes carrying a large number of repetition units are abnormal chromosomes longer than rat chromosome No. 1.     

Structural changes in the DNA of Marek's disease virus during serial passage in cultured cells

Two virulent strains, BC-1 and JM, of Marek's disease virus (MDV) were serially passaged in cultured chick embryo fibroblasts. The purified viral DNAs at different passage levels were compared by electrophoresis on 0.5% agarose gels of the products obtained by restriction endonuclease digestion. Although the patterns of the fragments of the DNAs at different passage levels were very similar, some fragments were lost during serial passage. These fragments were not found in the restriction endonuclease digestion products of two strains, C2(A) and GA(A), of attenuated, avirulent MDV. The data indicated that serial passage of virulent MDV strains resulted in the loss of the virulent strain-specific DNA fragments. These fragments could be responsible for the virulence of MDV.     

DNA replication in synchronized cultured mammalian cells. V. The temporal order of synthesis of component alpha DNA during monkey DNA synthesis induced by SV40 virus.

Purified simian virus 40 (SV40) was used to induce host DNA replication in contact-inhibited monolayer cultures of African green monkey kidney cells. Approximately 20% of the nuclear DNA of these cells is the simple sequence, component α DNA (Maio, 1971). At the beginning of DNA synthesis induced by SV40 viral infection, the average ratio of the specific radioactivity of component α DNA to that of bulk DNA was 0.18. Similar low specific activity ratios are observed early during the DNA synthetic period following the trypsin release of monkey cells from contact inhibition. These results indicate that when host DNA replication is induced by SV40 viral infection, component α DNA synthesis does not shift to precede the replication of bulk DNA.     

Inhibition of SV40 DNA synthesis by vesicular stomatitis virus in doubly infected monkey kidney cells

Vesicular stomatitis virus (VSV) inhibited SV40 DNA synthesis in doubly infected synchronized Vero cells. Gel-electrophoretic profiles demonstrated that SV40 DNA monomers accumulated in all stages of supercoiling, regardless of whether cells were superinfected with VSV early or late in the S phase. These gel profiles were indistinguishable from ones obtained from SV40-infected, cycloheximide-treated cells in the absence of VSV infection. Radiolabel in the partial supercoils could be chased into supercoils, but only by restoring protein synthesis. The relative rates of SV40 DNA chain elongation were determined in VSV-superinfected and nonsuperinfected cells. The gradients of 3H incorporation as a function of distance from the origin of replication in pulse-labeled form I DNA were unaffected by VSV. It is concluded that VSV inhibition of SV40 DNA synthesis is an indirect result of inhibition of host cell protein synthesis and it is suggested that incompletely supercoiled SV40 chromatin is not a good template for DNA synthesis.     

Common surface receptors on both mouse and rat cells distinguish different classes of mouse mammary tumor viruses

Cell surface receptors which bind mouse mammary tumor virus (MMTV) were detected on mouse and rat cells. Virus binding was quantitated by measuring ¹²⁵T-protein A binding to immune complexes composed of a C3H MMTV gp52 type-specific monoclonal antibody and receptor-bound MMTV. C3H MMTV binding to normal mouse mammary epithelial cells (NMuMG) was dose dependent and was ≥50% inhibited by GR MMTV, but not by endogenous C3Hf MMTV or Gross murine leukemia virus. These results were confirmed in [³H]leucine MMTV binding inhibition assays in which GR MMTV and C3H MMTV blocked C3H [³H]MMTV binding while C3Hf MMTV did not block. The affinities of C3H [³H]MMTV binding to receptors on NMuMG, NIH Swiss (SLP), and Fischer rat embryo (FRE) cells were identical by Scatchard analysis. C3Hf [³H]MMTV also bound these cells, but with an affinity approximately 10 times weaker than the C3H [³H]MMTV binding. Interference assays using a Kirsten sarcoma virus (C3H MMTV) pseudotype confirmed the importance of C3H MMTV specific binding to viral penetration and expression. Pretreatment of SLP or FRE cells with 100 μg of C3H MMTV or GR MMTV inhibited focus formation by ≥50% while C3Hf MMTV and RIII MMTV did not inhibit. Therefore, the functional C3H MMTV cell receptors on mouse and rat cells were related and were able to distinguish C3H MMTV and GR MMTV from C3Hf MMTV and RIII MMTV. These comparable receptors may represent evolutionarily conserved surface components of murine cells.     

Inhibition by acyclovir of cell growth and DNA synthesis of cells biochemically transformed with herpesvirus genetic information

Thymidine kinase-deficient LM cells (LMTK^?-) biochemically transformed to the TK⁺ phenotype with herpes simplex virus genetic information showed an increased uptake of and ability to phosphorylate the acyclic nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acyclovir, acycloguanosine, acyclo-Guo). In growth inhibition studies the TK⁺ transformants were much more sensitive to inhibition with acyclovir than the untransformed cells (13- to 90-fold more sensitive). The synthesis of DNA in the transformed cells was significantly reduced by acyclovir treatment, whereas acyclovir had little effect on the DNA synthesis of the untransformed cells. Alkaline sucrose gradient sedimentation analysis of cellular DNA synthesized in the presence of acyclovir showed that, in contrast to untreated untransformed cells, the DNA newly synthesized by transformed cells was considerably smaller in size. In pulse-chase experiments the small fragments of DNA synthesized in the presence of acyclo-Guo were not chased to high molecular weight DNA. Finally, acyclo-Guo was shown to be incorporated terminally at 3′-ends of growing DNA chains in replicating cells.     

Investigation of vaccinia virus DNA replication employing a conditional lethal mutant defective in DNA

After infection of L cells with the DNA-defective temperature-sensitive (ts) mutant 6389 of vaccinia virus, [3H]thymidine incorporation into cytoplasmic DNA is inhibited at 39 degrees, but resumes upon shiftdown to 32 degrees, the permissive temperature. Following a 30-min lag period DNA synthesis is linear and contingent upon continuous protein synthesis. Sedimentation analysis of nascent DNA labeled during 10 to 60-min pulses revealed that the mutant molecules are produced at a slower rate, but are approximately the same size as those of wild-type vaccinia, synthesized under the same circumstances. During more prolonged incubation beyond 60 min, labeled DNA molecules sedimenting more rapidly than mature, full-length virus genomes are observed. The integration of mutant DNA into mature virions is less rapid than that of the wide-type DNA. Upon extraction from the virosomes, the ts6389 DNA sediments as both genome-size and larger, faster sedimenting DNA. Upon treatment with restriction endonucleases, the ts6389 virosomal DNA exhibited an additional fragment after separation on agarose gels, perhaps as a consequence of fusion between the terminal fragments of the molecule. Taken together these observations suggest that concatemeric intermediates are formed during vaccinia DNA replication. By measuring the radioactivity incorporated into the fragments and subfragments of the molecules labeled during the first round of replication, the initiation site of replication can be localized to a region within the terminal 150 bp.     

Asymmetric replication of hepatitis B virus DNA in human liver: demonstration of cytoplasmic minus-strand DNA by blot analyses and in situ hybridization

In situ and blot hybridization techniques have been used with strand- and region-specific probes to characterize the forms of hepatitis B virus (HBV) DNA in the liver of a patient with chronic active hepatitis B. The hepatocytes contain a heterogeneous population of rapidly migrating DNA species in the 0.5-1.4 kb position that are localized predominantly in the cytoplasm and are of minus-strand polarity. The findings indicate that the replication is asymmetric, with separate pathways for plus- and minus-strand synthesis of HBV DNA; that viral DNA synthesis is initiated at a site near the nick in the minus strand of virion DNA; and that actively replicating forms of HBV DNA can be identified at the cellular level by in situ hybridization.     

Murine cytomegalovirus DNA synthesis in nuclear monolayers.

Nuclear monolayers of murine cytomegalovirus (MCMV)-infected 3T3 cells were examined for their ability to synthesize viral DNA. Infected S-phase and Go-phase nuclei incorporated more ³H-dTMP than did their uninfected counterparts, although the kinetics of incorporation were similar. Synthesis continued for at least 60 min at 37°. The majority of the viral DNA product (from S-phase nuclei) had a molecular weight of approximately 17 × 10⁶, about one-eighth genome size. Infected nuclei contained a new DNA-polymerase activity, which was stimulated by ammonium sulfate and specifically inhibited by antibodies to infected cell proteins. Although infected Go-phase nuclei also contained this new enzyme activity, they could not synthesize viral DNA, thus reflecting their inability to replicate MCMV-DNA in whole cells.