Damaging action of photodynamic treatment in combination with hyperthermia on transmembrane transport in murine L929 fibroblasts

Photodynamic treatment of murine L929 fibroblasts with hematoporphyrin derivative caused inhibition of the 2-aminoisobutyric acid transport system. This was reflected by an increase in the apparent Km with a constant Vmax, indicating impairment of the carrier function rather than a decrease of the number of transport sites. Hyperthermic treatment of these cells resulted in a moderate decrease of the activity of the 2-aminoisobutyric acid transport system. Overall protein synthesis was severely inhibited both by photodynamic treatment and by hyperthermia. Hyperthermia subsequent to photodynamic treatment resulted in an additive inhibition of 2-aminoisobutyric acid transport and of protein synthesis. After photodynamic treatment both 2-aminoisobutyric acid transport and protein synthesis were repaired. The repair of 2-aminoisobutyric acid transport depended on protein synthesis, as shown by the virtually complete blockage of repair by anisomycin. After hyperthermia (either alone or subsequent to photodynamic treatment), no recovery of 2-aminoisobutyric acid transport was observed, although protein synthesis was restored to the initial level. Apparently, hyperthermia subsequent to photodynamic treatment blocks the repair of photodynamically induced damage of this transport system. The experimental results further indicate that protein synthesis is not the rate-determining step for the repair of 2-aminoisobutyric acid transport, although it is necessary in this process. Cell survival was decreased both by photodynamic treatment and by hyperthermia. The combined effects of these two treatments were additive. It is discussed that these results indicate that photodynamic inhibition of 2-aminoisobutyric acid transport is not causally related to loss of clonogenicity, contrary to earlier suggestions.     

Inhibition of the initiation of leukemic transcription by N-trifluoroacetyladriamycin-14-O-hemiadipate in vitro. Impaired formation of RNA polymerase-DNA complex

The effect of N-trifluoroacetyladriamycin-14-O-hemiadipate (AD 143), a new derivative of adriamycin, on various steps of the enzymic reaction catalyzed by chicken myeloblastosis RNA polymerase II was studied. AD 143 inhibition of RNA synthesis, which was evident at the beginning of the reaction, could not be reversed by increasing the concentrations of any one of the four nucleoside triphosphate substrates of the reaction. Furthermore, the RNA synthesis inhibition was not affected by varying the concentrations of template DNA. The AD 143-induced inhibition caused a reduction of the frequency of RNA chain initiation, whereas the average chain length of RNA synthesized at the end of the reaction remained unaltered. The susceptible step in the initiation process was found to be the formation of stable complexes between RNA polymerase and the DNA template. While AD 143 causes no inhibition of Escherichia coli RNA polymerase activity, it was found not to affect the E. coli RNA polymerase-template DNA complex formation.     

Preferential uptake of cytotoxic porphyrins from hematoporphyrin derivative in murine L929 fibroblasts and Chinese hamster ovary K1 epithelial cells

Photodynamically induced loss of clonogenicity of murine L929 fibroblasts and Chinese hamster ovary K1 epithelial cells was determined with two different assays. It appeared that the loss of clonogenicity was much higher when 20 cells/cm2 were incubated with hematoporphyrin derivative (HPD) and illuminated, than when confluent cell layers were incubated with the same amount of HPD and illuminated prior to plating out. This dependency of cell killing on the experimental protocol was also observed when protoporphyrin (90-95% pure) was used as photosensitizer, but not when the cells were photodynamically treated with rose bengal or exposed to mitomycin C. Further, when cell layers were incubated with the residual solution that remained after the previous incubation of a confluent cell layer with HPD, illumination of these layers appeared to be almost non-toxic, although the overall porphyrin concentration in the residual solution was only slightly lower than in HPD. These results indicate that the porphyrins, responsible for loss of clonogenicity, are present in relatively small amounts in HPD and unpurified protoporphyrin and are preferentially taken up by the cells. Although 2-aminoisobutyric acid transport and DNA synthesis are among the most photosensitive targets with HPD, photodynamic treatment of L929 cells with the residual solution did not result in inhibition of the transport system and DNA synthesis. In contrast, the K+ content of the cells still decreased considerably, when utilizing the porphyrins, remaining in the residual solution as sensitizer. This indicates that under the present experimental conditions the disturbance of the membrane barrier function does not contribute to loss of clonogenicity of these cells and, moreover, that the photodynamically induced K+ leakage is caused by a component of HPD other than inhibition of 2-aminoisobutyric acid transport and DNA synthesis.     

Influence of Protein and Ribonucleic Acid Synthesis on the Replication of the Bacteriocinogenic Factor Clo DF13 in Escherichia coli Cells and Minicells

The influence of ribonucleic acid (RNA) and protein synthesis on the replication of the cloacinogenic factor Clo DF13 was studied in Escherichia coli cells and minicells. In chromosomeless minicells harboring the Clo DF13 factor, Clo DF13 deoxyribonucleic acid (DNA) synthesis is slightly stimulated after inhibition of protein synthesis by chloramphenicol or puromycin and continues for more than 8 h. When minicells were treated with rifampin, a specific inhibitor of DNA-dependent RNA polymerase, Clo DF13 RNA and DNA synthesis appeared to stop abruptly. In cells, the Clo DF13 factor continues to replicate during treatment with chloramphenicol long after chromosomal DNA synthesis ceases. When rifampin was included during chloramphenicol treatment of cells, synthesis of Clo DF13 plasmid DNA was blocked completely. Isolated, supercoiled Clo DF13 DNA, synthesized in cells or minicells in the presence of chloramphenicol, appeared to be sensitive to ribonuclease and alkali treatment. These treatments convert a relatively large portion of the covalently closed Clo DF13 DNA to the open circular form, whereas supercoiled Clo DF13 DNA, isolated from non-chloramphenicol-treated cells or minicells, is not significantly affected by these treatments. These results indicate that RNA synthesis and specifically Clo DF13 RNA synthesis are involved in Clo DF13 DNA replication and that the covalently closed Clo DF13 DNA, synthesized in the presence of chloramphenicol, contains one or more RNA sequences. De novo synthesis of chromosomal and Clo DF13-specific proteins is not required for the replication of the Clo DF13 factor. Supercoiled Clo DF13 DNA, isolated from a polA107 (Clo DF13) strain which lacks the 5' --> 3' exonucleolytic activity of DNA polymerase I, is insensitive to ribonuclease or alkali treatment, indicating that in this mutant the RNA sequences are still removed from the RNA-DNA hybrid.     

Effect of aflatoxin B1 on chromatin-bound ribonucleic acid polymerase and nucleic acid and protein synthesis in germinating maize seeds.

The treatment of germinating maize seeds (cv. Ganga 2) with aflatoxin B1 resulted in suppression of ribonucleic acid (RNA), protein, and deoxyribonucleic acid (DNA) synthesis at 3, 4, and 5 h, respectively. At or below the concentrations inhibitory for these in vivo syntheses, the toxin inhibited chromatin-bound DNA-dependent RNA polymerase activity. The synthesis of both polyadenylated and non-polyadenylated RNA was inhibited, but the effect on the former was more pronounced. Equilibrium dialysis and difference spectral and viscometric analyses showed a binding of aflatoxin B1 to DNA isolated from the seeds. It is proposed that the inhibition of RNA synthesis in maize seeds by the toxin is due to the interference with the RNA polymerase activity, which seems, at least partially, due to the impairment of DNA template functions.     

Effect of aflatoxin B1 on chromatin-bound ribonucleic acid polymerase and nucleic acid and protein synthesis in germinating maize seeds

The treatment of germinating maize seeds (cv. Ganga 2) with aflatoxin B1 resulted in suppression of ribonucleic acid (RNA), protein, and deoxyribonucleic acid (DNA) synthesis at 3, 4, and 5 h, respectively. At or below the concentrations inhibitory for these in vivo syntheses, the toxin inhibited chromatin-bound DNA-dependent RNA polymerase activity. The synthesis of both polyadenylated and non-polyadenylated RNA was inhibited, but the effect on the former was more pronounced. Equilibrium dialysis and difference spectral and viscometric analyses showed a binding of aflatoxin B1 to DNA isolated from the seeds. It is proposed that the inhibition of RNA synthesis in maize seeds by the toxin is due to the interference with the RNA polymerase activity, which seems, at least partially, due to the impairment of DNA template functions.     

Selective inhibition of tRNATyr transcription by guanosine 3'-diphosphate 5'-diphosphate.

Guanosine 3'-diphosphate 5'-diphosphate (ppGpp) selectively reduces the synthesis of su+III tRNA from omega 80 psu+III DNA relative to the synthesis of omega 80 RNA in a system in vitro containing DNA and Escherichia coli RNA polymerase holoenzyme as the sole macromolecular components. The response of su+III tRNA synthesis to increasing salt and to temperature in the presence of ppGpp suggests that the nucleotide may reduce the affinity of the enzyme for su+III promoters. The Ki for the selective inhibition of tRNA synthesis by ppGpp is 4 muM in contrast to the value of 150 muM for the inhibition of rRNA synthesis.     

Studies on the control of ribosomal RNA synthesis in HeLa cells.

In many eucaryotic systems protein synthesis is coupled to ribosomal RNA synthesis such that shut-down of the former causes inhibition of the latter. We have investigated this stringency phenomenon in HeLa cells. The protein synthesis inhibitors cycloheximide and puromycin cause inactivation of both processes but valine starvation totally inhibits only the processing of 45-S RNA. DNA-dependent RNA polymerases from A, B and C (or I, II and III respectively) were extracted, separated partially by DEAE-cellulose chromatography and their activity levels determined. These do not decrease significantly during inhibition of protein synthesis. To find out whether or not form A is bound to its template under these conditions, proteins were removed from chromatin with the detergent sarkosyl. This does not affect bound RNA polymerase. Inhibition of protein synthesis caused up to 50% reduction in endogenous alpha-amanitin-insensitive chromatin-RNA-synthesising activity. This reduced level of activity was not affected by sarkosyl treatment. Levels in normal cells were stimulated. This result indicates that the form A RNA polymerase is not bound to its template when protein synthesis is inhibited.     

Inhibition of chicken myeloblastosis RNA polymerase II activity by adriamycin.

In vitro RNA synthesis by isolated RNA polymerase II of chicken myeloblastosis cells was shown to be highly sensitive to adriamycin inhibition. The template activity of the single-stranded DNA, purified by chromatography of denatured calf thymus DNA through hydroxylapatite columns, was found to be equally as sensitive to the inhibition as denatured calf thymus DNA. However, contrary to denatured DNA, the single-stranded DNA thus purified showed no significant binding to adriamycin as analyzed by cosedimentation of the drug and DNA through a sucrose gradient. This indicated that inhibition of RNA synthesis on a single-stranded DNA template might involve a mechanism other than DNA intercalation. Kinetic studies of the inhibition showed that the inhibition of RNA synthesis by adriamycin could not be reversed by increasing the concentrations of RNA polymerase and four nucleoside triphosphates, but it could be reversed by increasing DNA concentrations. Analysis of the size of RNA synthesized indicated that the ultimate size of the product RNA was not altered by adriamycin, suggesting that the drug may inhibit RNA synthesis by reducing RNA chain initiation.     

Hematoporphyrin-induced photo-oxidation and photodynamic cross-linking of nucleic acids and their constituents

The pH-dependency of photo-oxidation of the physiological purine and pyrimidine bases and some of their derivatives was studied, with hematoporphyrin as sensitizer. At high pH these bases (adenine, guanine, uracil, thymine and cytosine) were photo-oxidizable. In the physiological pH range only guanine, and to a much less extent thymine, were sensitive to photo-oxidation. At physiological pH values a slow photo-oxidation of RNA and DNA took place. The photo-oxidation of nuclei acids was strongly augmented by perturbation of their structure in 8 M urea. In model experiments photodynamic cross-linking of tryptophan and cysteine to DNA was demonstrated. No covalent binding of purine or pyrimidine bases to DNA was observed. In similar model experiments covalent photodynamic coupling of guanosine and guanosine-monophosphate to proteins could be shown, whereas no coupling of the other bases occured. These studies confirm the preferential photo-oxidation of guanine in nucleic acids and demonstrate the possible photodynamic cross-linking of proteins to the guanine moiety in other molecules.     

Differentiated inhibition of DNA, RNA and protein synthesis in L1210 cells by 8-methoxypsoralen

The synthesis of DNA, RNA and protein was measured in L1210 cells following treatment with 8-methoxypsoralen in combination with long wavelength ultraviolet irradiation. The results show that the DNA synthesis is strongly inhibited (approximately 95%) at 200 ng/ml reaching a minimum within 2 hours while RNA synthesis is only weakly affected at this concentration (approximately 40% inhibition). At 2 micrograms/ml the RNA synthesis is inhibited approximately 90%. Even at this concentration only a moderate effect is seen on the protein synthesis. These results strongly indicate that the phototoxic action of 8-methoxypsoralen is primarily due to inhibition of DNA synthesis.     

Initiation of Bacillus subtilis Ribonucleic Acid Polymerase on Deoxyribonucleic Acid from Bacteriophages 2C, φ29, T4, and Lambda

Ribonucleic acid (RNA) synthesis primed by bacteriophage T4 or lambda deoxyribonucleic acid (DNA) with Bacillus subtilis RNA polymerase is severely inhibited by high ionic strength. In contrast, RNA synthesis on B. subtilis bacteriophage 2C, SPO1, or phi29 DNA is only moderately affected under similar conditions. The basis of this inhibition lies in the inability of the enzyme to initiate RNA chains with adenosine triphosphate or guanosine triphosphate (ATP, GTP). Binding to templates and the rate of catalysis in high salt after initiation do not seem to be affected. Incorporation of gamma-(32)P-ATP and GTP under a variety of conditions suggests that the specificity of B. subtilis RNA polymerase is different from that of the Escherichia coli enzyme and that it recognizes few promoters on T4 and lambda DNA. Although B. subtilis RNA polymerase initiates RNA chains primarily with ATP or GTP, initiations with pyrimidines can occur on DNA molecules in which hydroxymethyluracil replaces thymine. RNA synthesis on denatured DNA does not seem to be inhibited by high ionic strength, and on native T4 or lambda DNA the inhibition of initiation at constant ionic strength is inversely but not linearly proportional to the ionic radii of cations used to stabilize bihelical DNA to denaturation.     

Lomofungin as an inhibitor of nucleic acid synthesis in Saccharomyces cerevisiae

1. The antibiotic lomofungin was found to be a potent inhibitor of both DNA and RNA synthesis in Saccharomyces cerevisiae. Under selected growth conditions inhibition of DNA synthesis by the drug preceded inhibition of RNA synthesis. 2. Although in general lomofungin inhibited synthesis of ribosomal RNA and polydisperse RNA more effectively than that of low-molecular-weight RNA, under certain conditions the drug inhibited almost completely synthesis of both 4S and 5S RNA. 3. Inhibition of both RNA and DNA synthesis may be explained if RNA synthesis is required for DNA synthesis in yeast. Alternatively, lomofungin, in addition to interacting with DNA-dependent RNA polymerase, might interfere with a component(s) of the DNA-synthetic apparatus. The drug may thus prove to be of considerable value in studies of DNA synthesis in eukaryotes.     

Synthesis of Semliki-forest virus in polyamine-depleted baby-hamster kidney cells.

The role of polyamines in macromolecular synthesis has been studied using the synthesis of Semliki-Forest virus (SF virus) in normal and alpha-difluoromethylornithine-treated baby-hamster kidney (BHK21) cells as a model system. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, the rate-limiting enzymes in polyamine biosynthesis, decreased rapidly in mock- and SF-virus-infected cells, indicating that virus production in BHK21 cells was not dependent on polyamines formed after infection. A prolonged treatment of BHK21 cells with alpha-difluoro-methylornithine, a specific inhibitor of polyamine synthesis, resulted in a marked inhibition of the initial rate of virus production, which appeared 72 h after the beginning of the treatment. This inhibition was reversed by putrescine, spermidine and spermine, and at last partially by several other diamines and polyamine homologues. Polyamine-depletion also markedly reduced viral RNA polymerase activity in SF-virus infected cells. Addition of spermidine to the culture medium rapidly increased viral RNA polymerase activity in the inhibitor-treated cells but had no effect on the enzyme activity when added directly to the assay mixture. The results indicated that polyamines are needed for maximum initial rate of SF-virus replication and suggest that the inhibition of virus production in polyamine-depleted cells is at least partly due to malfunction of the protein-synthetic machinery of the host cell.     

Characterization of a DNA primase from rat liver mitochondria

A DNA primase was partially purified from rat liver mitochondria and separated from the bulk of DNA polymerase gamma and mtRNA polymerase by heparin-agarose chromatography. The primase was distinguished from mtRNA polymerase by its response to pH, monoand divalent cations, and ATP concentrations. In the absence of an active DNA polymerase and using poly(dT) as template, primase synthesized mixed polynucleotide products consisting of units of oligo(A) 1-12 alternating with units of oligo(dA)25-40. Contributions to these products by contaminating DNA polymerase gamma were eliminated by the addition of dideoxy-ATP. Addition of 50 microM dATP to the primase reaction caused a 50% inhibition of AMP incorporation as compared to reactions containing low levels of dATP present only as a contaminant of the ATP added. The inhibition was due primarily to a reduction of new chain initiations. The dATP did not "lock" the primase reaction into the DNA mode of synthesis since the proportion of internal and 3'-terminal RNA segments was little affected. However, the addition of both 50 microM dATP and exogenous DNA polymerase to the primase reaction greatly reduced the amount of internal and 3'-terminal RNA segments, presumably due to the displacement of primase by DNA polymerase. Our data are consistent with the hypothesis (Hu, S.-Z., Wang, T.S.-F., and Korn, D. (1984) J. Biol. Chem. 259, 2602-2609) that the physiologically significant primer is a mixed 5'-oligoribonucleotide-3'-oligodeoxyribonucleotide and that the formation of the RNA to DNA junction is inherently a primase function.