Effect of bleomycin on [3H]Thymidine 5'-Triphosphate incorporation into host liver and hepatoma nuclei.

The effect of bleomycin on [3H]thymidine 5'-triphosphate ([3H]TTP) incorporation into isolated sucrose nuclei from host liver and Morris hepatomas has been compared. Bleomycin stimulates [3H]TTP incorporation 13-fold in host liver and hepatoma 16 nuclei, 8-fold in hepatoma 7800 nuclei, and 3-fold in hepatoma 7777 nuclei. Differences in the nuclear membranes are not responsible for the different response of the nuclei. Nuclei, denuded of their membranes by Triton X-100 treatment, give similar results to sucrose nuclei. Analysis of DNA extracted from liver or hepatoma nuclei incubated with bleomycin indicates that bleomycin produces scissions in the nuclear DNA and that some repair synthesis takes place. Incubation of nuclei with 111indium-labeled bleomycin shows an equal binding capacity of liver and hepatoma nuclei for bleomycin. Bleomycin also stimulates incorporation of [3H]TTP in a system using chromatin or calf thymus DNA as primer. Host liver or hepatoma chromatin incubated with a DNA polymerase extracted from normal rat liver nuclei is stimulated approximately to the same extent by bleomycin. When DNA polymerase extracts from host liver and hepatoma nuclei are assayed with calf thymus DNA as primer, bleomycin has a greater stimulatory effect on [3H]TTP incorporation with host liver DNA polymerase than with hepatoma DNA polymerase in the system. We suggest that a defect in the repair system in hepatoma nuclei is responsible for the relatively lower response to bleomycin.     

DNA synthesis in membrane-denuded nuclei and nuclear fractions from host liver and Morris hepatomas.

Incorporation of [3H]TTP into membrane-denuded nuclei and fractions of these nuclei from host liver and Morris hepatomas has been compared. Treatment of sucrose nuclei with Triton X-100 removed 95% of the phospholipids and 15 to 20% of the protein. These membrane-denuded nuclei remained physically stable. The Triton X-100-extracted nuclei incorporated label into their DNA in nuclear-incorporating system similar to sucrose nuclei with their membranes intact. Triton X-100-treated nuclei from hepatoma 7777 incorporated six times more label and those from hepatoma 7800 incorporated three times more label than Triton X-100-treated host liver nuclei. Nuclei from the three sources incorporated more label when exogenous DNA was added to the incubation system, but the difference in incorporation between the hepatoma nuclei and the host liver nuclei disappeared. When Triton X-100-treated nuclei, prepared from a tumor-bearing animal given injections of [3H]thymidine for 10 min were fractionated on sucrose gradients after disruption by high Mg2+ concentration, the fractions from hepatoma 7777 nuclei contained six times as much label as the host liver nuclear fractions. Nuclear fractions prepared from unabeled hepatomas or host livers had DNA polymerase activity. The activity, however, is the same in fractions prepared from hepatoma 7777 or host liver nuclei. It is suggested that the nuclear membrane does not play an important role in nuclear DNA synthesis. It is further suggested that the increased incorporation found with hepatoma nuclei is dependent on a physical or chemical arrangement of components within the nucleus and not solely on different enzyme levels.     

Effects of N-acetoxy-2-acetylaminofluorene and N'-nitro-N-nitrosoguanidine on nuclear DNA synthesis in rat liver.

A system for the study of DNA synthesis in isolated nuclei is described for sham and regenerating rat liver. The system has been characterized with respect to nuclear purity, conditions for optimum incorporation of [5-methyl-3H]thymidine triphosphate, time course of incorporation, product analysis by neutral and alkaline sucrose gradients, and the effect of exogenously added DNA. No difference in the basal level of activity was detected between nuclei prepared from normal or regenerating liver when isolated 24 hr after operation. However, exogenous activated DNA preferentially stimulated [5-methyl-3H]thymidine triphosphate incorporation in nuclei from regenerating liver. Activated DNA caused to react with the carcinogen N-acetoxy-2-acetylaminofluorene was a less effective primer-template in this system and decreased in a dose-dependent fashion the incorporation of [5-methyl-3H]thymidine triphosphate to below basal levels in nuclei from both normal and regenerating liver. The carcinogen N-methyl-N'-nitro-N-nitrosoguanidine had no inhibitory effect when assayed in this fashion.     

The effect of several antitumor agents on 3H-TTP incorporation in host liver and hepatoma nuclei.

Six different tumor antibiotics have been investigated in a nuclear incorporating system for their ability to inhibit 3H-TTP incorporation. Both host liver nuclei and nuclei prepared from two different Morris hepatomas have been used in the investigation. Three of these anti-tumor agents inhibit 3H-TTP incorporation equally in host liver and hepatoma nuclei, two preferentially inhibit incorporation in hepatoma nuclei and one stimulates incorporation preferentially in host liver nuclei. The effects of these compounds on nuclear DNA has been analyzed on neutral and alkaline sucrose density gradients. The nuclear incorporation system appears to be useful as a screening test system for potential anti-tumor agents.     

Stimulatory effect of a serum factor on DNA synthesis in isolated hepatoma nuclei.

A serum protein present in normal rat serum and absent from the serum of hepatoma-bearing animals at advanced stages has a stimulatory effect on 3H-thymidine incorporation into hepatoma cells in suspension. Liver cells maintained in a similar suspension are not affected by the factor. The stimulation appears to be at the level of chromatin or DNA. Isolated membrane-denuded nuclei from Morris hepatoma 7777 incorporate more 3H-TTP when the factor is present in the incubation mixture. Nuclei from host liver are not stimulated. The factor also stimulates incorporation of 3H-TTP in a system using calf thymus DNA as primer and an extracted DNA polymerase. In this system incorporation is stimulated with DNA polymerase from both tissues, host liver and hepatoma 7777. It is concluded that the factor does not act on the DNA polymerase but on chromatin or DNA.     

Effects of nicotinamide, isonicotinamide, and bleomycin on DNA synthesis and repair in rat hepatocytes and hepatoma cells

Because inhibitors of poly (ADP-ribose) synthetase have been found to influence DNA synthesis in some systems, the possibility that nicotinamide or isonicotinamide might potentiate the effect of bleomycin on DNA replication and repair was examined. After a 30-minute incubation with bleomycin (200 micrograms/ml), tritiated thymidine ([3H]dThd) incorporation into DNA was stimulated during a subsequent 30-minute incubation with hepatocytes of inbred BUF rats but was decreased in HTC cells of BUF rats. When unscheduled DNA synthesis was measured in the presence of 10 mM hydroxyurea, bleomycin (200 micrograms/ml) increased [3H]dThd incorporation in both cell types. A dose of 20 mM nicotinamide and isonicotinamide caused an approximately 50% inhibition of total [3H]dThd incorporation in HTC cells. Significant inhibitory effects of 20 mM nicotinamide and isonicotinamide on unscheduled DNA synthesis were observed after preincubation of hepatocytes and HTC cells with bleomycin. When the effects of bleomycin on DNA structure were assessed fluorometrically with ethidium bromide after mild alkaline incubation, nicotinamide and isonicotinamide did not significantly affect the damage revealed with bleomycin alone. When HTC cells were incubated for 48 hours with bleomycin (20 micrograms/ml), the increase in cell numbers was about 50% of that in control cultures. Nicotinamide and isonicotinamide also inhibited the proliferation of HTC cells, but the effects were not additive with the effect of bleomycin.     

Soluble factors from liver and hepatomas which inhibit [3H]thymidine incorporation into DNA of Novikoff hepatoma cells.

The nature of soluble factors from liver and hepatomas which inhibit [3H]thymidine incorporation into DNA was studied in Novikoff hepatoma cells. The decreased activity in hepatoma preparations was due to loss of a high-molecular-weight heat-labile factor. Although this factor cochromatographed with arginase activity on Sephadex G-150, it does not appear to result from this activity as judged by the failure of arginine to prevent the inhibitory effect on [3H]thymidine incorporation. Both liver and hepatomas contained a heat-stable factor with inhibitory activity. Studies with ethanol-soluble material suggested that the action was not solely attributable to the presence of unlabeled thymidine, since the apparent molecular weight was too high and since the factor(s) inhibited [3H]leucine incorporation into protein in addition to inhibiting [3H]thymidine incorporation in DNA.     

Spermine-induced variations in the adenosine 5'-diphosphate ribosylation patterns of nuclear proteins from rat liver and hepatoma.

Rat liver and hepatoma nuclei were incubated in vitro with [3H]nicotinamide adenine dinucleotide to allow synthesis of a polymer of adenosine diphosphoribose subunits joined in an 1',2' ribose-ribose linkage. The addition of 1 mM spermine altered the adenosine 5'-diphosphate (ADP) ribosylation patterns of nuclear proteins in hepatoma, host liver, and regenerating liver. Spermine-treated nuclei showed a greater incorporation of ADP-ribose into H1 histones and nonhistone nuclear proteins with isoelectric points between pH 3.0 and 6.0 when separated on polyacrylamide gels. Conversely, a large reduction in ADP ribosylation was seen in core histones (H2A, H2B, and H3) from the same nuclei. The proportion of ADP-ribose incorporated into histones was reduced in the nuclei from proliferating cells relative to their respective control livers. These results imply that polyamines, which are higher in concentration in rapidly dividing cells, may elicit a regulatory function by causing the preferential ADP ribosylation of H1 histones, as well as the more acidic of the nuclear proteins.     

Mode of action of C-1027, a new macromolecular antitumor antibiotic with highly potent cytotoxicity,on human hepatoma BEL-7402 cells

Summary C-1027, a new macromolecular peptide antitumor antibiotic produced by aStreptomyces strain, was extremely cytotoxic to cultured cancer cells and markedly inhibited the growth of transplantable tumors in mice. As determined by tritium-labeled precursor-incorporation assay, C-1027 strongly inhibited DNA and RNA synthesis in hepatoma BEL-7402 cells without affecting protein synthesis. After incubation with the hepatoma cells for 4 h, IC₅₀ values for [³H]-thymidine and [³H]-uridine incorporation were 0.00012 and 0.00032 m, respectively. After 30 min incubation, C-1027 showed much stronger inhibition of [³H]-thymidine incorporation than did Adriamycin, mitomycin C or methotrexate, even at a concentration 10,000 times lower. The effect of C-1027 on pBR322 DNA suggested that the drug could cause single- or double-strand scission of DNA. As determined by flow cytometry, C-1027 delayed the progression of hepatoma cells through the S-phase and blocked the cells at G2+M. Cytological study showed that C-1027 caused a drastic reduction of the mitotic index within 1 h and that an overshot of the mitotic index occurred at 48 h. Our results indicate that C-1027 is an interesting compound with highly potent activity on cellular DNA.     

Effect of copper administration on the incorporation of [3H]-thymidine into the liver DNA of rats stimulated by dimethyl-nitrosamine and diethylnitrosamine

The incorporation of [³H]thymidine into liver DNA of rats increased6–8 times 48 h after a single injection of dimethylnitrosamine(DMN, 30 mg/kg) and diethyl-nitrosamine (DEN, 100 mg/kg). Totest the suppressive effect of copper, the incorporation of[³H]thymidine in to liver DNA in the DMN groups or DEN groupspretreated with copper was measured 48 h after the administrationof DMN or DEN. The incorporation of [³H]thymidine into liverDNA of rats stimulated by the injection of DEN was strikinglysuppressed by the injection of cupric acetate (20 mg Ci/kg),but that of rats simultated by the injection of DEN was notsuppressed by the injection of copper. Some other metal salts,silver nitrate (20 mg Ag/kg), nickel acetate (20 mg Ni/kg) andbasic lead acetate (20 mg Pb/kg) did not significantly suppressthe incorporation of [³H]thymidine stimulated by DMN or DEN.The accumulation of copper was much higher in the liver of copper-administeredrats than that of nickel or lead in the liver of nickel-administeredrats or lead-administered rats. The accumulation of silver wascomparatively high in the liver of silver-administered rats.     

Synergistic cytotoxicity and DNA strand break formation by bromodeoxyuridine and bleomycin in human tumor cells

5-Bromo-2'-deoxyuridine (BrdUrd) is a thymidine analogue whose cellular effects are related to its incorporation into DNA. BrdUrd is a known radiosensitizing agent that could potentially enhance the activity of chemotherapeutic agents that interact directly with DNA. Therefore we studied the interaction of BrdUrd and bleomycin in a human head and neck squamous carcinoma cell line, SQ20B. Using a colony-forming assay and analyzing results by the median-effect method, we have shown that there is synergistic cytotoxicity between BrdUrd and bleomycin. Synergism is evident when BrdUrd is administered prior to bleomycin or when the two drugs are applied simultaneously and is evident at a variety of BrdUrd:bleomycin concentration ratios. Alkaline elution of DNA from cells exposed to BrdUrd and bleomycin demonstrated greater single strand break formation than expected from the individual single strand break frequencies induced by each drug alone. BrdUrd did not affect the rate of repair of bleomycin-induced single strand breaks or the formation of double strand breaks. Although the mechanism of this interaction at the molecular level is unclear, our studies suggest that a direct interaction of bleomycin with BrdUrd-substituted DNA may be the cause of the synergism of these two agents.     

Potentiation of topoisomerase inhibitor-induced DNA strand breakage and cytotoxicity by tumor necrosis factor: enhancement of topoisomerase activity as a mechanism of potentiation

A combination of tumor necrosis factor (TNF) and the topoisomerase I inhibitor, camptothecin, or the topoisomerase II inhibitors, teniposide and amsacrine, produced dose-dependent synergistic cytotoxicity against the murine L929 fibrosarcoma cells. Similar synergy was not observed with a combination of TNF and bleomycin. To define the role of TNF in the augmentation of tumor cell killing by topoisomerase I or II inhibitors, the effect of TNF on the production of enzyme-linked DNA strand breaks induced in cells by topoisomerase inhibitors was investigated. L929 cells incubated for 1 h with the topoisomerase inhibitors contained protein-linked strand breaks. In contrast, TNF alone did not induce DNA strand breakage. However, when cells were incubated simultaneously with TNF and camptothecin, amsacrine, Adriamycin, actinomycin D, teniposide, or etoposide, increased numbers of strand breaks were produced. Preincubation of the cells with TNF for 30 min or 3 h before the addition of camptothecin or etoposide resulted in no more strand breaks than that observed in cells incubated with the drugs alone. TNF treatment of L929 cells produced a rapid and transient increase in specific activity of extractable topoisomerases I and II. These increases were maximum at 2-5 min of TNF treatment and by 30 min the activities of extractable enzymes were equal to or less than those detected in extracts from untreated cell controls. The transient nature of the increase in extractable topoisomerase activity may explain the kinetics and significance of the order of addition of TNF and inhibitors for maximal synergistic activity. These data are consistent also with a role for topoisomerase-linked DNA lesions in the TNF-mediated potentiation of killing of L929 cells by topoisomerase inhibitors.     

Molecular interactions of the combined effects of bleomycin and x-rays on mammalian cell survival.

The interactions between bleomycin and X-ray damage and repair have been examined in rat and human tumor cells. Bleomycin itself indices extensive DNA single-strand breaks but does not appear to inhibit the repair of X-ray-induced DNA single-strand breaks. Quantitative analysis of these interactions is complicated by the retention of active bleomycin within cells that remains capable of further DNA degradation even under the conditions of alkaline sucrose gradient cell lysis. DNA double-strand breaks and/or disruptions of DNA-lipid complexes also occur following bleomycin exposure. X-ray-induced excision repair replication is only minimally influenced by even high concentrations of bleomycin. A small amount of excision repair is demonstrable in nonirradiated cells treated with high concentrations of bleomycin consistent with repair of bleomycin-induced nucleotide damage in cellular DNA by a "cut and patch" repair mechanism. Repair of bleomycin-induced DNA single-strand breaks also occurs. The data indicate that bleomycin and X-ray damage are quite similar both in their induction and repair, but that lesions occur and are repaired independently. The enzymatic mechanisms appear similar in the two cell types despite substantial differences in their sensitivity to bleomycin.     

Bleomycin-induced chromosome breaks as a risk marker for lung cancer: a case-control study with population and hospital controls

Environmental exposure to carcinogens and individual susceptibility play significant roles in cancer risk. Suboptimal DNA repair capability, measured by quantifying mutagen-induced chromosome breaks, might explain variable host susceptibility to environmental carcinogens. In an ongoing lung cancer case-control study, we compared individual sensitivity to bleomycin-induced chromosome breaks in 152 non-small cell lung cancer patients with 94 population controls and 85 hospital controls with no history of cancer. Mutagen sensitivity was measured by mean number of chromatid breaks per cell in cultured peripheral blood lymphocytes treated with bleomycin. Non-parametric tests and chi(2) tests were used to determine the statistical significance of the crude case-control comparisons, followed by logistic regression to adjust for important covariates. The mean number of bleomycin-induced breaks per cell was 1.01 for the cases compared with 0.86 for hospital controls (P < 0.01) and 0.89 for population controls (P < 0.01). The mean number of breaks per cell was 1.01 for those >65 years old and 0.81 for those < or = 65 years old (P < 0.01) among population controls. Defining bleomycin sensitive as >0.84 break/cell (the median level in population controls), 67% of the cases were bleomycin sensitive compared with 49% of the hospital controls [adjusted odds ratio (OR) = 2.69, 95% confidence interval (CI) = 1.44, 5.04], and 51% of the population controls (adjusted OR = 2.18, 95% CI = 1.13, 4.21). Our data indicate that the increased number of bleomycin-induced chromosome breaks was significantly associated with an increased risk of lung cancer in the first 331 subjects.     

Reductive activation of diaziquone and possible involvement of free radicals and the hydroquinone dianion

To analyze the mode of action of diaziquone [AZQ] on DNA, we examined the activity of two AZQ analogues and N,N',N"-triethylenethiophosphoramide on three forms [supercoiled (Form I), open circular (Form II), and linear (Form III)] of PM-2 DNA. The AZQ analogues contained chlorine atoms which substituted either the carbethoxyamino groups or the aziridine groups of the parent compound. N,N',N"-triethylenethiophosphoramide is a triaziridine compound containing pentavalent phosphorus instead of a quinone group. We found that only when reduced with sodium borohydride did AZQ change the topology of the three forms of PM-2 DNA by introducing mainly single strand breaks. The AZQ analogue containing only aziridines (RQ2) was active in both its oxidized and its reduced forms, while the analogue containing only the carbethoxyamino groups (RQ14) or N,N',N"-triethylenethiophosphoramide were not active in either form. Under similar experimental conditions, Adriamycin alone altered the electrophoretic mobility of PM-2 DNA, while borohydride reduced Adriamycin did not. By using electron spin resonance spectroscopy, we showed that dihydrodiaziquone (AZQH2) oxidizes to the semiquinone in the presence of oxygen. Although AZQH2 was active against DNA, it was not active against cellular DNA synthesis as measured by [3H]thymidine incorporation into exponentially growing HEp-2 cells. However, AZQ alone prevented [3H]thymidine incorporation into HEp-2 cells. We found that HEp-2 cells have the ability to reduce AZQ to its free radical anion, but AZQH2 does not autoxidize to the free radical in the presence of cells. The reductive ability of HEp-2 cells may be responsible in part for preventing the oxidation of AZQH2 to the free radical. We found that under our conditions (1-h incubations) the aziridines are essential for the activity of aziridinyl quinones against PM-2 DNA and that in the case of AZQ the hydroquinone is also required.     

Effects of cyanate, thiocyanate, and amygdalin on metabolite uptake in normal and neoplastic tissues of the rat.

Thiocyanate was found to resemble cyanate in its inhibitory effects on [3H]thymidine incorporation and the uptake of [32P]phosphate and [3H]amino acids in transplanted tumors of the BUF rat. The capacity to inhibit metabolite uptake in hepatomas and a colon tumor under conditions in which uptake was unchanged or increased in host liver was concluded to be a common feature of the action of cyanate and thiocyanate. Inhibition of [32P]phosphate uptake and [3H]thymidine incorporation into DNA of tumors was also observed after treatment of rats with amygdalin. With this drug, however, the action on tumors and livers of host rats was similar.     

In vitro sensitivity testing of human breast cancer cells to hormones and chemotherapeutic agents

Summary The sensitivities of human breast cancer cells to hormones and chemotherapeutic agents were measured using a new in vitro assay. Tumor cells from individual patients were cultured on collagen-coated dishes in medium containing the patient’s serum. The rationale for use of the patient’s serum is that the components of this serum interact with the cells and therapeutic agents in vivo. Cells were incubated for the length of the assay in the presence or absence of estrogen (E₂) with or without tamoxifen (TAM) or in the presence or absence of cortisol (F). At 1 day after cell seeding, cells were exposed to a chemotherapeutic agent, Adriamycin, melphalan, or 5-fluorouracil, for 24 h. After a 48-h recovery period, [³H]-thymidine ([³H]-TdR) was added to the cultures for 24 h. Depending on the concentration, E₂ generally stimulated or inhibited incorporation of [³H]-TdR into the DNA of cells from estrogen-receptor (ER)-positive tumors. TAM eliminated the effects of E₂. F generally stimulated or inhibited incorporation in cells with no correlation to ER status. Stimulation of [³H]-TdR incorporation by hormones increased cell sensitivity to Adriamycin. In contrast, hormone inhibition of [³H]-TdR incorporation decreased cell responsiveness to this drug. This rapid assay, which can measure the sensitivities of breast carcinoma cells to hormones and drugs and identify effective combinations of therapeutic agents, should lead to a rational selection of treatment for the individual patient.     

13-Hydroxyoctadecadienoic acid is the mitogenic signal for linoleic acid-dependent growth in rat hepatoma 7288CTC in vivo.

Growth of hepatoma 7288CTC in male Buffalo rats is directly dependent on uptake of linoleic acid (LA) from the arterial blood. One to 5% of the LA taken up is converted to 13-hydroxyoctadecadienoic acid (HODE), an agent that enhances epidermal growth factor-dependent mitogenesis. The role of 13-HODE in LA-dependent growth of solid tumors is not known. In this study, we examined LA uptake and 13-HODE formation on growth of tissue-isolated hepatoma 7288CTC in vivo and on [3H]thymidine incorporation and DNA content during perfusion in situ. Fatty acid uptake and metabolite release were determined from arteriovenous difference measurements. Tumor-bearing and blood donor rats were fed either LA-sufficient or -deficient diets. Hepatoma 7288CTC removed LA from the arterial blood and released 13-HODE [and a small amount of 13-ketooctadecadienoic acid (KODE)] into the venous blood both in vivo and during perfusion. Treatment with the lipoxygenase inhibitor nordihydroguaiaretic acid (10 microM) did not affect tumor LA uptake, but inhibited release of 13-HODE and 13-KODE in vivo and during perfusion, suppressed growth in vivo, and inhibited [3H]thymidine incorporation during perfusion. The addition of 13-HODE to the nordihydroguaiaretic acid-containing whole blood perfusate increased the rate of [3H]thymidine incorporation 10 times and nearly doubled tumor DNA content; the addition of 13-KODE or 9-HODE had no effect. 13-HODE and 13-KODE were not released from tumors growing in rats fed a LA-deficient diet, and the rates of tumor growth in vivo and [3H]thymidine incorporation during perfusion were decreased. The addition of 13-HODE to the LA-deficient blood perfusate promoted tumor 13-HODE uptake and a dose-dependent increase in [3H]thymidine incorporation and tumor DNA content. These results provide strong evidence that 13-HODE is the mitogenic signal responsible for LA-dependent growth in hepatoma 7288CTC in vivo.     

Effects of nickel compounds on incorporation of [3H] thymidine into DNA in rat liver and kidney

In vivo incorporation of [³H] thymidine into DNA was determinedin rats at 28 h after partial hepatectomy. Administration ofnickel carbonyl (Ni(CO)₄) at 2 or 4 h before sacrifice inhibited[³H] thymidine uptake into liver and kidney DNA. For example,in rats killed 4 h after i.v. injection of Ni(CO)₄ (2 mg Ni/100g), [³H]-labelling of liver DNA averaged 54 (SE ± 10)%of controls (p<0.05), and [³H]-labelling of kidney DNA averaged53 (SE ± 6)% of controls (p<0.01). Injection of NiCI₂(2 mg Ni/100 g, i.m.) 4 h before death did not significantlyaffect [³H] thymidine uptake into liver DNA, but did inhibit[³H] thymidine uptake into kidney DNA (65 ± 6%, p<0.02).Binding of ⁶³Ni to DNA in liver and kidney of rats killed 4h after injection of ⁶³Ni(CO)⁴or ⁶³NiCl² ranged from 0.3 to2.2 mol ⁶³Ni/mol of DNA nucleotides. Ultracentrifugation ofDNA on alkaline sucrose gradients did not reveal any differencesbetween sedimentation profiles of hepatic DNA from Ni(CO)⁴-treatedrats versus paired control rats.