FEEDBACK INHIBITION OF HEPATIC DNA SYNTHESIS

α-Hexachlorocyclohexane (α-HCH) and some other xenobiotic inducers were used to elicit adaptive increases in mono-oxygenase activity, size and DNA content of rat liver. After elimination of the inducers, organ size and mono-oxygenase activity returned to normal whereas the DNA content of the liver remained increased. Upon renewed treatment with an inducer the adaptive responses uncoupled. While mono-oxygenase induction and liver enlargement did occur, DNA replication was largely suppressed. These findings show that in the hyperplastic state the liver is resistant to stimulation of DNA synthesis by the inducers. It is concluded that the DNA content of the liver (or the number of liver cells) is controlled by a feedback system which monitors an excess of DNA (cells) and suppresses cell replication if the content of DNA exceeds the normal level. Organ mass has little, if any, effect on the operation of this feedback system.     

Induction of benzo[a]pyrene Mono-oxygenase in liver cell culture by the photochemical generation of active oxygen species. Evidence for the involvement of singlet oxygen and the formation of a stable inducing intermediate.

1. The photochemical generation of excited states of oxygen in liver cell culture by the mild ilumination of culture medium containing riboflavin, results in stimulation of benzo[a]pyrene 3-mono-oxygenase, a cytochrome P-450-linked mono-oxygenase. 2. The same large increase in mono-oxygenase activity was found when medium containing riboflavin was illuminated in the absence of cells and then stored in the dark for 24h before contact with the cells. From this it may be inferred that stimulation is due to the formation of a stable inducer in the culture medium. Further experiments indicate that the stable inducer is due to the photo-oxidation of an amino acid. 3. Evidence that singlet oxygen is responsible for initiating the stimulation of the mono-oxygenase is based on the use of molecules that scavenge particular active oxygen species. Of all the scavengers tested, only those that scavenge single oxygen inhibited the stimulation. 4. A hypothesis is developed to relate the stimulation of the mono-oxygenase by singlet oxygen in cultured cells to the regulation of the cytochrome P-450 enzyme system in vivo. It is suggested that single oxygen generation within cells may be a common factor linking the many structurally diverse inducers of the enzyme system.     

The expression of the histone H1° gene in the human hepatoma cell line HepG2 is independent of the state of cell proliferation

The H1 histone subtype H1 (0) is a characteristic component of the chromatin of several mammalian tissues. Since H1 (0) is synthesized in nondividing cells upon terminal differentiation, it has been mostly considered either as a prerequisite for or as a consequence of an arrest of DNA replication during the process of differentiation. In several H1 (0)-expressing systems studied until now, inducers of differentiation or inhibitors of DNA synthesis cause an increase of the ratio between H1 (0) and the other H1 proteins. We have studied the steady-state levels of histone H1 (0) mRNA under varied growth conditions in the human hepatoma cell lines HepG2 and Hep3B, and we show in the HepG2 system that H1 (0) is not confined to resting cells, that the H1 (0) gene appears to be expressed throughout the cell cycle and that established inducers of de novo H1 (0) synthesis fail to cause a further increase of the high H1 (0) level. This constitutive expression of H1 (0) appears to reflect the chromatin structure of the liver cells, from which the HepG2 hepatoblastoma cells initially may have evolved. In contrast to the situation in nondividing adult liver cells, the H1 (0) gene is transcribed in HepG2 at a high level, and this expression is compatible with DNA replication.     

Control of DNA replication in a transformed lymphoid cell line: coexistence of activator and inhibitor activities

Proliferating lymphocytes contain an intracellular factor, ADR (activator of DNA replication), which can initiate DNA synthesis in isolated quiescent nuclei. Resting lymphocytes lack ADR activity and contain an intracellular inhibitory factor that suppresses DNA synthesis in normal but not transformed nuclei. In this study we describe a MOLT-4 subline that produces both the activator and inhibitory activities which can be separated by ammonium sulfate fractionation. The inhibitor is heat stable and inhibits ADR-mediated DNA replication in a dose-dependent manner. It does not inhibit DNA polymerase alpha activity. The inhibitor must be present at the initiation of DNA replication to be effective, as it loses most of its effectiveness if it is added after replication has begun. The presence of inhibitory activity in proliferating MOLT-4 cells, taken with the previous observation that inhibitor derived from normal resting cells does not affect DNA synthesis by MOLT-4 nuclei, suggests that failure of a down-regulating signal may play an important role in proliferative disorder.     

An image-based, high-throughput screening assay for molecules that induce excess DNA replication in human cancer cells

Previous studies have shown DNA re-replication can be induced in cells derived from human cancers under conditions in which it is not possible for cells derived from normal tissues. Because DNA re-replication induces cell death, this strategy could be applied to the discovery of potential anticancer therapeutics. Therefore, an imaging assay amenable to high-throughput screening was developed that measures DNA replication in excess of four genomic equivalents in the nuclei of intact cells and indexes cell proliferation. This assay was validated by screening a library of 1,280 bioactive molecules on both normal and tumor-derived cells where it proved more sensitive than current methods for detecting excess DNA replication. This screen identified known inducers of excess DNA replication, such as inhibitors of microtubule dynamics, and novel compounds that induced excess DNA replication in both normal and cancer cells. In addition, two compounds were identified that induced excess DNA replication selectively in cancer cells and one that induced endocycles selectively in cancer cells. Thus, this assay provides a new approach to the discovery of compounds useful for investigating the regulation of genome duplication and for the treatment of cancer.     

Tumor necrosis factor stimulates DNA synthesis in the liver of intact rats

TNF is cytotoxic to tumor cell lines but enhances growth of some nontransformed cells. Because animals administered TNF have an increase in liver size, we studied the [3H]thymidine incorporation into DNA in the liver of intact rats. A significant increase in [3H]thymidine incorporation is seen 20 hours following TNF administration and peaks at 24 hours. The lowest dose of TNF that increases DNA synthesis is 10 micrograms/200 g rat with a maximal increase occurring with 25 micrograms/200 g, considerably less than the dose required for maximally increasing plasma triglycerides. The increase in [3H]thymidine incorporation was shown to be due to an increase in DNA polymerase alpha activity (associated with the replication of DNA) rather than DNA polymerases beta (associated with DNA repair) plus gamma activity. These results indicate that TNF administration stimulates DNA replication in the liver of intact animals.     

A limited number of chromosomes makes a nucleus competent to respond to inducers of replication and mitosis in a plant.

Multinucleate tetraploid cells with unbalanced chromosomal distribution in aneuploid nuclei were obtained in Allium cepa L. root meristems. For this, their natural diploid cells were treated with a multipolarizing agent (1 h carbetamide) followed by an inhibitor of cytokinesis (1 h caffeine). Data from these multinucleate cells with aneuploid nuclei suggest that only four out of the thirty-two chromosomes of their autotetraploid complement possess DNA sequences making the nucleus competent to respond to inducers of replication and mitosis. Direct observation of cells where a single replicated chromosome had reached mitosis showed that this chromosome was the one bearing the nucleolar organizer. Six specific chromosomes would confer competence to the nucleus to respond to inducers of replication but not to those producing chromosome condensation. Another four different chromosomes would confer the nucleus with the ability to respond to mitotic inducers but not to replication inducers. The rest of the chromosomal complement seemed to lack any of the DNA sequences needed for these two important cycle transitions. In a nutshell, certain DNA sequences distributed in a few chromosomes of the onion complement are an intranuclear requirement to initiate replication and mitosis in these plant cells.     

Effects of Chromosome Underreplication on Cell Division in Escherichia coli

The key processes of the bacterial cell cycle are controlled and coordinated to match cellular mass growth. We have studied the coordination between replication and cell division by using a temperature-controlled Escherichia coli intR1 strain. In this strain, the initiation time for chromosome replication can be displaced to later (underreplication) or earlier (overreplication) times in the cell cycle. We used underreplication conditions to study the response of cell division to a delayed initiation of replication. The bacteria were grown exponentially at 39°C (normal DNA/mass ratio) and shifted to 38 and 37°C. In the last two cases, new, stable, lower DNA/mass ratios were obtained. The rate of replication elongation was not affected under these conditions. At increasing degrees of underreplication, increasing proportions of the cells became elongated. Cell division took place in the middle in cells of normal size, whereas the longer cells divided at twice that size to produce one daughter cell of normal size and one three times as big. The elongated cells often produced one daughter cell lacking a chromosome; this was always the smallest daughter cells, and it was the size of a normal newborn cell. These results favor a model in which cell division takes place at only distinct cell sizes. Furthermore, the elongated cells had a lower probability of dividing than the cells of normal size, and they often contained more than two nucleoids. This suggests that for cell division to occur, not only must replication and nucleoid partitioning be completed, but also the DNA/mass ratio must be above a certain threshold value. Our data support the ideas that cell division has its own control system and that there is a checkpoint at which cell division may be abolished if previous key cell cycle processes have not run to completion.     

Comparison of the photochemical and enzymic generation of excited states of oxygen on the induction of benzo(alpha)pyrene mono-oxygenase in liver cell cultures.

The photochemical generation of excited states of oxygen such as the superoxide ion(O-2) and singlet oxygen (1o2) by the mild illumination of culture medium containing riboflavin induces benzo(alpha)pyrene mono-oxygenase in 3 different cell lines derived from rat liver. Similar rates of O-2 generation can be produced by the action of xanthine oxidase on xanthine yet this system does not induce the mono-oxygenase. This result confirms that the mono-oxygenase induction is not mediated by O-2 is not mediated by O-2 and that 1O2 is the most likely candidate for stimulating the mono-oxygenase activity.     

Possible function of the c-myc product: promotion of cellular DNA replication.

We have recently cloned a plasmid, pARS65, containing the sequences derived from mouse liver DNA which can autonomously replicate in mouse and human cells (Ariga et al., 1987). In this report, we show that replication of pARS65 in HL-60 cells can be inhibited by co-transfection with anti-c-myc antibody. In an in-vitro replication system using HL-60 nuclear extract, pARS65 functioned as a template. This in-vitro replication was also blocked by addition of anti-c-myc antibody. Specific binding activity of the c-myc product to pARS65 was detected by an immunobinding assay, suggesting that the c-myc protein promotes DNA replication through binding to the initiation site of replication. This has been substantiated using the antibody to help isolate a human DNA segment that can autonomously replicate in the cells.     

Inhibition of hepatic aryl hydrocarbon hydroxylase by 3-methylcholanthrene, 7,8-benzoflavone and other inducers added in vitro.

A close correlation has been observed between the ability of aromatic polycyclic hydrocarbons and 7,8-benzoflavone (7,8-BF) to induce hepatic aryl hydrocarbon hydroxylase (AHH) in vivo and to inhibit the induced enzyme system in vitro. The activity of this mono-oxygenase was measured by the conversion of 14C-labeled dimethylbenz(a)anthracene (DMBA) or benzo(a)pyrene (BP) to water-soluble products by rat liver preparations (8000 X g supernatant). DMBA as substrate had the advantage over BP in giving a wider range of ethyl acetate-soluble metabolites and allowing the observation of changes in the pattern of these products following injection or addition of the inducing agents. This property was used to detect low concentration (0.1 muM) of polycyclic hydrocarbons which are strong AHH inducers and which may also be carcinogenic. The liver preparation was active for several months when stored at --20 degrees. A possible mechanism of action for the in vitro behaviour of polycyclic hydrocarbons and 7,8-BF towards AHH is proposed.     

Enrichment and visualization of small replication units from cultured mammalian cells

DNA from cultured Chinese hamster cells has been fractionated to yield a population of DNA enriched for replicating molecules. Molecules containing replication structures were analyzed by electron microscopy, and replicon size was estimated. The enrichment procedure takes advantage of single-stranded regions characteristic of replicating molecules, and the greater affinity of mercuric ion for single-stranded rather than native DNA. After interaction with low concentrations of HgCl2, DNA with bound mercury is separated from the bulk of the DNA by virtue of its increased buoyant density in an isopycnic Cs2SO4 gradient. When DNA from cells labeled with [3H]thymidine for 45 s is interacted with HgCl2 and banded in Cs2SO4, the DNA with the highest specific activity is found in a dense region of the gradient. The high specific activity DNA behaves kinetically like nascent DNA since the radioactivity can be chased into main band if the cells are incubated for a further 2 h in excess unlabeled thymidine. Electron microscope analysis of the DNA in the enriched fraction confirmed that it contains a substantial fraction of molecules with replication structures. The level of enrichment is about 25-fold compared to unfractionated DNA or DNA taken from the main band of the Hg++/Cs2SO4 gradient. Of the replicating molecules visualized, 85% possessed a single replication structure. All molecules with multiple replication forms contained replicon sizes less than 5 micron, ranging from 0.2 to 4.5 micron. Replicon size was determined by measuring the distance from the center of one replication structure to the center of the adjacent replication structure on the same molecule. The replicons observed in this study are far smaller than can be detected by DNA fiber autoradiography and are in the same size range as the very small replication units reported in embryonic systems.     

Trophoblast estrogen synthetase stimulation by dibutyryl cyclic AMP and theophylline: increase in cytochrome P-450 content.

The inclusion of both dibutyryl cyclic AMP and theophylline in the culture medium of human malignant trophoblast cells (JAr line) for 72 hours results in an enhanced estrogen secretion through the increased specific activity of estrogen synthetase (aromatase), a cytochrome P-450 mono-oxygenase enzyme system. The data described here suggest that this increased aromatase activity is due to an increase in the concentration of only one component of the mono-oxygenase system, cytochrome P-450.     

Association of 3'----5' exodeoxyribonuclease activity with DNA replitase complex from S-phase Chinese hamster embryo fibroblast cells

DNA replitase has been described as a complex of enzymes/proteins that are associated with both DNA precursor biosynthesis and DNA replication in mammalian cells [Reddy, G. P. V., and Pardee, A. B. (1980) Proc. Natl. Acad. Sci. USA 77, 3312-3316]. We demonstrate for the first time a 3'----5' exodeoxyribonuclease activity is associated with the replitase complex. As much as 60% of this exonuclease activity was similar to that associated with DNA polymerase delta based upon its sensitivity to inhibition by GMP and by butyl-phenyl-deoxyguanosine triphosphate (BuPdGTP). Association of 3'----5' exonuclease activity with the DNA polymerase in the replitase complex was also demonstrated by analyzing dTTP turnover to dTMP in an in vitro DNA polymerase assay system. The DNA polymerase activity in replitase complex exhibited a sensitivity to BuPdGTP which both was similar to that of DNA replication in permeable cells and was intermediate between the BuPdGTP inhibition of purified DNA polymerases alpha and delta. These studies suggest that the replitase complex contains 3'----5' exonuclease activity associated with the DNA polymerase activity responsible for nuclear DNA replication in mammalian cells. Further studies are required to determine if these activities are at least partially attributed to DNA polymerase delta.     

Mode of initiation of constitutive stable DNA replication in RNase H-defective mutants of Escherichia coli K-12.

The alternative pathway of DNA replication in rnh mutants of Escherichia coli can be continuously initiated in the presence of chloramphenicol, giving rise to constitutive stable DNA replication (cSDR). We conducted a physiological analysis of cSDR in rnh-224 mutants in the presence or absence of the normal DNA replication system. The following results were obtained. cSDR allowed the cells to grow in the absence of the normal replication system at a 30 to 40% reduced growth rate and with an approximately twofold-decreased DNA content. cSDR initiation was random with respect to time in the cell cycle as well as choice of origins. cSDR initiation continued to increase exponentially for more than one doubling time when protein synthesis was inhibited by chloramphenicol. cSDR initiation was inhibited during amino acid starvation in stringent (relA+) but not in relaxed (relA1) strains, indicating its sensitivity to ppGpp. cSDR initiation was rifampin sensitive, demonstrating that RNA polymerase was involved. cSDR functioned in dnaA+ rnh-224 strains parallel to the normal oriC+ dnaA+-dependent chromosome replication system.     

Induction of drug metabolism enzymes by dihalogenated biphenyls

The effects of pretreatment with symmetrically dihalogenated biphenyls (DXBs, X-F, Cl(C), Br(B) and I) on rat liver drug metabolism enzymes were investigated. 4,4'-DFB, -DCB, and -DBB as well as 2,2'-DFB appeared to be inducers of microsomal cytochrome P-450-linked monoxygenases (N-demethylases of aminopyrine and ethylmorphine). However, no structure-induction relationship was found. 4,4'-DXBs also induced a cytochrome P-448-linked mono-oxygenase (ethoxyresorufin O-deethylase), and their order of induction potential seemed to parallel the increase of the size of the halogen substituent. Therefore, 4,4'-DXB's may be categorized as mixed-type inducers, the cytochrome P-450 component being the more pronounced. Data on the cytochrome P-448 induction by dihalogenated biphenyls with only para substituents may be considered as a refinement of the previously described structure-activity relationship in this respect. All of the DXBs except 3,3'-DCB and 4,4'-DIB, enhanced, like phenobarbital, the activity of UDP-glucuronyltransferase toward 4-hydroxybiphenyl. Only 4,4'-DFB was able to induce the activity of glutathione S-transferase toward 1,2-epoxy-3-(p-nitrophenoxy)propane. Studies after 4,4'-DBB-treatment revealed, like phenobarbital, a preferential induction of ethylmorphine N-demethylase on rough endoplasmic reticulum-derived microsomes, whereas UDP-glucuronyltransferase activity toward 4-hydroxybiphenyl was induced to a larger extent on smooth endoplasmic reticulum microsomes, suggesting a dissimilar enzyme induction in microsomal subfractions.     

DNA Repair in Potorous tridactylus

The DNA synthesized shortly after ultraviolet (UV) irradiation of Potorous tridactylis (PtK) cells sediments more slowly in alkali than that made by nonirradiated cells. The size of the single-strand segments is approximately equal to the average distance between 1 or 2 cyclobutyl pyrimidine dimers in the parental DNA. These data support the notion that dimers are the photoproducts which interrupt normal DNA replication. Upon incubation of irradiated cells the small segments are enlarged to form high molecular weight DNA as in nonirradiated cells. DNA synthesized at long times (~ 24 h) after irradiation is made in segments approximately equal to those synthesized by nonirradiated cells, although only 10-15% of the dimers have been removed by excision repair. These data imply that dimers are not the lesions which initially interrupt normal DNA replication in irradiated cells. In an attempt to resolve these conflicting interpretations, PtK cells were exposed to photoreactivating light after irradiation and before pulse-labeling, since photoreactivation repair is specific for only one type of UV lesion. After 1 h of exposure ~ 35% of the pyrimidine dimers have been monomerized, and the reduction in the percentage of dimers correlates with an increased size for the DNA synthesized by irradiated cells. Therefore, we conclude that the dimers are the lesions which initially interrupt DNA replication in irradiated PtK cells. The monomerization of pyrimidine dimers correlates with a disappearance of repair endonuclease-sensitive sites, as measured in vivo immediately after 1 h of photoreactivation, indicating that some of the sites sensitive to the repair endonuclease (from Micrococcus luteus) are pyrimidine dimers. However, at 24 h after irradiation and 1 h of photoreactivation there are no endonuclease-sensitive sites, even though ~ 50% of the pyrimidine dimers remain in the DNA. These data indicate that not all pyrimidine dimers are accessible to the repair endonuclease. The observation that at long times after irradiation DNA is made in segments equal to those synthesized by nonirradiated cells although only a small percentage of the dimers have been removed suggests that an additional repair system alters dimers so that they no longer interrupt DNA replication.     

A Novel DDB2-ATM Feedback Loop Regulates Human Cytomegalovirus Replication

Human cytomegalovirus (HCMV) genome replication requires host DNA damage responses (DDRs) and raises the possibility that DNA repair pathways may influence viral replication. We report here that a nucleotide excision repair (NER)-associated-factor is required for efficient HCMV DNA replication. Mutations in genes encoding NER factors are associated with xeroderma pigmentosum (XP). One of the XP complementation groups, XPE, involves mutation in ddb2, which encodes DNA damage binding protein 2 (DDB2). Infectious progeny virus production was reduced by >2 logs in XPE fibroblasts compared to levels in normal fibroblasts. The levels of immediate early (IE) (IE2), early (E) (pp65), and early/late (E/L) (gB55) proteins were decreased in XPE cells. These replication defects were rescued by infection with a retrovirus expressing DDB2 cDNA. Similar patterns of reduced viral gene expression and progeny virus production were also observed in normal fibroblasts that were depleted for DDB2 by RNA interference (RNAi). Mature replication compartments (RCs) were nearly absent in XPE cells, and there were 1.5- to 2.0-log reductions in viral DNA loads in infected XPE cells relative to those in normal fibroblasts. The expression of viral genes (UL122, UL44, UL54, UL55, and UL84) affected by DDB2 status was also sensitive to a viral DNA replication inhibitor, phosphonoacetic acid (PAA), suggesting that DDB2 affects gene expression upstream of or events associated with the initiation of DNA replication. Finally, a novel, infection-associated feedback loop between DDB2 and ataxia telangiectasia mutated (ATM) was observed in infected cells. Together, these results demonstrate that DDB2 and a DDB2-ATM feedback loop influence HCMV replication.     

Molecular Weight of Deoxyribonucleic Acid Synthesized During Initiation of Chromosome Replication in Escherichia coli

Alkaline sucrose gradients were used to study the molecular weight of deoxyribonucleic acid (DNA) synthesized during the initiation of chromosome replication in Escherichia coli 15 TAU-bar. The experiments were conducted to determine whether newly synthesized, replication origin DNA is attached to higher-molecular-weight parental DNA. Little of the DNA synthesized after readdition of required amino acids to cells previously deprived of the amino acids was present in DNA with a molecular weight comparable to that of the parental DNA. The newly synthesized, low-molecular-weight DNA rapidly appeared in higher-molecular-weight material, but there was an upper limit to the size of this intermediate-molecular-weight DNA. This limit was not observed when exponentially growing cells converted newly synthesized DNA to higher-molecular-weight material. The size of the intermediate-molecular-weight DNA was related to the age of the replication forks, and the size increased as the replication forks moved further from the replication origin. The results indicate that the newly synthesized replication origin DNA is not attached to parental DNA, but it is rapidly attached to the growing strands that extend from the replication fork to the replication origin, or to the other replication fork if replication is bidirectional. Experiments are reported which demonstrate that the DNA investigated was from the vicinity of the replication origin and was not plasmid DNA or DNA from random positions on the chromosome.     

Xenobiotic stress induces hepatomegaly and liver tumors via the nuclear receptor constitutive androstane receptor

The constitutive androstane receptor (CAR, NR1I3) is a central regulator of xenobiotic metabolism. CAR activation induces hepatic expression of detoxification enzymes and transporters and increases liver size. Here we show that CAR-mediated hepatomegaly is a transient, adaptive response to acute xenobiotic stress. In contrast, chronic CAR activation results in hepatocarcinogenesis. In both acute and chronic xenobiotic responses, hepatocyte DNA replication is increased and apoptosis is decreased. These effects are absent in CAR null mice, which are completely resistant to tumorigenic effects of chronic xenobiotic stress. In the acute response, direct up-regulation of Mdm2 expression by CAR contributes to both increased DNA replication and inhibition of p53-mediated apoptosis. These results demonstrate an essential role for CAR in regulating both liver homeostasis and tumorigenesis in response to xenobiotic stresses, and they also identify a specific molecular mechanism linking chronic environmental stress and tumor formation.