An endogenous protein inhibitor of DNA polymerase alpha in normal and neoplastic rat mammary tissues

Extracts of whole tissue or isolated nuclei from lactating rat mammary gland that has diminished cell replication capacity were more active than the corresponding extracts of pregnant rat mammary gland that contains actively replicating cells in causing a dose-dependent inhibition of DNA polymerase alpha in vitro. Purification of the inhibitor from both tissue and nuclear extracts using a sequence of Sephacryl S200, DEAE-cellulose and CM52 columns confirmed the above assay results. Using the same assay and purification procedures, both tissue and nuclear extracts from the rapidly growing transplanted R3220AC mammary tumors exhibited very little or no inhibitor activity. The partially purified mammary inhibitor (mol. wt of 155kD, high A280 nm/A260 nm ratio, heat labile) was equally inhibitory to the purified DNA polymerase alpha from either R3230AC tumor or calf thymus, and to the nuclear matrix bound DNA polymerase alpha of R3230AC tumor.     

Intranuclear dynamics of DNA polymerase alpha differs between the transplanted R3230AC mammary adenocarcinomas and the host mammary gland depending on lactation cycle

DNA polymerase alpha activity was markedly higher in all nuclear subfractions, including nuclear matrix, from transplanted R3230AC mammary adenocarcinomas than in the analogous fractions from mammary gland of same tumor-bearing pregnant or lactating rats. Changes in host lactational status had no significant effect on subnuclear distribution of tumor DNA polymerase alpha activity, with the majority (60-75%) localized in soluble nucleoplasm and a significant amount (13-20%) retained in the nuclear matrix. In the host mammary gland, nuclear matrix-bound DNA polymerase alpha was highest, accounting for 48% of total nuclear activity, during late pregnancy when mammary cells undergo rapid raplication. During lactation, when cells in mammary gland cease to divide, only 8% of enzyme activity was in the nuclear matrix, while the majority (60-80%) of DNA polymerase alpha activity was localized in nucleoplasm. In both R3230AC tumor and mammary gland regardless of host's lactational status, the majority (60-80%) of DNA polymerase beta activity was localized in the high salt-soluble chromatin. These present data thus suggest that, regardless of host lactational status, R3230AC tumor has many cycling cells, each with a large pool of DNA polymerase alpha molecules maintaining maximal and constant replicative activity, while normal mammary gland cells have a smaller pool of DNA polymerase alpha which become primarily matrix-bound only during active cell replication during late pregnancy. A constant localization of nuclear DNA polymerase beta in chromatin in both mammary gland and the tumor suggest it is not important in mammary cell proliferation.     

Glucose-6-phosphate dehydrogenase from lactating rat mammary gland and R3230AC adenocarcinoma.

A number of properties of glucose-6-phosphate dehydrogenase from lactating rat mammary gland and R3230AC rat mammary adenocarcinoma are compared. The main electrophoretic forms of the enzyme from these sources are indistinguishable with respect to charge and molecular weight whereas the minor forms show differences in these properties. The subunit molecular weight and steroid inhibition of the enzymes from the lactating gland and tumor are not significantly different. These results are contrasted with similar studies in mice.     

Gene expression of PCNA/cyclin in adult tissues and the R3230AC mammary tumor of rat

We have investigated the gene expression of PCNA (Proliferating Cell Nuclear Antigen)/cyclin in rat tissues and the R3230AC mammary tumor. The steady-state mRNA level of PCNA/cyclin in a tissue is related to the proliferation of the tissue. The observation was confirmed with the results from the studies of the immunoblotting analyses and the DNA polymerase activity measurements. Furthermore, an overexpression of PCNA/cyclin was found in the R3230AC mammary tumor, which is accompanied by an altered PCNA/cyclin gene structure detected with the Southern blot analysis.     

Intranuclear dynamics of DNA polymerase α differs between the transplanted R3230AC mammary adenocarcinomas and the host mammary gland depending on lactation cycle

DNA polymerase α activity was markedly higher in all nuclear subfractions, including nuclear matrix, from transplanted R3230AC mammary adenocarcinomas than in the analogous fractions from mammary gland of same tumor-bearing pregnant or lactating rats. Changes in host lactational status had no significant effect on subnuclear distribution of tumor DNA polymerase α activity, with the majority (60–75%) localized in soluble nucleoplasm and a significant amount (13–20%) retained in the nuclear matrix. In the host mammary gland, nuclear matrix-bound DNA polymerase α was highest, accounting for 48% of total nuclear activity, during late pregnancy when mammary cells undergo rapid raplication. During lactation, when cells in mammary gland cease to divide, only 8% of enzyme activity was in the nuclear matrix, while the majority (60–80%) of DNA polymerase α activity was localized in nucleoplasm. In both R3230AC tumor and mammary gland regardless of host's lactational status, the majority (60–80%) of DNA polymerase β activity was localized in the high salt-soluble chromatin. These present data thus suggest that, regardless of host lactational status, R3230AC tumor has many cycling cells, each with a large pool of DNA polymerase α molecules maintaining maximal and constant replicative activity, while normal mammary gland cells have a smaller pool of DNA polymerase α which become primarily matrix-bound only during active cell replication during late pregnancy. A constant localization of nuclear DNA polymerase β in chromatin in both mammary gland and the tumor suggest it is not important in mammary cell proliferation.     

Induction of glutathione S-transferases A, B and C in rat liver cytosol by trans-stilbene oxide

RNAase H, which catalyzes the hydrolysis of the RNA moiety of an RNA-DNA hybrid, was measured in the mammary gland of virgin, pregnant, lactating, and weaning Fischer rats and in the R3230AC mammary tumor grown in the same animals. In the normal mammary gland when DNA levels were low, as in the virgin state or during involution, RNAase H activity was also low. During pregnancy and lactogenesis when DNA levels increased, RNAase H activity, either on the basis of mammary gland weight or DNA content, also increased. During lactation when cellular proliferation ceases but rates of RNA and protein synthesis continue to reach peak values, RNAase H activity decreased. Compared to the corresponding enzyme from host glands, RNAase H from the R3230AC mammary tumor grown in pregnant and lactating hosts changes similarly, but to a lesser extent. The RNAase H activity which, ona tissue weight basis, was higher than in normal tissue also increased during pregnancy and directly after parturition, but decreased during lactation. During pregnancy these changes were accompanied by an increase in tumor DNA values. During lactation the tumor DNA values returned to the level seen in virgin hosts. These results are consistent with a role for RNAase H in DNA replication in rat mammary gland and in R3230Ac mammary tumor.     

Further studies on partially purified calf thymus DNA polymerase a.

Attempts to prevent the urea conversion of a 200-230,000 molecular weight DNA polymerase alpha to a 150-170,000 molecular weight form by the inclusion of protease inhibitors have not been successful. No other method has been found capable of dissociating a 50-70,000 fragment or subunit from the DNA polymerase subunit. Addition of this 50-70,000 subunit to the polymerase subunit does not aid the binding of the enzyme to DNA, but does have an effect on the utilisation of synthetic template-initiator complexes by the polymerase subunit.     

Endogenous inhibitors of specific benzodiazepine binding in the cerebral cortex of the bull

Chromatography of an acidic extract from bovine brain cortex on a Sephadex G-10 column demonstrated 7 fractions capable of inhibiting specific binding of 3H-diazepam to brain cortex membranes. It was established by ultrafiltration that the molecular weights of the inhibitors were not higher than 5000 dalton. Scatchard analysis of 4 main fractions has revealed a type of inhibition similar to the competitive one. Activity of all the fractions remained unchanged after pronase treatment.     

DNA polymerase alpha from normal rat liver is different than DNA polymerases alpha from Morris hepatoma strains

To investigate whether DNA replication in rat hepatoma cells is altered compared with that in normal rat liver, the main replicative enzyme, i.e. the DNA polymerase alpha complex, was partially purified from a slow-growing (TC5123) and a fast-growing (MH3924) Morris hepatoma cell strain as well as from normal rat liver. The purified DNA polymerase alpha complexes contained RNA primase. DNA polymerase alpha activities of these complexes were characterized with regard to both their molecular properties and their dNTP and DNA binding sites. The latter were probed with competitive inhibitors of dNTP binding, resulting in Ki values, and with DNA templates, yielding Km values. The sedimentation coefficients of native DNA polymerases alpha from Morris hepatoma cells were found to be lower than that of polymerase alpha from normal rat liver. Consequently, when following the procedure of Siegel and Monty for determination of molecular mass considerably smaller molecular masses were calculated for polymerases of hepatoma strains (TC5123, 127 kDa; MH3924, 138 kDa; rat liver, 168 kDa). Similar differences were found when the dNTP binding site was probed with inhibitors. Ki values obtained with butylphenyl-dGTP were higher for polymerases of the hepatoma strains than for that of normal rat liver. However, Ki values measured with aphidicolin and butylanilino-dATP were lower for DNA polymerase alpha from the fast-growing hepatoma cell strain than for that from normal rat liver, indicating a reduced affinity of the dNTP binding sites for dATP and dCTP. This reduced affinity could be responsible for lowered specificity of nucleotide selection in the base-pairing process which in turn may cause an enhanced error rate in DNA replication in malignant cells. Furthermore, when the DNA binding site was characterized by Michaelis-Menten constants using gapped DNA as a template, Km values were similar for all three DNA polymerases. In contrast, the Km value measured with single-stranded DNA as a template was found to be lower for DNA polymerase alpha from the fast-growing hepatoma MH3924 than for that from normal rat liver. Thus, the DNA-polymerizing complex from MH3924 combines both higher binding strength to single-stranded DNA templates and decreased nucleotide selection, properties which may enhance replication velocity and may lower fidelity.     

Prolactin binding to dissociated cells from rat mammary tumors and mammary gland.

Prolactin binding activity was studied in suspensions of cells which had been enzymatically dissociated from R3230AC mammary tumors, 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors and lactating rat mammary glands. Prolactin bound specifically with high affinity (apparent binding affinity = 4.0 X 10(9) M-1) to R3230AC tumor cells. Hormone binding at room temperature was proportional to cell number and increased with time of incubation up to 120-180 min. Prolactin binding to R3230AC tumor cells from diabetic animals was reduced by about 50%. Specific prolactin binding activity was also demonstrated in preparations of cells from DMBA-induced tumors and lactating mammary gland. The levels of hormone binding in both dissociated cells and subcellular particles prepared from these tissues varied as follows: DMBA-induced tumors > lactating mammary gland > R3230AC mammary adenocarcinoma.     

Nuclease sensitivity of estradiol-charged estrogen receptor binding sites in nuclei isolated from normal and neoplastic rat mammary tissues

The interaction of partially purified calf uterine estradiol-charged estrogen receptor ([3H]ER) with rat nuclei was studied in vitro. We previously observed a significantly greater number of [3H]ER binding sites (at saturation) in nuclei of R3230AC mammary tumors from intact vs ovariectomized (ovex) rats with no difference in the affinity of [3H]ER binding for these nuclei. We now report on the nuclease sensitivity of [3H]ER binding sites in nuclei from these tumors and from normal rat tissues. Digestion of tumor nuclei with deoxyribonuclease I (DNase I) prior to incubation with [3H]ER in vitro resulted in a progressive loss of [3H]ER binding capacity, which was not accompanied by alterations in the affinity of [3H]ER for the nuclei (Kd = 1-3 nM). A significantly lower concentration (P less than 0.005) of DNase I eliminated 50% of the [3H]ER binding sites in nuclei of tumors from intact hosts (8 unit.min/ml) compared to tumors from ovex hosts (22 unit.min/ml). These results indicate that DNA regions capable of binding ER are more susceptible to DNase I digestion in tumors from intact rats than those from ovex hosts, suggesting that the endogenous hormonal milieu is responsible, at least in part, for maintenance of nuclease-sensitive DNA conformations in this hormone-responsive mammary tumor. The amount of DNase I required to eliminate 50% of [3H]ER binding to nuclei from lactating mammary gland, liver, and kidney ranged from 14 to 56 unit.min/ml. Therefore, accessibility of [3H]ER binding sites to nuclease digestion in normal rat tissue is generally less than that of R3230AC tumors.     

Variable effects of DNA-synthesis inhibitors upon DNA methylation in mammalian cells.

Post-synthetic enzymatic hypermethylation of DNA was induced in hamster fibrosarcoma cells by the DNA synthesis inhibitors cytosine arabinoside, hydroxyurea and aphidicolin. This effect required direct inhibition of DNA polymerase alpha or reduction in deoxynucleotide pools and was not specific to a single cell type. At equivalently reduced levels of DNA synthesis, neither cycloheximide, actinomycin D nor serum deprivation affected DNA methylation in this way. The topoisomerase inhibitors nalidixic acid and novobiocin caused significant hypomethylation indicating that increased 5-mCyt content was not a necessary consequence of DNA synthesis inhibition. The induced hypermethylation occurred predominantly in that fraction of the DNA synthesized in the presence of inhibitor; was stable in the absence of drug; was most prominent in low molecular weight DNA representing sites of initiated but incomplete DNA synthesis; and occurred primarily within CpG dinucleotides, although other dinucleotides were overmethylated as well. Drug-induced CpG hypermethylation may be capable of silencing genes, an effect which may be relevant to the aberrantly expressed genes characteristic of neoplastic cells.     

Heterogeneous localization of adenylate and guanylate cyclases in R3230AC rat mammary adenocarcinoma cells

Adenylate and guanylate cyclase activities were demonstrated in R3230AC rat mammary adenocarcinomas by electron microscopic cytochemistry. Adenylate (AC) and guanylate (GC) cyclases were detected on plasma membrane of tumor epithelial cells, but not on fibroblasts and endothelial cells in the perivascular space. Both AC and GC activities were enriched in tumor epithelial cells at the periphery of the tumor lobular parenchyma rather than in cells in central core of the lobular parenchyma. Furthermore, the tumor cell plasma membranes facing the connective tissue stroma were in paucity or devoid of either enzyme activity. These heterogeneous distributions of both AC and GC among tumor epithelia suggest that R3230AC epithelial cells in different parts of the tumor mass may vary significantly in their regulation of cellular physiology.     

The relationship between two murine DNA-dependent DNA polymerases from the cytosol and the low molecular weight DNA polymerase.

After aqueous subcellular fractionation and partial purification by phosphocellulose chromatography, murine cells are found to contain a low molecular weight DNA-dependent DNA polymerase (beta) in the nuclear fraction and two distinguishable DNA-dependent DNA polymerases (C-I and C-II) in the cytosol. Both C-I and C-II are found in testis, liver, and regenerating liver; the amount of C-I being several fold increased in the regenerating liver and in immature testis. C-I and C-II are distinguishable by the criteria of salt sensitivity, inhibition by single-stranded DNA, elution from phosphocellulose, inhibition by 0.3 mM N-ethylmaleimide, template preference, and sedimentation coefficient. C-II is dissociated by 0.25 M KC1 to an active form of DNA polymerase of sedimentation coefficient 3.5 S while C-I is not dissociated, maintaining its sedimentation coefficient of 7.2 S. Many similar chemical and physical properties of C-II and the low molecular weight nuclear DNA polymerase (beta) suggest that C-II may represent an aggregate state of beta monomers, The size, reaction properties and the increase in enzyme activity under conditions of rapid cellular proliferation suggest C-I is analogous to the alpha DNA polymerase.     

Induction of DNA polymerase activities in the regenerating rat liver.

The levels of DNA polymerase alpha, DNA polymerase delta, and its accessory protein, proliferating cell nuclear antigen (PCNA) were examined in the regenerating rat liver. The levels of DNA polymerase alpha and delta activities in regenerating liver extracts were determined by the use of the DNA polymerase alpha specific inhibitor, BuAdATP [2-(p-n-butylanilino)-9-(2-deoxy-beta-D-ribofuranosyl) adenine 5'-triphosphate], and monoclonal antibodies. These reagents showed that the total DNA polymerase activities increased ca. 4-fold during regeneration and that the fraction of DNA polymerase delta activity at the peak was 40% of the total DNA polymerase activity. Immunoblots and inhibition studies using specific antibodies showed that DNA polymerase delta and epsilon and PCNA were concomitantly induced after partial hepatectomy. The levels of both DNA polymerase delta and epsilon and PCNA reached their maxima at 24-36 h post hepatectomy, i.e., at the same time that in vivo DNA synthesis reached its peak. Partial purification and characterization of DNA polymerases delta and epsilon from the regenerating rat liver were also performed. These observations suggest that the variation of DNA polymerase delta and epsilon and PCNA during liver regeneration is closely related to DNA synthesis and is consistent with their involvement in DNA replication.     

Plant sterols as selective DNA polymerase beta lyase inhibitors and potentiators of bleomycin cytotoxicity

In a survey of crude plant extracts for DNA polymerase beta lyase inhibitors, the hexanes extracts of Cladogynus orientalis, Hymenache donacifolia, and Heteropsis integerrima, and the methyl ethyl ketone extract of Acacia pilispina were found to exhibit good inhibition of the dRP lyase activity of DNA polymerase beta. Bioassay-guided fractionation of these extracts led to the isolation of three DNA polymerase beta lyase inhibitory phytosterols, namely stigmasterol (1) and beta-sitosterol (2), isolated from the hexanes extracts, and beta-sitosterol-beta-d-glucoside (3), isolated from the methyl ethyl ketone extract. Compounds 1-3 inhibited the DNA polymerase beta lyase activity with IC(50) values of 43.6, 43.3, and 72.4 microM, respectively. Compounds 1 and 2 were found capable of potentiating the action of bleomycin in cultured human tumor cells, consistent with the possibility that lyase inhibitors may find utility in vivo.     

Replication of antibiotic resistance plasmid R6K DNA in vitro.

A soluble extract prepared from cells of an Escherichia coli strain carrying the antibiotic resistance plasmid R6K is capable of carrying out the complete process of R6K DNA replication. DNA synthesis in vitro is dependent on the four deoxyribo- and ribonucleotide triphosphates and is sensitive to rifampin and streptolydigin, inhibitors of DNA-dependent RNA polymerase. The incorporation of deoxyribonucleotides into R6K DNA also is sensitive to actinomycin D, novobiocin, arabinofuranosyl-CTP, and N-ethylmaleimide. Kinetics of synthesis are linear for 60 to 120 min. Replication proceeds semiconservatively and supercoiled closed-circular DNA molecules are synthesized. Analysis by alkaline sucrose gradient centrifugation indicated that the early R6K DNA products contain DNA fragments of approximately 18 S in size, corresponding to the length between the R6K alpha origin of replication and the terminus of replication observed in vivo. Addition of exogenous supercoiled R6K DNA is inhibitory to the in vitro system, whereas the addition of R6K DNA in the form of relaxation complex stimulates R6K DNA synthesis to a small extent.     

Effects of diabetes and insulin on phosphoinositide metabolism in R3230AC mammary tumors.

The effects of diabetes and insulin administration on certain aspects of phosphoinositide metabolism in R3230AC mammary tumors were studied in vivo. Three weeks after diabetes was induced by streptozotocin, [3H]myoinositol incorporation into PI, PIP and PIP2 was increased in R3230AC tumors, whereas the formation of [3H]IP, [3H]IP2 and [3H]IP3 was decreased. Administration of protamine zinc insulin (3IU, twice daily, for 3 days) to diabetic rats decreased [3H]myoinositol incorporation into phosphoinositides and inositol phosphates in these mammary tumors. The R3230AC tumor from insulin-treated diabetic hosts had lower levels of unmetabolized [3H]-myoinositol compared to tumors from diabetic animals. Enzymatically-dissociated tumor cells from insulin-treated animals displayed decreased myoinositol transport in vitro. These findings suggest that the insulin-induced decrease in the turnover of inositol lipids in vivo in R3230AC mammary tumors could have resulted from the decreased level of [3H]myoinositol in these cells.