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.     

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.     

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.     

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.     

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.     

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.     

Inhibition of DNA polymerase alpha activity by proteins from rat liver

We determined that there is a protein in rat liver capable of inhibiting DNA polymerase alpha. To assay for this inhibitor, DNA polymerase alpha was purified from R3230AC rat mammary tumor, a rich source of this enzyme. Protein fractions from Sephacryl S200 gel filtration of total soluble liver extract showing inhibition of DNA polymerase alpha were further chromatographed on DEAE-cellulose. This step revealed two inhibitor protein populations with the major form corresponding to a molecular weight of 143,000 dalton. Soluble extract from isolated rat liver nuclei also showed the presence of at least two inhibitors; the major form was 200,000+ dalton in molecular weight. Both the 143,000 and 200,000+ dalton inhibitor proteins were capable of inhibiting the R3230AC tumor DNA polymerase alpha in a dose-dependent manner. These inhibitors exhibited similar inhibition of nuclear matrix-associated DNA polymerase alpha from either the R3230AC tumor or from regenerating rat liver.     

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.     

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.     

Effect of estrogen on synthesis of glucose-6-phosphate dehydrogenase in R3230AC mammary tumors and uteri.

The effect of estrogen on synthesis of glucose-6-phosphate dehydrogenase (D-Glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) in the R3230AC mammary adenocarcinoma of ovariectomized Fischer rats was investigated. Enzyme synthesis was estimated by techniques using immunochemica precipitation and isolation of enzyme protein from tissues of rats that had been given radioactive leucine prior to sacrifice. The antibody-enzyme complex was dissociated and glucose-6-phosphate dehydrogenase was isolated after electrophoresis on sodium dodecyl sulfate-acrylamide gels. Administration of estradiol-17beta produced a two-fold increase in glucose-6-phosphate dehydrogenase activity, which was preceded by a five-fold increase in specific synthesis of glucose-6-phosphate dehydrogenase in R3230AC tumors. At least a 15-fold increase in enzyme synthesis was observed in the uterus. The rate of enzyme degradation (t 1/2) in the tumor was estimated at 17 h. These data indicate that the estrogen-induced increase in glucose-6-phosphate dehydrogenase activity was due to a de novo increase in enzyme synthesis.     

Effects of diabetes on the dietary lipid alteration of R3230AC mammary carcinoma growth in rats

To examine the effects of diabetes on the alteration of R3230AC mammary tumor growth by dietary lipids, streptozotocin-induced diabetic rats were fed diets containing either 20% corn oil (HF), 20% hydrogenated cottonseed oil (HCTO), or 0% fat (FF). Diabetes resulted in lower tumor weights and body weights compared to those of intact animals. Unlike intact animals, relative tumor weight (g tumor/100 g body wt) of diabetic animals fed HF diets were not greater than those from animals fed FF diets. However, in these diabetic animals, growth of tumors in HF-fed rats was faster than in HCTO-fed rats, a relationship similar to that seen in intact rats. A surprising result was the almost twofold greater tumor weight/100 g body wt observed in diabetic FF-fed rats compared to those fed HCTO diets. Insulin binding to tumor plasma membranes from diabetic animals was higher in rats fed HF diets than in rats fed FF or HCTO diets. The tumor plasma membrane fatty acid composition of diabetic rats fed FF and HCTO diets displayed higher proportions of the monounsaturates (C18:1 and C21:1) and decreased amounts of the polyunsaturates (C18:2 and C20:4) compared to the levels observed in membranes from HF-fed rats. These results, as well as the insulin binding data, were similar to those obtained using intact animals. The data presented here indicate that the more rapid growth of the R3230AC mammary tumor seen in intact animals fed high polyunsaturated fat vs fat-free diets did not occur in diabetic animals.     

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.     

Dual secretory and myoepithelial differentiation in the transplantable R3230AC rat mammary carcinoma

The hormone-responsive R3230AC mammary carcinoma, serially transplantable in Fisher rats, shows striking functional and morphological similarities to the normal mammary gland. We have studied its cellular composition by both light and electron microscopy, employing markers of myoepithelial and epithelial cells. We identified two cell types: the major cellular component corresponded to epithelial milk-protein secreting cells, while a second component showed immunocytochemical and ultrastructural characteristics of the myoepithelial cells. These cells were positive with a monoclonal antibody detecting alpha smooth muscle actin. The dual differentiation which normally occurs in breast ducts is therefore reproduced in a malignant experimental tumor. The coexistence of neoplastic cell populations, divergent in morphology and function, that persist in a tumor despite many transplant generations, leads to reconsideration of the relationship between cellular differentiation and malignant transformation.     

Milk-specific RNase as a marker of differentiation of rat mammary tumors

Various rat mammary tumors were analyzed for the presence of a milk-specific Ca2+-stimulated RNase (Ca2+-RNase). When crude extracts of some differentiated tumors--adenocarcinomas of MT/W9, MT/W9a, R3230AC, DMBA-1, DMBA-8, and DMBA-14 and 3MN squamous cell carcinoma--were assayed for RNase activity under various ionic conditions, it was always highest in the presence of Ca2+/EDTA than under any other ionic condition. The opposite was true in invasive MT/W449a and 13762 adenocarcinomas, poorly differentiated SMT/2A carcinomas, MAMF2/TC fibrosarcoma, and MT/A fibroadenoma. Sephacryl S-200 chromatography separation of tumor extracts confirmed the presence of Ca2+-RNase in those differentiated tumors and absence of the enzyme from other tumors. Expressing the activity as a ratio of Ca2+/EDTA to either Mg2+/EDTA or EDTA alone to more clearly represent the relative level of Ca2+-RNase activity further illustrates the distinct differences between tumor classes. Thus Ca2+-RNase is a sensitive marker for use in the characterization of rat tumors with respect to differentiated mammary functions.     

Acyl specificity in triacylglycerol synthesis by mammary adenocarcinoma R3230AC in Fischer rats

Activities and acyl specificities of both sn-glycero-3-phosphate and diacylglycerol acyltransferases in microsomal fractions isolated frorn homogenates of the mammary adenocardinoma R3230AC carried by Fischer rats were compared to those from normal mammary glands of lactating Fischer rats. Although the neoplasm exhibited lower activities for these two enzyme reactions, the specificities for acyl-CoAs as donors were quite similar to those found in the normal tissue counterpart. Long-chain acyl-CoAs were preferred substrates for the sn-glycero-3-phosphate acyltransferase reaction while acyltransferase with diacylglycerol as acceptor showed much less preference. With both normal and neoplastic tissues, the products of each reaction were the same i.e., phosphatides with sn-glycero-3-phosphate and triacylglycerol with diacylglycerol as acyl acceptors, respectively. All results support the concept of a non-random distribution of fatty acids in the triacylglycerol of this mammary adenocarcinoma in virgin rats which is the same as that from the normal tissue in lactating animals.     

Effects of laser photodynamic therapy on tumor phosphate levels and pH assessed by 31P-NMR spectroscopy

The effects of photodynamic therapy using 632 nm photoradiation emitted from an ion pumped dye laser system on the phosphate metabolite levels of rat mammary tumors were monitored by 31P-NMR spectroscopy. A dramatic decline to almost undetectable levels, in the ratio of whole tumor beta-ATP (NTP) to Pi was observed after systemic administration of 5 mg/kg Photofrin II 24 h prior to exposure of R3230AC rat mammary tumor to laser irradiation at 180 and 360 J/cm2 total fluence. This decline in ATP was accompanied by a concomitant increase in the levels of Pi relative to the total observable phosphate signals. Whole tumor pH was calculated from the chemical shift in inorganic phosphate using the water proton signal as reference. Under the same treatment conditions used to monitor the phosphate metabolites following Photodynamic Therapy, the pH of the tumor as a whole decreased approximately 0.35 units at the time when the beta-ATP to Pi ratios were lowest. This maximal decrease in whole tumor ATP levels and pH, which occurred at 4-6 h post irradiation, was followed by a gradual return to pre-treatment levels over a 24 h period. These results demonstrate that Photodynamic Therapy employing porphyrin photosensitization and monochromatic laser irradiation is effective in reducing both tumor high energy phosphate levels and pH. Depending on sensitizer dose and light fluence, metabolic inhibition, represented by depleted nucleoside triphosphates and elevated Pi, may be reversible.     

Cytoplasmic dye transfer between metastatic tumor cells and vascular endothelium

Metastatic colonization of a secondary organ site is initiated by the attachment of blood-borne tumor cells to organ-specific adhesion molecules expressed on the surface of microvascular endothelial cells. Using digital video imaging microscopy and fluorescence activated cell sorting techniques, we show here that highly metastatic cells (B16-F10 murine melanoma and R3230AC-MET rat mammary adenocarcinoma cells) previously labeled with the fluorescent dye BCECF begin to transfer dye to endothelial cell monolayers shortly after adhesion is established. The extent of BCECF transfer to endothelial cell monolayers is dependent upon the number of BCECF-labeled tumor cells seeded onto the endothelial cell monolayer and the time of coculture of the two cell types, as visualized by an increase in the number of BCECF-positive cells among cells stained with an endothelial cell-specific mAb. Dye transfer to BAEC monolayers proceeds with a progressive loss of fluorescence intensity in the BCECF-labeled tumor cell population with time of coculture. The transfer of dye is bidirectional and sensitive to inhibition by 1-heptanol. In contrast, poorly metastatic B16-F0 melanoma cells and non-metastatic R3230AC-LR mammary adenocarcinoma cells do not efficiently couple with vascular endothelial cells. It is inferred from these experiments and from the amounts of connexin43 mRNA expressed by tumor cells that tumor cell/endothelial cell communication is mediated by gap junctional channels and that this interaction may play a critical role in tumor cell extravasation at secondary sites.     

Molecular cloning of cDNA coding for rat proliferating cell nuclear antigen (PCNA)/cyclin.

The 'proliferating cell nuclear antigen' (PCNA), also known as cyclin, appears at the G1/S boundary in the cell cycle. Because of its possible relationship with cell proliferation, PCNA/cyclin has been receiving attention. PCNA/cyclin is a non-histone acidic nuclear protein with an apparent mol. wt of 33000-36000. The amino acid composition and the sequence of the first 25 amino acids of rabbit PCNA/cyclin are known. Using an oligonucleotide probe corresponding to the sequence of the first five amino acids, a cDNA clone for PCNA/cyclin was isolated from rat thymocyte cDNA library. The cDNA (1195 bases) contains an open reading frame of 813 nucleotides coding for 261 amino acids. The 3'-non-coding region is 312 nucleotides long and contains three putative polyadenylation signals. The mol. wt of rat PCNA/cyclin was calculated to be 28 748. The deduced amino acid sequence and composition of rat PCNA/cyclin are in excellent agreement with the published data. Using the cDNA probe, two species of mRNA (1.1 and 0.98 kb) were detected in rat thymocyte RNA. Southern blot analysis of total human genomic DNA suggests that there is a single gene coding for PCNA/cyclin. The deduced amino acid sequence of rat PCNA/cyclin has a similarity with that of herpes simplex virus type-1 DNA binding protein.     

Core-modified porphyrins. Part 5: Electronic effects on photophysical and biological properties in vitro

21,23-Dithiaporphyrins 2-11 were prepared as analogues of 5,20-diphenyl-10,15-bis(4-carboxylatomethoxy)phenyl-21,23-dithiaporphyrin 1 to examine the impact of electronic properties at the 5- and 20-meso-positions. The effects of the electronic properties at the meso-rings were not significant with respect to absorption spectra, quantum yields for the generation of singlet oxygen and for fluorescence. While some differences were noted in the n-octanol/pH 7.4 buffer partition coefficient, log D(7.4), among the compounds, log D(7.4) did not critically influence the cellular uptake or phototoxicity. None of the dithiaporphyrins 1-11 displayed dark toxicity at concentrations up to 1 x 10(-5) M. Once irradiated with 5 J cm(-2) of 350-750 nm light, five porphyrins 2, 3, 5, 6, and 8 killed over 80% of R3230AC rat mammary adenocarcinoma cells at 5 x 10(-7) M photosensitizer. Among these five, compound 3 bearing 5-phenyl and 20-(4-fluorophenyl) substituents was the most potent photosensitizer toward R3230AC cells showing 67% cell kill at 1 x 10(-7) M 3. Bulky substituents at the 5- and 20-positions gave photosensitizers with minimal phototoxicity.     

Synthesis, properties, and photodynamic properties in vitro of heavy-chalcogen analogues of tetramethylrosamine

Thio and seleno analogues of tetramethylrosamine were prepared by the directed-metalation/cyclization of the corresponding N,N-diethyl 2-(3-dimethylaminophenylchalcogeno)-4-dimethylaminobenzamide to the 2,7-bis-(N,N-dimethylamino)-9H-chalcogenoxanthen-9-one followed by the addition of phenylmagnesium bromide, dehydration, and ion exchange to the chloride salt. The thio and seleno tetramethylrosamines had longer wavelengths of absorption and higher quantum yields for the generation of singlet oxygen than tetramethylrosamine. Both the thio and selenoanalogues of tetramethylrosamine were efficient photosensitizers against R3230AC rat mammary adenocarcinoma cells in vitro.