Effect of different doses of a chalone-containing alcohol precipitate of Ehrlich ascitic tumor on the mitotic activity and DNA synthesis in this tumor

Chalone-containing preparation has been obtained from ascitic Ehrlich's tumour by alcohol precipitation and the effect of various preparation doses on mitotic activity and DNA synthesis in the tumour has been studied. The preparation was shown to suppress tumour cell proliferation, acting on mitosis initiation and mitotic S phase as well as on DNA synthesis in the cells at S phase of mitotic cycle. The effect of the preparation on DNA synthesis in phase S cells was more pronounced than on cells entering DNA synthesis phase. The changes in all the parameters examined were dose-dependent. The preparation effect was tissue-specific.     

Effect of different fractions obtained by gel filtration of a chalone-containing preparation from Ehrlich ascites tumor on mitotic activity and DNA synthesis in this tumor

The effect of various fractions of chalone-containing preparation from ascite Ehrlich's tumour obtained by gel filtration on ultragel Ac-A-44 on mitotic activity and DNA synthesis in the tumour has been studied. The chalone-containing preparation (alcohol precipitate) was shown to suppress entering of tumour cells into M- and S-phase and DNA synthesis. After gel filtration, the partial division of active chalone component which inhibits entering of cells into M- and S-phase took place. The component inhibiting DNA synthesis eluted with G1-chalone.     

Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells. Properties of the purified polymerase.

Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells, partially purified by chromatography on DNA-agarose, was obtained as a more than 80% homogeneous preparation by isoelectric focusing in a sucrose gradient. The polymerase activity was shown to be associated with the major protein in the preparation. Results obtained by electrophoresis in the presence of sodium dodecyl-sulfate indicated that poly(ADP-ribose) polymerase consists of a polypeptide chain with a molecular weight of 130 000. Ultracentrifugation at non-denaturating conditions indicated that the active enzyme may be an oligomeric form of this polypeptide chain. The isoelectric point of the polymerase was 9.40. The effects of various additions to the assay mixture on the synthesis of poly(ADP-ribose) as well as some kinetic data, are given. It is shown that poly(ADP-ribose) is a highly efficient inhibitor of its own synthesis, and results are presented which suggest that the well-known stimulatory effect of DNA on the synthesis is due to reduction of this inhibitory effect of the product.     

Sphingosylphosphorylcholine is a remarkably potent mitogen for a variety of cell lines

The effect of spingosylphosphorylcholine on cellular proliferation was investigated in a variety of cell types. Spingosylphosphorylcholine at low concentrations greatly stimulated DNA synthesis and cell division in quiescent Swiss 3T3 fibroblasts. The increased DNA synthesis was also accompanied by pronounced morphological alterations. Spingosylphosphorylcholine was remarkably more potent than other known growth factors and also acted synergistically with insulin, epidermal growth factor, fibroblast growth factor, and the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, to induce cellular proliferation. Sphingosylphosphorylcholine was less effective in stimulating DNA synthesis in rapidly growing normal and transformed cells. Spingosylphosphorylcholine appears to be a new type of potent, wide-spectrum growth promoting agent.     

Effect of sulfochloranthine on protein and nucleic acid biosynthesis in Escherichia coli

Sulfochloranthine was shown to be bacteriostatic for Escherichia coli B cells grown in a chemically defined medium at a concentration of 0.002%, sublethal at a concentration of 0.005%, and lethal at 0.01% (0.000312, 0.00078 and 0.00156% of active chlorine, respectively). Protein synthesis by E. coli B cells was noticeably inhibited when the concentration of the preparation was 0.002%, and stopped completely at a 0.01% concentration of the preparation. Biosynthesis of nucleic acids, in particular DNA, was inhibited to a lesser extent. The bacteriostatic concentration of the preparation had virtually no effect on DNA biosynthesis, but inhibited RNA biosynthesis by 50%. Sulfochloranthine used at sublethal doses inhibited synthesis of both DNA and RNA by 75%; DNA and RNA biosynthesis ceased at the lethal concentration of the preparation.     

Mechanisms of the antitumor action of spirobromine in mice with leukemia P-388

The influence of new antitumor drug, spirobromine, a derivative of dispirotripiperazine, on DNA synthesis in tumor cells and organs at different times after its injection into mice with P388 leukemia has been studied. The duration of DNA synthesis inhibition in tumor cells was found to correlate with spirobromine antitumor activity. A certain selectivity of action of the studied compound on DNA synthesis in P 388 leukemia cells as compared to the action on DNA synthesis in bone marrow, small intestine, spleen and liver of tumor animals was observed.     

Heliomycin suppression of RNA synthesis in a cell-free system]

Heliomycin inhibited in vitro the RNA-polymerase reaction catalyzed by the preparation of DNA-dependent RNA-polymerase from E. coli. The blocking effect increased with a rise in the antibiotic concentration. The inhibitory effect of heliomycin decreased, when the amount of RNA-polymerase in the system increased. Yet, it did not depend on the content of DNA and the nature of the DNA preparation. Preincubation of RNA-polymerase with DNA resulting in formation of the enzyme-matrix complex did not prevent blocking RNA synthesis by heliomycin. Suppression of the RNA-polymerase reaction did not depend on the time of the antibiotic addition to the polymerizing system. Heliomycin had a significant activity not only with respect to the bacterial RNA-polymerase, but also in the system containing the enzyme isolated from the cells of Crithidia oncopelti.     

Action of a chalone-containing esophageal preparation on its epithelial cell proliferation

The effect of esophageal chalone on epithelial cell reproduction in the esophagus was studied. Lyophilized aqueous extract from the esophagus was used. The following properties of the esophageal preparation have been revealed: it is water-soluble; it is present in the same tissue where it acts; it has tissue-specific effect (the preparation does not act on the mitotic and radioactive index in the epithelial crypts of the small intestine); its action is short-term and reversible; its effect on DNA division and synthesis in the esophageal epithelial cells is dose-dependent. Therefore, it is suggested that the esophageal preparation contains a chalone.     

Purification of poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells by chromatography on DNA-agarose.

Poly(ADP-ribose) polymerase with a high specific activity was obtained from Ehrlich ascites tumor cells by extraction of nuclei with 175 mM potassium phosphate, followed by chromatography on DNA-agarose. Electrophoretic analysis indicated that the preparation contained two proteins, one of which was shown to catalyze the synthesis of poly(ADP-ribose). As expected from results obtained by other workers, the synthesis was inhibited by nicotinamide and thymidine, and stimulated by DNA. Addition of histones gave inhibition of the synthesis, unless DNA was present in the reaction mixture.     

Fractionation of a chalone-containing substance from Ehrlich's ascites tumor using high pressure chromatography]

The effect of various fractions of chalone--containing preparation from ascyte Ehrlich's tumour obtained by high performance liquid chromatography (HPLC) on mitotic activity and DNA synthesis in the tumour has been studied. After filtration the division of active chalone component which inhibits entering cells into M-phase and S-phase took place. The component inhibiting DNA synthesis eluated with G1-chalone.     

Effect of actinomycin D on nucleic acid hybridization: the cause of erroneous DNA elongation during DNA synthesis of RNA tumor viruses in vitro

Actinomycin D, known for its suppression of cellular RNA synthesis and for the reduction of the rate of synthesis of double-stranded DNA by the RNA tumor virus RNA-dependent DNA polymerase, was found to interact with single-stranded DNA in such a way as to inhibit DNA . DNA and DNA . RNA hybridizations. This finding is discussed in the light of the observation that DNA elongation during DNA synthesis of RNA tumor viruses is blocked in vitro in the presence of actinomycin D. It thus supports the model that hybridization is a necessary step during RNA tumor virus DNA synthesis.     

Participation of the SSB protein in the repair of the DNA of Ehrlich ascites carcinoma cells following UV irradiation

Synchronous changes were detected in the SSB-protein content of the chromatin and in the rate of repair DNA synthesis at different time intervals after UV-irradiation of Ehrlich ascites tumor cells. The amount of SSB-protein in the extra-chromatin fraction was in an inverse relation to its content in the chromatin, whereas the cumulative SSB-protein content remained invariable. Similar changes in the SSB-protein content of the chromatin and in repair synthesis were also registered after the effect of various doses of UV-light. The increase of the SSB-protein content in the chromatin was not connected with the postirradiation accumulation of single-strand sites in DNA.     

Mode of antitumor action of recombinant human tumor necrosis factor on the sarcoma Meth A transplanted in the mouse

The mode of antitumor action of rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation with respect to time course, dose-response relationships and selectivity of the effects. The maximal cytotoxic effect on tumor cells revealed by inhibition of DNA synthesis and maximal lesional effect on tumor vasculature revealed by change in blood pool-size in the tissue were detected at 30 min and 1 h after administration of rHu-TNF, respectively. The dose-response relationship between cytotoxic and tumoricidal effects of rHu-TNF was irrespective of administration route. ED50s of these antitumor effects after i.v. administration of rHu-TNF were about 50 times as high as ED50s after i.t. administration. ED50 of i.t. given rHu-TNF for vascular effect was about 20 times as high as that for cytotoxicity while ED50 of i.v. rHu-TNF for vascular effect was only 2-3 times as high as that for cytotoxicity. The whole body autoradiographies with [125I]HSA given i.v. to see the blood influx into tumor tissue and [14C]thymidine given i.v. to see DNA synthesis in the whole body after administration of rHu-TNF revealed that the distribution of radioactivity was markedly changed in the tumor alone without any detectable change in other whole body tissues. In conclusion, the in vivo antitumor effect of rHu-TNF given i.t. or i.v. appears to be exerted through the direct action on Meth A sarcoma rather than indirectly on tumor vasculature. Under present conditions, the effect of rHu-TNF in the whole body tissues seems rather selective on cells and vasculature of the tumor.     

Effect of cell density on the inhibition of tumor cell attachment and nucleic acid synthesis by selenite

The effect of cell density on the sensitivity of tumor celles to selenite has been examined. The inhibitory effect of selenite on cellular DNA and RNA synthesis was significantly greater in higher density cultures of HeLa cells and A2780 ovarian tumor cells. High-density cells were also more sensitive to the inhibitory effect of selenite on cell attachment. This difference could not be accounted for by a higher intracellular level of glutathione, since there was no significant difference between the cells at high or low density. The high-density cells were found to take up more selenium per cell during the exposure period; the resulting higher level of intracellular Se could explain their greater sensitivity to selenite. This hypothesis is supported by the observation that DNA synthesis in nuclei isolated from high-density cells did not exhibit higher sensitivity to inhibition by selenite than synthesis in nuclei isolated from low-density cells.     

Synthesis of Protein and Nucleic Acid by Disrupted Spheroplasts of Pseudomonas schuylkilliensis

Osmotically shocked spheroplasts obtained from Pseudomonas schuylkilliensis strain P contained about 54, 32, 28, and 82% of the total cellular protein, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and phospholipid, respectively. This preparation was capable of incorporating (32)P-orthophosphate into RNA and DNA, (3)H-adenosine or (3)H-uridine into RNA, and (3)H-leucine or (14)C-phenylalanine into protein. These activities were not found in the cytoplasmic fraction which contained most of the glucose-6-phosphate dehydrogenase activity. The synthesis of RNA by intact and disrupted spheroplast preparations was sensitive to actinomycin D, chromomycin A(3), streptovaricin, rifampin, Lubrol W, Triton X-100, and sodium deoxycholate, whereas RNA synthesis by intact cells was insensitive to these agents. Ethylenediaminetetraacetic acid, porcine pancreatic lipase, the protoplast-bursting factor, high concentrations of salts, and washing the preparation inhibited the synthesis of RNA by disrupted spheroplasts but had little or no effect on intact spheroplasts. Most of the newly synthesized RNA made by disrupted spheroplasts had the characteristics of messenger RNA. The DNA present in this preparation functioned as a template for RNA synthesis; continued protein synthesis was dependent on concomitant RNA synthesis. An unusual feature of the preparation was the finding that the synthesis of macromolecules was completely dependent on oxidative phosphorylation.     

Effects of inhibin on the in vitro synthesis of desoxyribonucleic acid in the rat testis

Three preparations of inhibin extracted from ram rete testis fluid (RTF) and from human seminal plasma (HSP) reduce tritiated thymidine incorporation into testicular desoxyribonucleic acids (DNA) in vitro. Effect of low molecular inhibin from RTF is dose-dependent. Castrated ram serum does not modify testicular DNA synthesis in vitro. Besides their suppressive action on follicle stimulating hormone (FSH) secretion in vivo and in vitro, these inhibin preparation display a direct inhibiting effect on testicular DNA synthesis and, thus, on mitotic activity. Identity between inhibin and testicular chalones are discussed.     

Stimulation of DNA synthesis by tumor promoters in primary rat hepatocytes is not mediated by arachidonic acid metabolites

Studies in vivo using inhibitors of eicosanoid synthesis suggested that prostaglandins may play a role in mediating tumor promotion in liver by agents such as phenobarbital (PB). However, it is not clear whether any stimulation of arachidonic acid metabolism/prostaglandin formation results directly from the action of tumor promoters on hepatocytes or indirectly from effects of promoters on Kupffer cells or other non-hepatocytes. Our laboratory has been utilizing relatively pure populations of rat hepatocytes under the defined conditions of primary cultures, to investigate growth-stimulatory actions of tumor promoters, an important element in the promotion stage of carcinogenesis. It has been shown that most if not all liver tumor promoters tested stimulate hepatocyte DNA synthesis when added in combination with factors such as EGF, insulin, and glucocorticoid. In the present study, we sought evidence for a role of prostaglandins (PGs) in the direct growth-stimulatory actions of tumor promoters on hepatocytes. PGE(2), PGF(2 alpha), and PGD(2) cause concentration-dependent stimulation of hepatocyte DNA synthesis, while arachidonic acid was without any effect. PGE(2) and PGF(2 alpha) required the presence of dexamethasone to exert significant effects. These PGs did not further augment the stimulatory effect of EGF. In contrast, PGD(2) stimulated DNA synthesis in the presence or absence of insulin, dexamethasone, or EGF. The effect of tumor promoters on arachidonic acid metabolism, as measured by [(3)H]arachidonic acid release and PGE(2) production, was determined. The phorbol ester TPA significantly increased [(3)H]arachidonic acid release as well as PGE(2) formation in hepatocytes in line with known effects in other cell types. However, liver tumor promoters phenobarbital (PB), alpha-hexachlorocycohexane (HCH), 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), and pregnenolone-16 alpha-carbonitrile (PCN) were without effects. Finally, inhibitors of arachidonic acid metabolism were tested for effects on the ability of TPA or liver tumor promoters to stimulate DNA synthesis by direct action on cultured hepatocytes. In all cases, lack of selective inhibition was observed. Taken together, the results show that while prostaglandins may directly stimulate DNA synthesis in hepatocytes, they are unlikely to mediate the direct growth-stimulatory actions of liver tumor promoters.     

The effect of saffron on intracellular DNA, RNA and protein synthesis in malignant and non-malignant human cells.

Extract of saffron (Crocus sativis) has previously been shown to inhibit colony formation and cellular DNA and RNA synthesis by HeLa cells in vitro. In order to compare the sensitivity of malignant and non-malignant cells to saffron, we examined the effect of the extract on macromolecular synthesis in three human cell lines: A549 cells (derived from a lung tumor), WI-38 cells (normal lung fibroblasts) and VA-13 cells (WI-38 cells transformed in vitro by SV40 tumor virus). We found that the malignant cells were more sensitive than the normal cells to the inhibitory effects of saffron on both DNA and RNA synthesis. There was no effect on protein synthesis in any of the cells.     

Initiation of DNA synthesis in the parotid salivary gland of adult mice by a factor isolated from acellular fluid of the Ehrlich ascites carcinoma

The rate of DNA synthesis in the parotid salivary gland of adult mice is very low. We have purified about 5 000-fold a mitogen from the aceIlular ascitic fluid of the Ehrlich ascites carcinoma which stimulates DNA synthesis in the parotid salivary gland in vivo. This stimulation of DNA synthesis was produced with a protein concentration of this mitogen of 3 μg per 25 g of body weight. The purification procedure included ammonium sulfate fractionation and DEAE Sephacel column chromatography. This potent, heat-labile mitogen is presumed to be a protein with a mol.wt, of 3.5×10³ to 1.3×10⁴. The data indicate that this new factor is quite different from epidermal growth factor and tumor growth factor. Hypophysectomy did not prevent the stimulatory effect of this mitogen on the parotid salivary gland, indicating that the pituitary gland is not involved directly in mediating the mitogenic effect.