Inhibition of DNA synthesis and oncogene expression in tumor cells by the biological factor DIF.

A regulatory factor isolated from the maternal part of bovine placentas (decidua inhibitory factor, DIF) inhibits the incorporation of thymidine into the DNA of a variety of animal and human tumors. The degree of inhibition is dependent on the concentration of the factor. Results indicate that signal transduction occurs via a Ca2+ mobilizing pathway after specific binding of the inhibitor to tumor cell surface receptors. On of the main consequences of DIF action is the inhibition of c-fos and c-myc expression and/or degradation.

Inhibition of tumor cell proliferation by a novel growth regulator from placenta: modulation of membrane phosphorylation and oncogene expression.

The tumor specific inhibition of thymidine uptake by a negative growth regulator isolated from the maternal part of the bovine placenta is in concert with an inhibited expression of ras oncogenes. The results indicate that primary processing is blocked in case of Ha-ras, while a lower transcription rate or a stimulated degradation of mRNA is more likely for N-ras. These reactions are preceded by a specific binding of the inhibitor to tumor cell surface membranes. A modulated phosphate incorporation into some of the surface components is observed as a result of the binding process.

Tumor-specific inhibition of DNA synthesis and cell proliferation by a factor from bovine placenta--possible mechanism.

A polypeptide isolated from the maternal part of bovine placentas inhibits significantly the incorporation of thymidine into the DNA of tumor cells. When normal cells are used, this effect is found only to a very limited degree. Surface membrane components have been identified which are enriched on tumor cells and which are responsible for a better binding of the inhibitor to tumor cells than to normal cells. After internalization of the receptor-inhibitor complex, a decrease in the nuclear content of two proteins is observed, which might be a requirement for the inhibition of the DNA synthesis.

The evaluation of an average nucleotide composition from melting curves.

A new formula has been derived for the calculation of the average G + C content - X of DNAs from different origins using thermal melting data. As compared to existing formulas the new method gives highly accurate results, although being much easier to use than similar equations.

Changes in chromatin structure during rat liver regeneration as demonstrated by thermal melting analyses.

Chromatin was isolated from regenerating rat livers at different times after 2/3 hepatectomy and enriched with respect to active sequences. Thermal melting analyses of the material resulted in profiles reflecting the specific structural organization of the chromatin during different phases of the regeneration process: The high transcriptional activity of the chromatin 1, 16, and 36 hours after hepatectomy is demonstrated by a high proportion of DNA-protein complexes melting between 80 degrees C and 85 degrees C, while the period of active DNA synthesis 24 hours after hepatectomy is reflected by high amounts of components with Tm around 90 degrees C.

Early alterations in rat liver chromatin structure after a single dose of diethylnitrosamine.

In the chromatin of 24-h regenerating rat livers, derivative melting profiles are characterized by a high proportion of transitions above 90 degrees C. After the injection of diethylnitrosamine there is a rapid shift to lower melting temperatures. This is due to a rearrangement of the chromatin to higher amounts of nucleosomal components but possibly also a consequence of chemical modifications and conformational alterations of the DNA. In the nonregenerating liver essentially the same observations can be made, although reactions proceed significantly slower. These results are in good agreement with the observation that carcinogens are more active in tissues stimulated to rapid proliferation as compared to resting tissues.

Isolation and characterization of the growth-stimulating factor in supernatants of herpes simplex virus type 2 infected cells.

Lytic infection of CV1 cells with herpes simplex virus type 2 does not stimulate ornithine decarboxylase activity and there is no correlation between polyamines and a growth-stimulating factor (GSF) which is present in the supernatant of the cultures. The factor was partially purified by gel filtration on Sephadex G-50 and polyacrylamide gel electrophoresis. Gel filtration as well as dialysis through membranes with different pore diameters indicated a molecular weight between 3,500 and 10,000 daltons. GSF is not extracted from aqueous solutions by diethyl ether. The activity is partially sensitive to trypsin digestion and is completely destroyed by periodate oxidation. These results suggest that GSF is a glycoprotein or a glycopeptide whose mitogenic activity resides mainly in the sugar moiety.

Inhibition of thymidine incorporation into the DNA of normal and neoplastic cells by a factor from bovine maternal placenta: interaction of the inhibitor with cell membranes.

An inhibitor was isolated from the maternal part of bovine placentas which inhibits the incorporation of [3H]thymidine into the DNA of a variety of tumor cells to a significantly higher degree as compared to normal cells. This protein-type component was labeled by reaction with N-succinimidyl[2,3-3H]propionate, and interactions with receptors on cell membranes were investigated. Results indicated that receptors on tumor cell surfaces have higher binding capacities versus the inhibitor than those of normal cells. Moreover, an additional type of receptor was detected on tumor membranes. Obviously one of the reasons for the higher inhibitory capacity of the factor in tumor cells is the better internalization of this component.

[Attempts to characterize a tumor specific inhibitor extracted from the maternal part of the bovine placenta]. / Versuche zur Charakterisierung eines tumorspezifischen Inhibitors aus dem mütterlichen Anteil der Rinderplazenta.

A fraction was isolated from the maternal part of bovine placenta, which significantly inhibits the incorporation of thymidine into the DNA of tumour cells. This factor has, however, only a limited effect on the same reaction in bone marrow cells or in fibroblasts. It is suggested that the factor enters the cell by an active transport mechanism and that the active part thereof is a protein. The protein pattern of the chromatin is not influenced by the factor as is the phosphorylation or acetylation of nuclear proteins.

The phosphorylation of histones in herpes virus infected Vervet-monkey kidney cells (CV-1).

The phosphorylation of histones of the F1 group and of fraction F2a2 is stimulated in monkey kidney cells (CV-1) to almost two times the control values 14 hours after their infection with herpes virus, type 2. At the same time high amounts of viral DNA are produced. It seems very likely that the stimulated phosphorylation of these histone fractions is a prerequisite for the enhanced synthesis of DNA and possibly also RNA.

The phosphorylation of histones in rat liver undergoing malignant transformation.

The incorporation of inorganic phosphate into H1-histones of rat liver was stimulated after the repeated s.c. administration of diethylnitrosamine. This stimulation was observed as early as after the fifth daily injection of the carcinogen and amounts to approximately 3 times the control value on the 60th day of the experiment. The effect was reversible when the application of the carcinogen was not extended beyond 4 weeks. A correlation was observed between these phenomena and alterations in the morphological structure, although the latter required a higher amount of single doses before the first signs of a forthcoming malignant transformation were seen. There was a difference in distribution of phosphate between the individual phosphorylation sites of the H1 molecule as compared to stimulated phosphate incorporation induced by adenosine cyclic 3':5'-monophosphoric acid or liver regeneration. The stimulated phosphorylation was not due to the inflammatory action of the carcinogen.

The tumor enhancing property of herpes simplex virus type-2 (HSV-2).

The s.c. infection of DBF-1 mice with HSV-2 has a tumor enhancing effect on simultaneously i.m. implanted MCA-induced syngeneic spindle cell sarcoma. Thus with the dosage used here, the first palpable tumors appeared on day 6 in the virus treated group as compared to day 9 p.i. in the sham treated batch. Further, more tumors were formed: 75% among the infected and 40% among the sham injected. The tumor yield could be modulated: no difference between the 2 groups was found when the tumor cell dose was increased sufficiently to advance the first appearance of tumors to less than 6 days. The same result was obtained when the neoplastic cells were implanted 3 days ahead of injection of the virus. When the dose of the cells was decreased, no tumors were found in the sham treated batch, whereas there were some in the infected group. Previous in vitro mixing of the neoplastic cells with virus completely prevented tumor formation, probably due to their lysis by the virus. In fact, in tissue cultures, MCA cells become lytically infected with HSV-2. It is speculated that a similar situation occurs in acutely ill mice which die before the 13th day p.i., i.e., the tumor cells are destroyed by the virus. In fact, those animals, as a rule are free of neoplasmas. Thus it appears that an acute HSV-2 infection prevents MCA sarcoma whereas a latent infection promotes its growth. The possible reasons for this tumor enhancement caused by HSV-2 infection are discussed.

The phosphorylation of nuclear proteins in the regenerating and premalignant rat liver and its significance for cell proliferation.

In liver regeneration or neoplastic transformation, phosphorylation of nuclear proteins is stimulated. In the regenerating liver all main histone fractions are involved in this process. The type of histone phosphorylated seems to be dependent on the position of the partially synchronized cells within the generation cycle. At a time when most cells are exhibiting maximum HnRNA-synthesis, histone F2a2 belongs to those fractions with highly stimulated phosphate incorporation. Phosphorylation of this fraction alone is stimulated by cyclic AMP in parallel to a stimulation of HnRNA-synthesis. The preneoplastic liver is characterized by oscillating phosphorylation and dephosphorylation reactions of nearly all histone fractions during the first days of N-nitroso-diethylamine administration. After 2 months of carcinogen feeding a 50-150% stimulation of the phosphorylation of Fl subfractions is observed. The phosphate content of the other histones, however, has returned to the original level. A series of further proteins, isolated together with the histones, show very similar phosphorylation characteristics. These proteins are mostly of non-histone origin. It is suggested that some of them are responsible for the transport of RNA with messenger properties within the cell.