Characterization of acidic actin in mouse sarcoma 180 cells.

Mouse sarcoma 180 cells have a polypeptide that has the same molecular weight as actin but it is more acidic than alpha-actin. Its tryptic peptide pattern on reversed-phase HPLC was very similar to that of beta + gamma-actin, an actin sample prepared by affinity chromatography on DNase I-Sepharose contained the acidic polypeptide, and monoclonal anti-actin antibody reacted with it; therefore, the polypeptide is considered an actin isoform. The mRNA for this variant actin was identified by analyzing the polypeptides translated in vitro, which indicated that the variant actin is not a post-translationally modified form of any known actin. The variant actin was not stained by polyclonal anti-gizzard actin antibody which reacts with gamma-cytoplasmic, alpha-smooth and gamma-smooth muscle actins, nor by polyclonal anti-skeletal muscle actin antibody which reacts with skeletal, cardiac and alpha-smooth muscle actins. These results suggest that this variant actin is related to beta-cytoplasmic actin or, is a novel species whose N-terminal amino acid sequence is not Glu-Glu-Glu.

Inhibition of tumor-induced migration of bovine capillary endothelial cells by mouse and rabbit tumor necrosis factor.

The effect of tumor necrosis factor (TNF) on the tumor-induced endothelial migration was evaluated with the use of a phagokinetic track assay. TNF from both ddy mice and Japanese albino rabbits (sp act, 3-5 X 10(5) U/mg, respectively) was found to inhibit the migration of bovine capillary endothelial (BCE) cells stimulated by factors from tumor cells, such as the medium conditioned with mouse sarcoma 180 cells or human meningioma extract. These TNF preparations, however, did not affect the spontaneous migration of the BCE cells. When mouse TNF was further fractionated by polyacrylamide gel electrophoresis, only TNF-positive fractions showed an inhibitory activity on the tumor-induced endothelial motility. Moreover, monoclonal antibody against rabbit TNF completely neutralized its migration-inhibitory activity. These findings indicate that the observed inhibitory effect of TNF preparations on the endothelial motility evoked by tumor is exclusively ascribed to the function of TNF. This activity presumably is involved in the suppression of tumor angiogenesis in vivo.

Flow cytofluorometric analysis of choriogonadotropin-like material on the surface of human and mouse malignant cells.

Quantitation by flow cytofluorometry of the distribution of human choriogonadotropin (hCG)-like material on the surface of various human and mouse tumor cells grown in tissue culture and as solid tumors has been done using fluorescein-tagged rabbit antisera (IgG fraction) to intact hCG and, in one experiment, by use of two monoclonal antibodies specific for hCG. Fibroblasts were used as a negative (nontumorigenic) cell control, and a rabbit antiserum to human hemoglobin was used as reagent control. All malignant cells tested stained more intensely with the anti-hCG serum than with the antihuman hemoglobin serum. Positive reaction with the monoclonal antibodies specific for hCG provided strong evidence that the material stained was identical to hCG. Heterogeneity of the expression of the hCG-like material was notable both within each cell line and between different cell lines. This heterogeneity was not associated with cell-cycle phase. 3T3 fibroblast-like cells in vitro were originally negative for hCG but acquired reactivity with anti-hCG serum after ten passages.

[Distribution of 125I-DNA in mice bearing cancer].

Following intravenous administration, the distribution of 125I-DNA in mice bearing cancer showed that the 125I-DNA accumulated more in soft tissue tumors than the surrounding tissues, but it did not accumulate in inflammatory tissue. It shows that 125I-DNA has the excellent localizing potential for the soft tissue tumors.

Biological and biophysical properties of the tumor-localizing component of hematoporphyrin derivative.

Reverse-phase chromatography, aqueous gel exclusion, and nonaqueous gel exclusion were assessed as procedures for preparative fractionation of the tumor-localizing product hematoporphyrin derivative. Porphyrin accumulation, fluorescence, and photodynamic cytotoxicity were monitored using the murine Sarcoma 180 tumor. Aqueous gel exclusion chromatography can provide a hematoporphyrin derivative fraction enriched in the tumor-localizing component. A further enrichment occurs when this procedure is carried out at 55 degrees C, but nonlocalizing porphyrins could not be eliminated. While providing a better separation, reverse-phase chromatography cannot provide a tumor-localizing fraction free from contaminating protoporphyrin. However, this and other contaminants can be eliminated from the tumor-localizing fraction via nonaqueous gel exclusion chromatography. This latter separation provides two tumor-localizing products: a fast-eluting fraction enriched in the major photosensitizing component(s); and a more complex slowly eluting fraction enriched in fluorescence localizers.

Biological and biochemical properties of nerve growth factor synthesized by mouse S-180 cells in culture.

Nerve Growth Factor (NGF), a protein involved in the maintenance and differentiation of sensory and sympathetic neuronal cells [1], is synthesized by several different types of cells in culture [2-7]. In this paper, the biochemical and biological properties of NGF synthesized by a mouse S-180 sarcoma cell line were examined. These cells do not appear to produce the 7S-NGF molecule, a form of NGF found in high concentrations in the mouse submandibular gland [8]. The 7S-NGF is comprised of three distinct protein subunits named beta-NGF, alpha and gamma [9]. Although the S-180 cells do not produce 7S-NGF, the cells do synthesize one of the component subunits of 7S-NGF, the beta-NGF subunit. Biological, electrophoretic, immunological and molecular weight criteria were used to establish that the beta-NGF synthesized by the S-180 cells is very similar to the submandibular gland beta-NGF. The S-180 beta-NGF was bound to an unidentified binding component(s) which is not immunologically similar to either the alpha- or gamma-subunit. The functional significance of this interaction is not known.

Negatively charged RNase-susceptible molecules on the surface of ascites tumor cells.

RNase-susceptible ionogenic groups on the cell surface membranes of two leukemic and two nonleukemic strains of ascites tumor cells were studied by cell electrophoresis, DEAE-Sephadex A-25 column and paper chromatography, and indirect membrane immunofluorescence. RNase treatment of the nonleukemic ascites tumor cells (Ehrlich ascites tumor and Sarcoma 180) produced a significant reduction in their electrophoretic mobilities. When the cells were labeled with [3H]uridine then incubated with RNase, there was a marked increased in the radioactive nucleotides present in the incubation medium as compared to the results of the experiment with RNase-untreated controls. Indirect membrane immunofluorescence studies of nonleukemic ascites tumor cells suggest that the sites that react with anti-RNA antibody are distributed diffusely on their surfaces. RNase treatment of these cells markedly reduced their ability to react with the antibody. It thus appears that RNAs are present on the surface membrane of nonleukemic ascites tumor cells and that RNase digests these RNAs, removing negatively charged nucleotides from their electrophoretic surfaces. This results in a reduction in mobility. In contrast, leukemic ascites cells (L1210 and C1498) incubated with RNase showed no significant change in mobility or in the amount of nucleotides released into the incubation medium. Moreover, no fluorescence was found on the surface of cells examined by indirect membrane immunofluorescence. This suggests that leukemic ascites cells are devoid of RNAs on their surface.

Isolation of alpha-actinin from sarcoma 180 ascites cells plasma membranes and comparison with smooth muscle alpha-actinin.

alpha-Actinin from Sarcoma 180 ascites cell plasma membranes was purified after extraction in 10 mM Tris, 1 mM EDTA, 1 mM mercaptoethanol, pH 8.5, by chromatography on DEAE and hydroxyapatite for comparisons with smooth muscles alpha-actinin purified from turkey gizzard by the same procedure. The two proteins were found to be very similar by sedimentation analysis, gel filtration and dodecyl sulfate gel electrophoresis. Direct comparisons of smooth muscle, skeletal muscle and ascites alpha-actinin by amino acid analysis indicated a closer relationship between the smooth muscle and ascites proteins than between the smooth and skeletal muscle proteins. Both smooth muscle and ascites alpha-actinins cross-link F-actin filaments. The results suggest that the smooth muscle protein is a better system for understanding properties of non-muscle alpha-actinins than is the skeletal muscle protein.

[Quantitative determination of sulfhydryl groups with "mercurochrome" (author's transl)]. / Quantitative Bestimmung von Sulfhydryl gruppen mit "Mercurochrom".

Dibrommercuryfluoresceine (DBMF) reacts stoichiometrically and quantitatively with the thiol group of cysteine, glutathione and thioglycolic acid respectively, at pH 7.0. Polarographical and spectrometrical titrations clearly show that in the spectra of the investigated mercaptides the wave length of the first absorption maximum of DMBF (507 nm) remains unchanged but the molar extinction coefficient increases by approximately 20%. Serum albumin, ovalbumin, beta-lactoglobulin and glyceraldehydephosphatedihydrogenase, after incubation with DBMF, form adducts with the dye from which the pure mercaptide complexes were separated by means of column chromatogrphy. These complexes were separated by means of column chromatography. These complexes show a bathochromic shift (520 nm) of the dye band which is decreased now by 50%. The molar extinction coefficient epsilon 520 has been determined from 32,000 to 33,850. On the basis of these values SH-contents of the four proteins were obtained which are in good accordance with data previously published in the literature. No selective reaction, f.i. with more accessible or/and reactive SH-groups was observed. After 30 min incubation with DBMF and washing with isotonic phosphate buffer, native animal tumor cells show in the main absorption band the bathochromically shifted dye maximum. A first temptative estimation of the protein SH-groups yielded 1.7-2.1 X 10(-14) mole SH/single cell. This result lies between the SH-content determined microspectrometrically on cells stained with DDD-Fast Blue B (1.1-1.55 X 10(-14)) and macroscopically on cell homogenates with DTNB (3.1 X 10(-14)). Up to now, no certain information can be given whether or to what extent unspecific absorption effects possibly might be involved in the data obtained with DBMF treated cells, but interaction with nucleic acids can be excluded with certainty on the basis of relevant model experiments.

The effect of cancer on the spin-lattice relaxation time of mouse blood and tissue.

Spin-lattice relaxation time measurements were made on the muscle, heart, kidneys, spleen, liver, brain and blood of both normal mice and those injected with Sarcoma-180. As well as a marked increase in the relaxation time of the tumour itself, mice injected with Sarcoma-180 showed a rise in the kidney relaxation time, along with blood relaxation times which were often either above or below the range found in normal mice. Correlation coefficients for the relaxation times of the various organs were calculated and found to be very sensitive to the physiological state of the animal. For healthy mice, the correlations were reasonably high, but even mild stress decreased the correlations, whilst the presence of cancer almost completely destroyed the correlations. These findings are all consistent with the hypothesis that cancer affects the water regulatory system of the animal as a whole.

A major species of mammalian messenger RNA lacking a polyadenylate segment.

Translation of total polysomal RNA from sarcoma 180 ascites cells in a wheat germ cell-free system produces two major polypeptides, A and B, with molecular weights of 50,000 and 45,000, respectively. Fractionation on Millipore filters or on oligo(dT)-cellulose leads to retention of the mRNA specific for protein A in the poly(A)-containing fraction and to accumulation of the B mRNA in the unadsorbed poly(A)-deficient fraction. The mRNA for B sediments at approximately 18 S; it is released as a 50S ribonucleorprotein upon EDTA treatment of polysomes. Its translation is particularly sensitive to an inhibitor present in the polysomal RNA. The poly(A)-deficient mRNA for the 45,000 dalton polypeptide is also present in mouse myeloma MPC-11 cells, where it seems to be localized in membrane-bound polysomes.

[Sarcoma 180: growth and regression. Comparative investigations using flow-through and scanning cytophotometers as well as histological, cytological and autoradiographic techniques (author's transl)]. / Sarkom 180: Wachstum und Regression. Vergleichende Messungen mit dem Impuls- und Scanningzytophotometer sowie histologische, zytologische und autoradiographische Untersuchungen

MATERIAL AND METHODS: The growth and the regression of the experimental tumor S 180 was investiaged by volume measurements and by cytophotometric studies on 50 mice in each of a pilot and in the present main experiment. In addition, histological, cytological and cytokinetic examinations were performed. RESULTS AND DISCUSSION: Beginning on day 18, a spontaneous regression of the tumor was found in 20% and 38% of the animals, respectively. DNA-measurements were performed with a scanning-microspectrophotometer on tumor tissue imprints and tumor cell suspensions and were also carried out with a flow-through cytophotometer ICP on suspensions. DNA-histograms were plotted on days 4, 7, 12, 18, 20, 22, 26 and 29 after the transplantation. These revealed a constant position of the maxima at 2C for normal cells and at 4C and 8C for the tumor cells. In particular, by measuring a large total number of 4,968,000 nuclei with the ICP, distinct changes were found in the proportion of the single nuclei classes during growth and spontaneous regression. This technique also enables a quantitative measurement to be made of the DNA of cell debris from necrosis. With growing tumos, the proportion of the tumor cells increased to a maximum of 65% on day 18 and decreased to 37% on day 29. The DNA of cell debris grew from 8% to 47%. The proportion of normal cells was only 10% to 15% in the final phases. In tumors with a spontaneous regression the proportion of the tumor cell nuclei was 20% on day 18 and 11% on day 29. The proportion of the DNA of cell debris was again about 45%. The proportion of normal nuclei was greatly increased to 45%. Histological and cytological evidence together with measurements of the areas of nuclei and incorporation of 3H-TdR confirmed that the normal cells, the number of which increased during spontaneous regression were cells of a fibrovascular granulation tissue. While the rate of the DNA-synthesis of growing tumors continously decreased with age there was also a rapid decrease of the labelling index of spontaneously regressing tumors between days 12 and 18. In the final phase only the nuclei of the granulation tissue were labelled.

Association of the polyadenylate segment of messenger RNA with other polynucleotide sequences in mouse sarcoma 180 polyribosomes.

Limited digestion of polysomal RNA with pancreatic ribonuclease releases a structure consisting of poly(A) associated with other polyribonucleotide sequences. This complex can be purified by oligo(dT)-cellulose chromatography. Heating for formamide treatment causes the dissociation of fragments free of poly(A) from the poly(A)-containing components. The two types of fragments tend to reassociate under annealing conditions, and this association is prevented by poly(U). Control experiments indicate that this structure is not an artifact generated during the manipulations. The same structure can be obtained by limited RNase digestion of polyribosomes, followed by deproteinization. The results suggest that the mRNA in polyribosomes may have a defined configuration caused by the interaction of the poly(A) sequence with another segment of the RNA.