Cell surface constituents of sarcoma 180 ascites tumor cells

The cell surface protein components of Sarcoma 180 ascites tumor cells have been investigated by a combination of plasma membrane isolation techniques and lactoperoxidase iodination. For plasma membrane isolation cells were homogenized in the presence or absence of Zn²⁺ and fractionated by sucrose density gradient centrifugation or a two-phase partition to give large membrane fragments or membrane envelopes. Membrane purification was monitored by phase contrast microscopy and chemical and enzyme marker assays. The membrane preparations were analyzed by acrylamide gel electrophoresis in sodium dodecylsulfate. Each preparation showed a common protein pattern of about 15 bands ranging in molecular weights from 33 000 to >300000. Two carbohydrate-containing bands were also present in all preparations. Membranes prepared with Zn²⁺ were much less fragmented and showed much greater amounts of three high molecular weight components than those prepared in the absence of Zn²⁺. This might suggest a role for these components in membrane stabilization.The tumor cells were also subjected to iodination with lactoperoxidase, followed by membrane isolation and acrylamide gel electrophoresis in sodium dodecylsulfate in order to identify polypeptides accessible to the cell surface. The major radioactive band coincided with the major carbohydrate-containing band, presumably a surface glycoprotein. A second carbohydrate-containing band showed variable labeling behavior between different cell preparations. This material had a high molecular weight, as indicated by both acrylamide gel electrophoresis and gel permeation chromatography in dodecylsulfate. Several other components are labeled to a lesser extent in the intact cell.     

Adriamycin-induced changes in the surface membrane of sarcoma 180 ascites cells

Adriamycin increases (a) the rate of agglutination of Sarcoma 180 cells by concanavalin A after brief exposure of 2–3 h and (b) membrane fluidity as measured by ESR within 30 min of exposure at concentrations of the anthracycline of 10^?7–10^?5 M. The effect of adriamycin on agglutination is not due to an increase in the number of surface receptors for concanavalin A, since the extent of binding of the lectin is not altered by adriamycin and no change occurs in the rate of occupancy of the concanavalin A binding sites by the lectin in cells treated with the antibiotic. The order parameter, a measurement of membrane fluidity, decreases in cells exposed to adriamycin and is dose-related. The results indicate that adriamycin can induce changes in the surface membrane of Sarcoma 180 cells within a brief period of exposure to a low but cytotoxic level of this agent.     

Zinc and copper accumulation and isometallothionein induction in mouse ascites sarcoma S180A cells.

To investigate Zn and Cu accumulation and isometallothionein (iso-MT) induction in ascites-sarcoma S180A cells, 5 micrograms of Zn2+ or Cu2+/g body weight was administered to tumour-bearing mice intraperitoneally. In the tumour cells the Zn or Cu concentration increased more than in the host liver, which is the target organ for those metals; the maximum Zn or Cu level was about 2-3 times that in the host liver. The amounts of Zn-MT or Cu-MT accumulated in the tumour cells and host liver were proportional to such dose accumulation levels in the each cytosol; the maximum level of Zn-MT or Cu-MT was 4 or 2 times higher than in the host liver. MT accumulated in the tumour cells showed two subfractions (MT-1 and MT-2); the ratio of Zn (or Cu) bound to MT-1 to that bound to MT-2 in the host liver and tumour cells was 1.0 (or 1.0) and 0.7 (or 0.25) respectively, suggesting that the induction level of MT-2 in the tumour cells is more than that of MT-1. The h.p.l.c. profiles (using an anion-exchange column) of the isolated MT-1 and MT-2 subfractions from Zn-treated normal-mouse liver showed a single peak (MT-1-1) and two peaks (MT-2-1 and MT-2-2) respectively; mouse MTs were separated into three isoforms. In the ascites cells, the MT fraction obtained by a gel filtration was also separated into three isoforms; however, the amount of MT-2-1 isoform was 3 times that in the Zn-treated normal-mouse liver.     

A cytological study of the yoshida sarcoma. An ascites tumor op white rats

Summary In the Yoshida sarcoma a strain of tumor cells is present which have their own characteristic chromosome constitution and multiply by regular mitosis. The well-balanced complement of ±40 chromosomes consists of two distinct groups: one is represented by 22 to 24 rodshaped elements which probably come directly and without change from the original normal cell, the other group comprises 16 to 18 Vand J-shaped chromosomes which are specific for the tumor cells. Their exact origin is unknown, but must be mutational in character. Because of this morphological peculiarity, the chromosomes of the tumor cells are markedly different from those of the host cells for which 42 rodshaped elements are typical. No transitional types bridging the gap between ordinary and tumor cells occur. The individuality of the chromosomes in the strain cells remains unchanged during successive transplant generations from rat to rat. The growth of the tumor is primarily caused by the proliferation of these strain cells. In the course of multiplication part of the proliferating cells become abnormal and undergo aberrant mitotic processes owing probably to an alteration of the spindle mechanism, structural changes of the chromosomes, and some other causes. The frequently occurring tumor cells showing mitotic abnormalities are, therefore, derivatives of the sub-diploid strain cells. Destruction of the derivative cells by chemical treatment (podophyllin, CaCl₂) is followed by multiplication of the resistant strain cells.Comparable evidence has been found in two new strains of ascites tumor similar to the Yoshida sarcoma. Their strain cells have the same total chromosome number as the Yoshida strain cells and, within the set, the same two groups of rod-shaped and of Vor J-shaped chromosomes, but differ from each other as well as from the Yoshida sarcoma in the number of Vand J-shaped chromosomes.Contribution No. 263 from the Zoological Institute, Faculty of Science, Hokkaido University, Sapporo, Japan.     

IMP dehydrogenase. II. Purification and properties of the enzyme from Yoshida sarcoma ascites tumor cells

The preceding paper showed that IMP dehydrogenase [IMP:NAD+ oxidoreductase, EC 1.2.1.14] tended to form a precipitable complex(es) through ionic and hydrophobic interactions. On the basis of these observations, a method was developed for purification of IMP dehydrogenase from Yoshida sarcoma ascites cells. On SDS-polyacrylamide gel electrophoresis, the purified preparation (1.19 U/mg protein) appeared homogeneous and its minimum molecular weight was estimated to be 68K daltons. Amino acid analyses indicated a subunit molecular weight of 68,042. Molecular sieve chromatography in the presence of 10% (NH4)2SO4 showed that the molecular weight of the native enzyme was 127K daltons. These values indicate that the native enzyme is composed of two identical subunits. However, the purified enzyme gave 4 protein bands on polyacrylamide gel electrophoresis under non-denaturing conditions, and appeared as a single fraction in the vicinity of the void volume on Ultrogel AcA 34 column chromatography at low salt concentration, indicating that its molecular weight exceeded 200K daltons. These findings indicate that the enzyme tends to aggregate owing to its own physicochemical characteristics. The Km values for IMP and NAD were calculated to be 12 and 25 microM, respectively, and the Ki values for XMP, GMP, and AMP to be 109, 130, and 854 microM, respectively. The purified enzyme showed full activity in the presence of K+, and K+ could be partially replaced by Na+. PCMB inactivated the enzyme, but the activity was completely restored by the addition of DTT. Cl-IMP also inactivated the enzyme and IMP prevented this inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)