Characterization of 83-kilodalton nonmuscle caldesmon from cultured rat cells: stimulation of actin binding of nonmuscle tropomyosin and periodic localization along microfilaments like tropomyosin

Nonmuscle caldesmon purified from cultured rat cells shows a molecular weight of 83,000 on SDS gels, Stokes radius of 60.5 A, and sedimentation coefficient (S20,w) of 3.5 in the presence of reducing agents. These values give a native molecular weight of 87,000 and a frictional ratio of 2.04, suggesting that the molecule is a monomeric, asymmetric protein. In the absence of reducing agents, the protein is self-associated, through disulfide bonds, into oligomers with a molecular weight of 230,000 on SDS gels. These S-S oligomers appear to be responsible for the actin-bundling activity of nonmuscle caldesmon in the absence of reducing agents. Actin binding is saturated at a molar ratio of one 83-kD protein to six actins with an apparent binding constant of 5 X 10(6) M-1. Because of 83-kD nonmuscle caldesmon and tropomyosin are colocalized in stress fibers of cultured cells, we have examined effects of 83-kD protein on the actin binding of cultured cell tropomyosin. Of five isoforms of cultured rat cell tropomyosin, tropomyosin isoforms with high molecular weight values (40,000 and 36,500) show higher affinity to actin than do tropomyosin isoforms with low molecular weight values (32,400 and 32,000) (Matsumura, F., and S. Yamashiro-Matsumura. 1986. J. Biol. Chem. 260:13851-13859). At physiological concentration of KCl (100 mM), 83-kD nonmuscle caldesmon stimulates binding of low molecular weight tropomyosins to actin and increases the apparent binding constant (Ka from 4.4 X 10(5) to 1.5 X 10(6) M-1. In contrast, 83-kD protein has slight stimulation of actin binding of high molecular weight tropomyosins because high molecular weight tropomyosins bind to actin strongly in this condition. As the binding of 83-kD protein to actin is regulated by calcium/calmodulin, 83-kD protein regulates the binding of low molecular weight tropomyosins to actin in a calcium/calmodulin-dependent way. Using monoclonal antibodies to visualize nonmuscle caldesmon along microfilaments or actin filaments reconstituted with purified 83-kD protein, we demonstrate that 83-kD nonmuscle caldesmon is localized periodically along microfilaments or actin filaments with similar periodicity (36 +/- 4 nm) as tropomyosin. These results suggest that 83-kD protein plays an important role in the organization of microfilaments, as well as the control of the motility, through the regulation of the binding of tropomyosin to actin.     

Differential expression of multiple forms of arginase in cultured cells

Summary Arginase (EC 3.5.3.1), the final enzyme in the urea cycle, catalyzes the cleavage of arginine to orthinine and urea. At least two forms of this enzyme, Al and All, have been described and are probably encoded by discrete genetic loci. The expression of these separate genes has been studied in mammalian cells grown in culture. The permanent rat-hepatoma line H4-II-E-C3 contained exclusively the Al enzyme; the form in mammals comprising about 98% of the arginase activity in liver and erythrocytes but catalyzing only about one half of that reaction in kidney, gastrointestinal tract, and brain. By contrast, human-embryonic-kidney and -brain cells, after transformation with the human papovavirus BK, contained only the All species of arginase, which form contributes the remaining half of that catalysis in those mammalian tissues in vivo. We report here the results of an extensive study on the properties of these two forms of arginase in the three cell lines, including K_m values for arginine, behavior on polyacrylamide gels under non-denaturing conditions, and cross-reactivity with lapine antibodies against the arginases from either rat or human liver.[/p]Presented in part at the annual meeting of the Society for Pediatric Research, Washington, D.C., May, 1982. Pediatr. Res. 16:195A.     

Differential localization of tropomyosin isoforms in cultured nonmuscle cells

We have previously shown that chicken embryo fibroblast (CEF) cells and human bladder carcinoma (EJ) cells contain multiple isoforms of tropomyosin, identified as a, b, 1, 2, and 3 in CEF cells and 1, 2, 3, 4, and 5 in human EJ cells by one-dimensional SDS-PAGE (Lin, J. J.-C., D. M. Helfman, S. H. Hughes, and C.-S. Chou. 1985. J. Cell Biol. 100: 692-703; and Lin, J. J.-C., S. Yamashiro-Matsumura, and F. Matsumura. 1984. Cancer Cells 1:57-65). Both isoform 3 (TM-3) of CEF and isoforms 4,5 (TM-4,-5) of human EJ cells are the minor isoforms found respectively in normal chicken and human cells. They have a lower apparent molecular mass and show a weaker affinity to actin filaments when compared to the higher molecular mass isoforms. Using individual tropomyosin isoforms immobilized on nitrocellulose papers and sequential absorption of polyclonal antiserum on these papers, we have prepared antibodies specific to CEF TM-3 and to CEF TM-1,-2. In addition, two of our antitropomyosin mAbs, CG beta 6 and CG3, have now been demonstrated by Western blots, immunoprecipitation, and two-dimensional gel analysis to have specificities to human EJ TM-3 and TM-5, respectively. By using these isoform-specific reagents, we are able to compare the intracellular localizations of the lower and higher molecular mass isoforms in both CEF and human EJ cells. We have found that both lower and higher molecular mass isoforms of tropomyosin are localized along stress fibers of cells, as one would expect. However, the lower molecular mass isoforms are also distributed in regions near ruffling membranes. Further evidence for this different localization of different tropomyosin isoforms comes from double-label immunofluorescence microscopy on the same CEF cells with affinity-purified antibody against TM-3, and monoclonal CG beta 6 antibody against TM-a, -b, -1, and -2 of CEF tropomyosin. The presence of the lower molecular mass isoform of tropomyosin in ruffling membranes may indicate a novel way for the nonmuscle cell to control the stability and organization of microfilaments, and to regulate the cell motility.     

Differences in DNA-binding proteins isolated from normal and transformed human cells

When the DNA-binding proteins (DBPs) of WI38 normal human fibroblasts and their SV40-transformed counterpart were compared, two DBPs were present in greater amounts in the transformed cells. These two DBPs, P5a and P6b, were also present in greater amounts in HeLa cells versus WI38 cells and in chemically transformed human liver cells versus normal liver cells. Therefore, these DBP differences do not appear to be specific for transformation by SV40. Increased amounts of P5a were present in 7 of 9 transformed cell lines examined. The two tumor cell lines lacking the P5a change were sensitive to density-dependent inhibition of replication, whereas the other seven cell lines were not. This correlation suggests that the increase in P5a may play a role in the release from density-dependent inhibition of replication observed in most transformed cells.     

Purification and characterization of multiple isoforms of tropomyosin from rat cultured cells

We have previously shown that rat cultured cells contain five isoforms of tropomyosin (Matsumura, F., Yamashiro-Matsumura, S., and Lin, J. J.-C. (1983) J. Biol. Chem. 258, 6636-6644) and that these tropomyosins are differentially expressed upon cell transformation (Matsumura, F., Lin, J. J.-C., Yamashiro-Matsumura, S., Thomas, G. P., and Topp, W. C. (1983) J. Biol. Chem. 258, 13954-13964). To examine functions of tropomyosin in microfilament organization, we have purified and partially separated the multiple isoforms of tropomyosin by chromatography on hydroxylapatite. Analyses of cross-linked dimers produced by air oxidation have revealed that all isoforms except the tropomyosin isoform with apparent Mr of 35,000 form homodimers. Although these tropomyosins share many properties characteristic of tropomyosin, structural analyses at a peptide level and immunological analyses have shown that the five isoforms can be classified into two groups, i.e. tropomyosins with higher apparent Mr (Mr = 40,000, 36,500, and 35,000) and tropomyosins with lower apparent Mr (Mr = 32,400 and 32,000). The low Mr tropomyosins show less ability for head-to-tail polymerization and lower affinity to actin than the high Mr tropomyosins. We suggest that these differences in properties may be related to the changes in microfilament organization observed in transformed cells.     

Procoagulant activity of mouse transformed cells: Different expression in freshly isolated or cultured cells

The online version of the original article can be found at     

Procoagulant activity of mouse transformed cells: Different expression in freshly isolated or cultured cells

Summary This study was originally designed to investigate wheter there is any correlation between the type of procoagulant activity (PCA) and the tumorigenicity of transformed cells. The data obtained are relevant to this question and to defining the differences in the expression of cellular activities depending on the in vitro system used. PCA was measured and characterized in normal, immortalized, and tumorigenic mouse fibroblasts. In all the lines studied the activity was of tissue factor type, as established with functional, enzymatic, and immunochemical criteria. However, the PCA of cells freshly isolated from the tumors induced by tumorigenic cell lines was of cancer procoagulant type, i.e. a cysteine protease with direct factor X activator activity. The same cells, when cultured in vitro, expressed again PCA of tissue-factor type. These results suggest that either a tumor-host interaction is required for the expression of cancer procoagulant or the latter activity, produced by tumor cells under in vitro conditions, is destroyed or inactivated during the culture period. Our findings caution against defining the procoagulant activity of tumors based on experiments on cultured cells. This work was partially supported by the Italian National Research Council and by the Italian Association for Cancer Research.     

Isolation and characterization of tropomyosin-containing microfilaments from cultured cells

We have developed a new method for the rapid isolation of tropomyosin-containing microfilaments from cultured cells using anti-tropomyosin monoclonal antibodies. Anti-tropomyosin monoclonal antibodies induce the bundle formation of microfilaments, which can be easily collected by low speed centrifugation. Electron microscopic studies of the isolated microfilaments show periodic localization of tropomyosin along the microfilaments of nonmuscle cells with a 33-34 nm repeat. Furthermore, the isolated microfilaments have the ability to activate the Mg2+-ATPase activity of skeletal muscle myosin to almost the same extent as skeletal muscle F-actin (filamentous actin). This microfilament isolation method is applicable to a variety of cell types, including REF-52 cells (an established rat embryo line), L6 myoblasts, 3T3 fibroblasts, Chinese hamster ovary cells, baby hamster kidney (BHK-21) cells, mouse neuroblastoma cells, gerbil fibroma cells, and chicken embryo fibroblasts. Sodium dodecyl sulfate-polyacrylamide gel analysis shows that, in addition to actin, microfilaments isolated from REF-52 cells contain five species of tropomyosin with apparent Mr = 40,000, 36,500, 35,000, 32,400, and 32,000, alpha-actinin, and as yet unknown proteins with apparent Mr = 83,000 and 37,000. The molar ratio of total tropomyosin (dimer) to actin in the isolated microfilaments is 1:8. The patterns of these multiple forms of tropomyosin were found to change when REF-52 cells were transformed with SV40 or adenovirus type 5.     

Alkaline phosphatase activity of normal and transformed cultured human cells

A study was made of the relationship between the activity of alkaline phosphatase and the proliferation of cultured human cells with different replicative potentials. It is shown that alkaline phosphatase plays a role as one of endogenic stimulators of cellular proliferation. The ageing of diploid cells is accompanied by a decrease in the enzyme activity. Maximum activity was observed during a period of logarithmic cell growth. Addition of placental alkaline phosphatase to the synchronized diploid cells stimulated DNA synthesis in the S-phase of the cell cycle. Heteroploid cells with a high growth rate possessed a 30-100 times higher alkaline phosphatase activity than in the diploid cells. Under certain conditions alkaline phosphatase may presumably function as a proteinkinase.     

Differential gene expression in normal and transformed human mammary epithelial cells in response to oxidative stress

Oxidative stress plays a key role in breast carcinogenesis. To investigate whether normal and malignant breast epithelial cells differ in their responses to oxidative stress, we examined the global gene expression profiles of three cell types, representing cancer progression from a normal to a malignant stage, under oxidative stress. Normal human mammary epithelial cells (HMECs), an immortalized cell line (HMLER-1), and a tumorigenic cell line (HMLER-5) were exposed to increased levels of reactive oxygen species (ROS) by treatment with glucose oxidase. Functional analysis of the metabolic pathways enriched with differentially expressed genes demonstrated that normal and malignant breast epithelial cells diverge substantially in their response to oxidative stress. Whereas normal cells exhibit the up-regulation of antioxidant mechanisms, cancer cells are unresponsive to the ROS insult. However, the gene expression response of normal HMECs under oxidative stress is comparable to that of the malignant cells under normal conditions, indicating that altered redox status is persistent in breast cancer cells, which makes them resistant to increased generation of ROS. We discuss some of the possible adaptation mechanisms of breast cancer cells under persistent oxidative stress that differentiate them from normal mammary epithelial cells as regards the response to acute oxidative stress.     

Lack of tropomyosin correlates with the absence of stress fibers in transformed rat kidney cells

We have utilized epithelial rat kidney cells and their Kirsten viral transformant (442) to examine the role of actin-binding proteins in cellular morphogenesis. Normal rat kidney cells are well spread while the transformed cells are more spherical, poorly adherent, and lack actin stress fibers (Rubin, R.W., Warren, R.H., Lukeman, D.S. and Clements, E. (1978) J. Cell Biol. 78, 28-35). By immunofluorescence, antitropomyosin prominently stains normal rat kidney cell stress fibers while only a weak, nonspecific fluorescence is observed in 442 cells. Using two-dimensional gel electrophoresis, tropomyosin can be detected in normal rat kidney cells homogenates. The tropomyosin subunits are enriched in Triton-extracted filamentous normal rat kidney cell models, and in extracts of normal rat kidney cell homogenate produced by using a rapid myosin affinity technique to isolate actin and actin-associated proteins. The identity of the tropomyosin subunits has been confirmed by electrophoretic mobility, lack of proline, and the peptide map generated by limited proteolysis. None of these techniques have detected tropomyosin in the corresponding 442 preparations. Our results suggest that the transformation of normal rat kidney cells has led to an overall reduction in tropomyosin content. This may be related to the inability of 442 cells to organize filamentous actin stress fibers.     

Organization of tubulin in normal and transformed rat kidney cells

We have carried out a quantitative biochemical and ultrastructural study of tubulin and microtubules in a normal rat kidney (NRK) cell line and its viral transformant (442) in culture. Under equivalent culture conditions, both cell lines contain the same amount of tubulin according to a colchicine-binding assay. The normal and transformed cells differ significantly, however, with respect to the state of organization of their tubulin. Counts of microtubules in sectioned cells indicate that NRK cells have almost twice as many microtubules per unit area of cytoplasm as the 442 cells. Centrifugation studies, on the other hand, show that 442 cells have almost twice as much pelletable tubulin as the NRK cells. We propose, therefore, that the transformed cells contain a large amount of tubulin which is in some alternative aggregate form that is not morphologically detectable as microtubles in the cytoplasm     

1-Methylnicotinamide and NAD metabolism in normal and transformed normal rat kidney cells

NAD, 1-methylnicotinamide, S-adenosylmethionine, and S-adenosylhomocysteine levels were analyzed in different clones of untransformed normal rat kidney cells and in cells transformed by different viruses. No consistent changes in the levels of these metabolites were apparent as a result of malignant transformation, and also differences in the levels of metabolites did not correlate with growth rate in the various cell lines. 3-Deazaadenosine prevented synthesis of 1-methylnicotinamide but not of NAD. The S-denosylmethionine/S-adenosylhomocysteine ratio did not change in serum-starved, growth-arrested cells although 1-methylnicotinamide synthesis increased about twofold. These results were used to consider possible physiological roles for 1-methylnicotinamide. Its intracellular levels did not correlate with growth rate and were not altered by transformation. No evidence was obtained that its synthesis is involved with maintenance of nicotinamide of S-adenosylmethionine levels. Thus the biological function for 1-methylnicotinamide remains a mystery.     

Polyribosomes associated with microfilaments in cultured lens cells

Epithelial hamster lens cells, transformed by SV40 can be grown in suspension culture. Triton X-100 extraction of these cells grown under conditions when ribosome run off is blocked releases about 40% of the total amount of polyribosomes, designated as free- and loosely-bound polyribosomes. The Triton ghosts retain the remaining polysomal population which can be released by a combined treatment with deoxycholate and Nonidet P 40. Electron microscopic examination of the ghosts reveals microfilament-associated ribosome clusters next to a fraction of polysomes still attached to membranes. Preincubation of the cells with cytochalasin D prior to polyribosome isolation enables us to discriminate between these two latter polysome populations. The experiments indicate that about 25% of the polyribosomes are attached to microfilaments, while the remaining 35% are tightly bound to the membranes of the endoplasmic reticulum. When the different polyribosome classes were translated in a reticulocyte lysate, no significant differences could be observed in the patterns of the newly synthesized polypeptides. In all cases actin was one of the major products synthesized de novo.     

Nitrobenzylthioinosine-sensitive and -resistant nucleoside transport in normal and transformed rat cells

Cultured Novikoff rat hepatoma and Walker 256 carcinoma cells have previously been reported to express only nitrobenzylthioinosine (NBTI)-resistant uridine transport and to lack high affinity NBTI-binding sites, whereas the latter are common on all other types of cultured mammalian cells from different species [1-7) X 10(5) sites/cell) which have been investigated with the exception of a transport-deficient cell variant which lacks high-affinity NBTI-binding sites. The present study shows that lack of NBTI sensitivity of transport and of NBTI-binding sites in Novikoff and Walker 256 cells are not related to the species or tissue origin of these cells. Uridine transport in a variant (NRM) of Novikoff hepatoma cells, in HTC rat hepatoma cells, normal rat kidney (NRK) cells, rat erythrocytes and rat hepatocytes was inhibited 15-60% by 10-500 nM NBTI and the cells expressed high-affinity NBTI-binding sites (Kd = 0.1-0.6 nM). The apparent turnover numbers for the NBTI-sensitive nucleoside carriers fell into two classes, with those for transformed cells about 10-times higher than those for the normal rat cells.     

The role of microfilaments and of microtubules in taurocholate uptake by isolated rat liver cells

The role of microfilaments and microtubules on bile salt transport was studied by investigating the influence of a microfilament and a microtubule inhibitor, cytochalasin B and colchicine, respectively, on taurocholate uptake by isolated hepatocytes in vitro. Hepatocytes were prepared by the enzyme perfusion method and [¹⁴C]taurocholate uptake velocity was determined by a filtration assay. Taurocholate uptake obeyed Michaelis-Menten kinetics, maximal uptake velocity and apparent half-saturation constants averaging $\text{0.87 ±}\text{SD}\text{0.05}\text{nmol}\text{· s}{}^{\mathrm{?1}}\text{· 10}{}^{\mathrm{?6}}\text{cells}$ and $\text{10.9 ± 1.8 μ}\text{M}$, respectively. Cytochalasin B ($\text{4.2–420 μ}\text{M}$) inhibited taurocholate uptake in a competitive fashion; $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ being $\text{33 ± 7 μ}\text{M}$. At concentrations above 100 μM the compound decreased ³⁶Cl membrane potential and intracellular K⁺ concentration. Other parameters of cell viability were not affected by cytochalasin B. Colchicine (0.1–1.0 mM), by contrast, inhibited taurocholate uptake non-competitively, $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ being $\text{0.47 ± 0.07}\text{mM}$. The inhibition brought about by colchicine was considerably smaller than that induced by cytochalasin B. None of the parameters of cell viability tested was affected by colchicine. These results suggest that microfilaments may be involved in the carrier-mediated hepatocellular transport of bile salts. This could, at least in part, account for cytochalasin B-induced cholestasis. The contribution of the microtubular system, if any, is less important quantitatively. The mechanisms whereby these two components of the cytoskeleton partake in bile salt transport remain to be elucidated.     

Less membrane-associated actin in transformed versus normal rat kidney cells

Plasma membranes were isolated by adhering normal rat kidney cells (NRK) and their Kirsten viral transformants (442 cells) to a poly-L-lysine treated surface, and lysing them with hypotonic buffer. Scanning and transmission electron micrographs of substrate-adhered membranes verified the presence of a membrane-microfilament “lawn” attached to the substrate. These microfilaments bind heavy meromyosin in a uniform polarity which demonstrates that the microfilament network is composed primarily of actin, and that the spatial organization of the membrane was not disrupted during isolation. The actin to total protein ratio of the substrate-attached membrane was measured on one dimensional sodium dodecyl sulfate polyacrylamide gels. Our results demonstrate that the membrane-associated actin to total membrane-associated protein ratio is significantly greater for NRK than for 442 cells. However, no unique changes in the 180 most prominent polypeptides could be detected by 2 dimensional gel electrophoresis.