Regulation of polyamine biosynthesis in normal and transformed hamster cells in culture

Several aspects of polyamine biosynthesis were compared in low-passage hamster embryo fibroblasts and transformed hamster fibroblasts. Earlier studies had demonstrated a larger and longer-lasting induction of ornithine decarboxylase activity in transformed cells than in hamster embryo fibroblasts. The increases in intracellular polyamine concentrations after serum stimulation were much greater in chemically transformed HE68BP cells than in normal hamster fibroblasts. Treatment of confluent cultures with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, greatly potentiated ornithine decarboxylase induction by fresh medium in HE68BP cells, but not in hamster fibroblasts. A similar synergistic effect was observed when transformed cells, but not normal cells, were treated with the combination of insulin and promoter. HE68BP cells were capable of growth in medium containing serum concentrations as low as 0.5%, whereas only concentrations of 5% or more supported the growth of hamster embryo fibroblasts. Low serum concentrations induced ornithine decarboxylase in HE68BP cells but not in normal cells, and a given serum concentration always produced a greater induction of ornithine decarboxylase in transformed than in normal cells.Another enzyme involved in polyamine synthesis, $\text{S-}\text{adenosyl-}\text{L}\text{-methionine}$ decarboxylase was induced in normal and transformed cells by serum-containing medium or tetradecanoylphorbol acetate, but in contrast to ornithine decarboxylase, no synergistic effect was seen in transformed cells exposed to the combination of fresh medium and the tumor promoter. A macromolecular inhibitor of ornithine decarboxylase was readily detected in hamster fibroblast cultures treated with high concentrations of putrescine, but little or none of this inhibitor was found in HE68BP cultures. In both cell types, however, serum induction of ornithine decarboxylase was inhibited under conditions of excess putrescine.The results demonstrate several differences between normal and transformed hamster cells in the regulation of polyamine synthesis.     

Polyamines and tumor cells: effect of transformation of chick embryo fibroblasts by Rous sarcoma virus on polyamine levels

The effect of transformation of chick embryo fibroblasts, by Rous sarcoma virus, on intracellular polyamine levels has been studied. A good correlation between spermidine and cellular protein content has been demonstrated. Upon changing the medium, a sharp increase in spermidine level was noticed both in normal and transformed cells. This increase was accompanied by enhanced protein synthesis. The intracellular concentrations of spermine and spermidine were very similar in normal and transformed cells. On the other hand, significant differences in putrescine levels were demonstrated: in normal cultures the intracellular concentration of putrescine reached a plateau approximately 6 days after seeding, whereas a continuous rise of the diamine in transformed cells was noticed. These differences, which were observed in cultured cells, may explain the known accumulation of polyamines during neoplastic growth.     

Effect of 5''-deoxy-5''-isobutylthioadenosine on putrescine uptake and polyamine biosynthesis by chick embryo fibroblasts.

The effect of 5'-deoxy-5'-S-isobutylthioadenosine (SIBA) on polyamine biosynthesis has been studied by using cultured chick embryo fibroblasts. It has been shown that the drug inhibits the uptake of [14C]putrescine and its conversion into labelled spermidine or spermine. The inhibitory effect is reversed by removing the inhibitor after exposing the cells to the drug for 24 h. SIBA also caused a significant decrease in cellular spermine levels and an accumulation of putrescine. These changes are reversed by removing the inhibitor. SIBA had the same effect on chick embryo fibroblasts transformed by Rous sarcoma virus; a decrease in cellular spermine levels in SIBA-treated cells was observed. In all the experiments SIBA caused a reduction in the spermine/putrescine and spermidine/putrescine ratios. It is suggested that SIBA is not only an inhibitor of transmethylation but also interferes with polyamine biosynthesis, probably by blocking aminopropyltransferase.     

Low resistance junctions between normal and between virus transformed fibroblasts in tissue culture

The occurrence of low resistance junctions between normal chick embryo fibroblasts and between fibroblasts transformed with Rous sarcoma virus in tissue culture was studied with intracellular microelectrodes. The results showed that these junctions were present between normal chick fibroblasts in proliferating cultures as well as between cells in ‘density dependent inhibited cultures’. Mechanical injury to a fibroblast within a small group of coupled cells caused the injured fibroblast to uncouple immediately from its neighboring cells without interrupting coupling between the healthy uninjured cells. In the case of fibroblasts transformed with a Rous sarcoma virus, the results further showed that low resistance junctions were present when the transformation appeared in the infected cells and remained present thereafter.     

Changes in the synthesis and phosphorylation of cellular proteins in chick fibroblasts transformed by two avian sarcoma viruses

35S- and 32P-labeled proteins from control chick embryo fibroblasts and from fibroblasts transformed by UR2 sarcoma virus, or by a temperature-sensitive mutant (tsLA29) of Rous sarcoma virus, were separated by two-dimensional electrophoresis on giant gels to detect transformation-specific changes in protein synthesis and total phosphorylation. A nontransforming avian retrovirus, UR2-associated virus (UR2AV), was also studied. Virus-coded proteins appear in whole cell lysates of all infected cells. The structural proteins can be identified by comparison with proteins immunoprecipitated with antivirus serum. The transforming proteins pp60src and p68ros, present in cells transformed with Rous sarcoma virus and UR2, respectively, are phosphorylated in vivo. Eighteen increases and eight decreases in cellular phosphoproteins are associated with transformation, and revert toward normal levels when cells infected with tsLA29 are incubated at 42 degrees C. These changes are more extensive than previously reported, but none represent new phosphorylations, since all phosphoproteins seen in transformed cells also appear to be phosphorylated to a certain extent in control cells. Fifteen cellular proteins show increased relative rates of synthesis apparently related either to transformation or to growth at 42 degrees C. Four other proteins are increased exclusively in cells incubated at 42 degrees C, but not at 37 degrees C, whether transformed or not. Eleven additional increases in the synthesis of cellular proteins, many quite large, and one seemingly a de novo induction, appear to be specific for transformation. These changes occur in cells transformed by either UR2 or Rous sarcoma virus at 37 degrees C, do not occur with UR2AV infection, and tend to revert in cells infected with tsLA29 incubated at 42 degrees C. These 11 changes may represent increases in cellular gene expression that are related specifically to the maintenance of the transformed state.     

Growth control in chick embryo fibroblasts; no evidence for a specific role for cyclic purine nucleotides

Cyclic AMP and cyclic GMP concentrations were measured in cultures of normal chick embryo fibroblasts and those transformed by Rous sarcoma virus under different growth conditions. No significant and reproducible correlation between the nucleotide levels and the rate of proliferation was observed. Neither release of normal cells from density dependent inhibition of growth nor transformation of the cultures by different strains of Rous sarcoma virus affected the concentrations of cyclic AMP or cyclic GMP. Activities of cellular cyclic nucleotide phosphodiesterases, enzymes involved in regulating the level of the nucleotides, were not directly affected by growth-stimulating concentrations of insulin or neuraminidase. Growth stimulation by insulin did not alter the activities of cellular cAMP-dependent protein kinase. These results do not support the hypothesis that cyclic AMP or cyclic GMP has a specific role in the growth control of chick embryo fibroblasts.     

Polyamines in mycoplasmas and in mycoplasma-infected tumour cells.

Three out of four different mycoplasma strains analysed for the polyamine contents contained relatively high concentrations of putrescine, cadaverine, spermidine and spermine. In addition to ornithine decarboxylase (EC 4.1.1.17) activity, the mycoplasmas also exhibited comparable or higher lysine decarboxylase (EC 4.1.1.18) activity fully resistant to the action of 2-difluoromethylornithine, an irreversible inhibitor of eukaryotic ornithine decarboxylase. 2-Difluoromethylornithine did not modify the polyamine pattern of actively growing mycoplasmas. Ehrlich ascites carcinoma cells and L1210 mouse leukemia cells infected with any of the four mycoplasma strains contained, in addition to putrescine, spermidine and spermine, and also easily measurable concentrations of cadaverine; the latter diamine was absent in uninfected cultures. When the infected cells were exposed to difluoromethylornithine, the accumulation of cadaverine was markedly enhanced. The modification of cellular polyamine pattern by mycoplasmas, especially in the presence of inhibitors of eukaryotic ornithine decarboxylase, could conceivably be used as an indicator of mycoplasma infection in cultured animal cells.     

Calmodulin and Ca2+ in normal and transformed cells

Numerous lines of evidence implicate calcium and calmodulin (CaM) as regulators of cell growth and functional differentiation. In light of this evidence, several studies of the possible involvement of the CaM system in cellular transformation by RNA and DNA tumor viruses have been carried out. This paper summarizes the evidence linking calcium and CaM to the regulation of cell growth and critically examines the evidence that increases in CaM levels occur in transformed versus normal cells. A nontraumatic method for synchronizing both normal and transformed chick fibroblasts is presented. This method is utilized in a comparison of CaM level throughout the cell cycle of Rous sarcoma virus transformed and normal chick embryo fibroblasts. These studies best support the hypothesis that the observed differences in CaM levels between transformed and normal cultures under optimal growth conditions may largely reflect differences in the proportion of cells in a dividing versus a nondividing state.     

Effect of microinjected amine and diamine oxidases on the ultrastructure of eukaryotic cultured cells

Diamine oxide and serum amine oxidase, which catalyse the oxidation of diamines and polyamines, respectively, were trapped within reconstituted Sendai virus envelopes. These loaded envelopes were incubated with cultured normal chick fibroblasts or with fibroblasts transformed by Rous sarcoma viruses. The binding of the reconstituted envelopes to the cultured cells was confirmed by scanning electron microscopy. It has been shown that the reconstituted envelopes (1-3 microns diameter) were attached to the eukaryotic cells. No significant changes in the morphology of the normal chick embryo fibroblasts were noted upon treatment with enzyme-loaded envelopes. On the other hand, chick embryo fibroblasts transformed by Rous sarcoma virus were affected by the microinjected amine oxidases. Scanning electron microscopy demonstrated the formation of holes in the microinjected cells. Similar morphological changes were also observed when diamine oxidase was microinjected into cultured glioma cells. These holes may be the result of the ejection of the nucleus. These findings are in line with the observed effect of the injected amine oxidases on macromolecular synthesis in normal and transformed chick embryo fibroblasts.     

Characterization of a temperature-sensitive membrane alteration in chick embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus

The intramembrane particles of freeze-fractured chick embryo fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (TS68) are distributed differently at the permissive and non-permissive temperatures if, and only if, the cells are treated with glycerol before fixation. Few aggregates of intramembrane particles are present in glycerol-treated cells grown at the permissive temperature for transformation (36 degrees C), while numerous large aggregates of particles are present at the non-permissive temperature (41 degrees C). Changes in the distribution of particles after cells are shifted from 36 to 41 degrees C are observed after 20 min, while a temperature shift from 41 to 26 degrees C causes changes in glycerol-induced redistributions after 1 h. The changes observed in temperature shifts from 36 to 41 degrees C and from 41 to 36 degrees D do not require protein synthesis or RNA synthesis.     

Activation of ornithine decarboxylase in monolayer cells treated with trypsin

When monolayer Chinese hamster cells are treated with trypsin for short periods of time, ornithine decarboxylase (ODCase) activity increases two- to fourfold. This increase can be blocked by aprotinin, a protease inhibitor, and is not observed when cultures are dislodged from substrate mechanically prior to contact with exogenous trypsin. The trypsin-induced increase in ornithine decarboxylase activity is not due to degradation of enzyme or inhibitor molecules or to new enzyme synthesis. Immunoprecipitable protein, radiolabeled with [3H]alpha-difluoromethylornithine in vitro, is the same molecular weight in cells harvested with or without trypsin. Protein-bound levels of this specific enzyme-activated irreversible inhibitor of ornithine decarboxylase are unchanged by trypsin treatments that increase enzyme activity. Trypsin treatment of rat embryonic fibroblasts, transformed by a temperature-sensitive mutant of Rous sarcoma virus, increases ODCase activity in cells growing at the nonpermissive, but not at the permissive, temperature for the transformed phenotype. These results suggest that ornithine decarboxylase can be activated by exogenous trypsin treatment in a manner that is dependent on cell adhesion properties, which are modified in transformed cells.     

Polyamine depletion enhances the roscovitine-induced apoptosis through the activation of mitochondria in HCT116 colon carcinoma cells

Small molecule inhibitors of cyclin-dependent kinases (CDKs) show high therapeutic potential in various cancer types which are characterized by the accumulation of transformed cells due to impaired apoptotic machinery. Roscovitine, a CDK inhibitor showed to be a potent apoptotic inducer in several cancer cells. Polyamines, putrescine, spermidine and spermine, are biogenic amines involved in many cellular processes, including apoptosis. In this study, we explored the potential role of polyamines in roscovitine-induced apoptosis in HCT116 colon cancer cells. Roscovitine induced apoptosis by activating mitochondrial pathway caspases and modulating the expression of Bcl-2 family members. Depletion of polyamines by treatment with difluoromethylornithine (DFMO) increased roscovitine-induced apoptosis. Transient silencing of ornithine decarboxylase, polyamine biosynthesis enzyme and special target of DFMO also increased roscovitine-induced apoptosis in HCT116 cells. Interestingly, additional putrescine treatment was found pro-apoptotic due to the presence of non-functional ornithine decarboxylase (ODC). Finally, roscovitine altered polyamine catabolic pathway and led to decrease in putrescine and spermidine levels. Therefore, the metabolic regulation of polyamines may dictate the power of roscovitine induced apoptotic responses in HCT116 colon cancer cells.     

Cell surface changes accompanying viral transformation: N-acetylneuraminic acid ectotransferase system activity.

The activity of the sialyl ectotransferase system of normal chick embryo fibroblasts (CEF) and chick embryo fibroblasts transformed with the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) have been compared. Neuraminidase treatment of the intact cells increased the sialyl ectotransferase system activity of control and transformed cells two to three times. The ectotransferase system activity increased as the pH was decreased from 7.8 to 6.0. The temperature optimum for both systems was 40 degrees C. Approximately 60% of the 14C-sialic acid incorporated at pH 6.5 or above could be removed with neuraminidase. The activity of the transformed cell system with or without neuraminidase treatment was more stimulated by addition of Mn2+ ions, particularly above pH 7.0. This difference in ion sensitivity indicates that a different cell surface phenomenon is being studied after transformation.     

Action of cobra venom cardiotoxin on chick embryonal fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus

The cytolytic action of cardiotoxin analogue III from the venom of the Formosan cobra on chick embryonal fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus was investigated. The 50% effective dose of the toxin for the cells cultured at a non-permissive temperature (41 degrees C) or for noninfected normal cells was about 8 micrograms/ml whereas the value was 2 micrograms/ml for the cells cultured at a permissive temperature (36 degrees C). This indicates that the transformed cells became more susceptible to the cytolytic action of the toxin than the non-transformed cells.     

Regulation or procollagen messenger ribonucleic acid levels in Rous sarcoma virus transformed chick embryo fibroblasts

Using cloned cDNAs for pro-alpha 1 and pro-alpha 2 collagen messenger ribonucleic acid (mRNA), we have investigated the regulation of collagen mRNA levels in Rous sarcoma virus (RSV) transformed chick embryo fibroblasts (CEF). We find that both pro-alpha 1 and pro-alpha 2 mRNA levels are decreased approximately 10-fold in CEF transformed by either the Bryan high-titer strain or the Schmidt-Ruppin strain of RSV. Using temperature-sensitive mutants in the transforming gene src, we also investigated the rate of change in the levels of the two mRNA species. We employed mutants of both the Bryan high-titre strain (BHTa) and the Schmidt-Ruppin strain (ts68). With both mutants the results were similar. Upon shift from the permissive temperature (35 degrees C) to the non-permissive temperature (41 degrees C), collagen mRNA synthesis, did not increase until more than 5 h had passed, suggesting that action of src on collagen gene expression is indirect. Upon shift from 41 to 35 degrees C, collagen mRNA levels fell with a half-life of 10 h. Whether this fall reflects the half-life of procollagen mRNA or an effect of src on procollagen RNA stability is unclear. Both pro-alpha 1 and pro-alpha 2 mRNA levels were coordinately controlled.     

The extracellular matrix of normal chick embryo fibroblasts: its effect on transformed chick fibroblasts and its proteolytic degradation by the transformants

Extracellular matrix (ECM), prepared from chick embryo fibroblasts, contains fibronectin as the major structural protein along with collagen and other polypeptides as less abundant protein components. When Rous sarcoma virus-transformed chick embryo fibroblasts are cultured on the ECM in the presence of the tumor promoter tetradecanoyl phorbol acetate, the transformed cells lose their characteristic rounded morphology and align on and within the ECM fibrillar network. This restrictive aspect of ECM is only temporary, however, and with time (24-72 h) the transformed cells progressively degrade the ECM fibers and resume their rounded appearance. The matrix degradation can be monitored by employing biosynthetically radiolabeled ECM. The addition of purified chicken plasminogen to the Rous sarcoma virus- transformed chick embryo fibroblast cultures enhances the rate and extent of ECM degradation, due to the elevated levels in the transformed cultures of plasminogen activator. Plasminogen-dependent and -independent degradation of ECM has been characterized with regard to sensitivity to various natural and synthetic protease inhibitors and to the requirement of cell/ECM contact. Plasminogen-dependent degradation of ECM occurs rapidly when ECM and cells are in contact or separated, whereas plasminogen-independent degradation is greatly reduced when ECM and cells are separated, which suggests that cell surface-associated proteolytic enzymes are involved. A possible role in ECM degradation has been indicated for cysteine proteases, metallo enzymes, and plasminogen activator, the latter as both a zymogen activator and a direct catalytic mediator.     

Membrane lipid dynamics and density dependent growth control in normal and transformed avian cells

Membrane fluidity of normal chick embryo fibroblasts and normal Japanese quail fibroblasts and their Rous sarcoma virus and methylcholanthrene transformed counterparts was investigated using the technique of fluorescence depolarisation of 1,6-diphenylhexatriene incorporated in the whole cells and in their isolated plasma membrane vesicles. Normal cells and isolated plasma membranes of normal cells showed significant changes in fluidity as a function of population density while neither Rous sarcoma virus transformed nor methylcholanthrene tumor cells or their isolated plasma membrane showed this effect. Stimulation of growth by addition of calf serum to cultures of quiescent, density-inhibited normal cells was accompanied by rapid changes in the direction of increased membrane lipid fluidity. Neither sparse normal cells, nor sparse or dense transformed cells showed any significant fluidity change in their membrane lipids upon addition of serum. Enzyme and electron microscopic analysis of the ratios of different membrane types in each cell type showed that this ratio was invariant with respect to cell population density but different between transformed and normal cells. Hence, the fluidity changes observed, measured as the mean rotational correlation time of the fluorescene probe in the membrane lipids, truly reflect organisational differences, occurring as a function of population density in cultures of cells which retain density-dependent growth control.