Increase in plasma membrane phosphodiesterase activity in contact-inhibited 3T3 cells and in phenotypically reverted SV-3T3 cells

Summary Contact-inhibited 3T3 mouse fibroblast cells, in contrast to logarithmically growing 3T3 cells and SV-3T3 transformed cells, have increased levels of plasma membranebound phosphodiesterase (oligonucleotidase, E.C. 3.1.4.19; nucleotide pryrophosphatase, E.C. 3.6.1.9) activity. The increase in enzyme, recorded as increased specific activity, is reversible, as evidenced by the return to normal values following dilution of confluent 3T2 cells and re-initiation of growth. Increased enzyme activity is induced again when the cells regain the confluent state. Transformed SV-3T3 cells can be induced to mimic the contact inhibited state, including increased plasma membrane phosphodiesterase activity, by exposure to a combination of: (i) agents that are known to induce increased intracellular cAMP levels and (ii) additions of purified 3T3 or SV-3T3 plasma membranes. Additions of either alone fails to induce the increase in membrane phosphodiesterase activity, although each alone can significantly suppress cell growth, as measured by incorporation of³H amino acids.We suggest that the elevation of plasma membrane phosphodiesterase activity may serve as a measure of conversion to the contact-inhibited state in both normal cells and phenotypically reverted transformed cells.     

Affinity chromatography of hyaluronate on glutaraldehyde-fixed SV-3T3 cells

A simple, rapid colorimetric assay for plasma heparin is presented. The assay employs the metachromasia of azure A when heparin is added. It is useful for 0 to 10 units/ml and does not depend on heparin's anticoagulant activity. Heparin concentrations determined with this assay are not exactly the same as those determined with coagulation assays. This is probably because azure A determines chemical heparin, not anticoagulant active heparin.     

Protein turnover and proliferation. Failure of SV-3T3 cells to increase lysosomal proteinases, increase protein degradation and cease net protein accumulation

The contrasting control of lysosomal proteinases, protein turnover and proliferation was studied in 3T3 and SV-3T3 (SV-40-virus-transformed 3T3) cells. 1. In 3T3 cells, net protein accumulation proceeded from 5%/h (doubling time, T(d)=14h) in growing cells to 0%/h as cells became quiescent. SV-3T3 cells never ceased to gain protein, but rather decreased their protein accumulation rate from 6-7%/h (T(d)=10-12h) to 2%/h (T(d)=35-40h) just before culture death in unchanged medium. 2. In both cell types the rates of protein synthesis per unit of protein (a) were proportional to the initial serum concentration from 0 to 6%, and (b) declined under progressive depletion of undefined serum growth factors. In depleted growth medium, leucine incorporation per unit of protein in 3T3 and SV-3T3 cells declined to almost equal synthetic rates while the 3T3 cell existed in a steady state of zero net gain, and the SV-3T3 cell continued to gain protein at a rate of 2%/h. 3. Whereas a large fraction of the control of 3T3-cell net protein accumulation can be accounted for by an increase in degradation from 1%/h to 3%/h, the SV-3T3 cell did not exhibit a growth-related increase in degradation appreciably above 1%/h. 4. Thus, by using first-order kinetics, the continued net protein accumulation of the transformed cell can be accounted for by a failure to increase protein degradation, whereas fractional synthesis can be made to decline to a rate similar to that in the quiescent non-transformed cell. 5. Upon acute serum deprivation, both cell types similarly exhibited small rapid increases in proteolysis independent of cell growth state or lysosomal enzyme status. 6. The 3T3 cell increased its lysosomal proteinase activity in conjunction with increase in the growth-state-dependent proteolytic mechanism; however, the SV-3T3 cell failed to increase lysosomal proteinases or the growth-state-dependent proteolytic mechanism.     

Characterization and identification of the hyaluronate binding site from membranes of SV-3T3 cells

Previous research has shown that binding sites for hyaluronate are present on the surfaces of a number of different cell types. To further characterize these binding sites, membranes were prepared from SV-3T3 cells and dissolved in a solution of sodium deoxycholate. Hyaluronate binding activity was detected by mixing the sodium deoxycholate extract with [3H]hyaluronate and then adding an equal volume of saturated (NH4)2SO4, which precipitated the binding protein and any [3H]hyaluronate associated with it, but left free [3H]hyaluronate in solution. Following partial purification by hydroxylapatite chromatography, the binding site was examined by molecular sieve chromatography and by rate-zonal centrifugation, which revealed that it has a Stokes radius of 6.5 nm and a sedimentation coefficient of 4.8 S. From these values, it was possible to calculate that the sodium deoxycholate-solubilized binding site has a frictional coefficient of 1.87 and a molecular weight of 132,000. Since this latter value applies to the complex of both detergent and protein, the binding protein by itself must have a molecular weight lower than 132,000. To determine the molecular weight of the hyaluronate binding site itself, the protein was purified by the sequential application of hydroxylapatite chromatography, molecular sieve chromatography, rate-zonal centrifugation, and finally lectin-affinity chromatography on concanavalin A-agarose. Analysis of the purified material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an 85,000 Mr protein which has been identified as the binding site. This protein was also detected on nitrocellulose blots which had been specifically stained for concanavalin A binding material, suggesting that the binding site is a glycoprotein.     

Induction of lysosomal enzymes in contact inhibited 3T3 cells

The pattern of changing activities of three lysosomal enzymes, N-acetyl-β-D-glucosaminidase, aryl sulfatase, and DNase II, and that of DNA polymerase was followed in homogenates of 3T3 cells during the logarithmic phase of growth and in stationary cultures. The change in activities of the polymerase and the lysosomal enzymes is antiparallel. DNA polymerase exhibits highest activity in growing cultures, and shows a three-fold decline of the specific activity in stationary cultures. The lysosomal enzymes show a very marked increase in their specific activity after density saturation is reached, which can be prevented by the addition of cycloheximide. Colcemid added to logarithmically growing cultures also causes an increase in the specific activities of lysosomal enzymes.     

Increase in S-adenosylmethionine decarboxylase in SV-3T3 cells treated with S-methyl-5''-methylthioadenosine.

We previously have shown [Takahashi & Kobayashi (1982) Hepatology 2, 249-254] that the administration of concanavalin A to mice with schistosomiasis caused liver collagen content to be reduced by 50%. Here we report the effects of concanavalin A and aggregated mouse myeloma IgG on liver lysyl oxidase activity and present further evidence concerning the possible mechanism by which the liver collagen content was decreased in infected-treated mice. The lysyl oxidase activity at 8 weeks after infection in both treated mice and untreated infected controls was about 28-fold greater than in the age-matched uninfected controls. The specific radioactivity of intracellular free [14C]proline, the rate of collagen synthesis, the ratio of collagenase-sensitive, protein-bound, hydroxyproline to proline of collagen and the intracellular degradation of newly synthesized collagen were similar in treated animals and in untreated infected controls. In contrast, the extracellular degradation of newly secreted collagen and the specific radioactivity of protein-bound [14C]hydroxyproline in the agent-treated groups were about 2-fold greater than those in the untreated infected controls. These results suggest that the observed 50% decrease in content of liver collagen of mice treated with the agents apparently was due to the increased extracellular degradation of newly secreted collagen.     

Transformed SV3T3 cells have a reduced lysosomal compartment and lower levels of enzyme activity than 3T3 cells.

The secreted and intracellular activities of a number of lysosomal hydrolases were higher in 3T3 cells than in SV40-transformed cells. The number of lysosomes and their total volume were also much larger in 3T3 cells and the surface area of their lysosomal membranes was almost twice that of SV3T3 cells. These differences alone were not sufficiently large, however, to account for the disparity seen in activity of some enzymes. Gel electrophoresis showed that a number of protein components present in lysosomal membranes purified from 3T3 cells were absent from SV3T3 membrane preparations. The absence of these components may be correlated with the reduced enzyme activity of SV3T3 cells particularly with respect to beta-glucosidase and acid phosphatase, both of which are normally found associated with lysosomal membranes.     

Effect of 1,3,6-triaminohexane and 1,4,7-triaminoheptane on growth and polyamine metabolism in SV-3T3 cells treated with 2-difluoromethylornithine

The possibility that one or both of the synthetic triamines, 1,3,6-triaminohexane and 1,4,7-triaminoheptane, could substitute for the naturally occurring polyamines in the growth of SV-3T3 cells was investigated. It was found that these triamines did lead to a restoration of growth in cells in which spermidine content had been depleted by exposure to the ornithine decarboxylase inhibitor 2-difluoromethylornithine. This resumption of a normal growth rate occurred prior to the reduction in the content of cellular decarboxylated S-adenosylmethionine, suggesting that this nucleoside (which increases in concentration several hundred-fold in cells treated with 2-difluoromethylornithine) does not cause the reduction of cell growth. However, unlike the increase in cell growth brought about by spermidine, which continued indefinitely, the increase produced by 1,3,6-triaminohexane or 1,4,7-triaminoheptane was transient. Cell growth in the presence of 2-difluoromethylornithine and these triamines stopped after about three or four population doublings. This corresponded to the time at which the intracellular spermine content of the cells was reduced to values less than 20% of normal. It is suggested that the increased growth rate of spermidine-depleted cells in response to these triamines is due to their uptake into the cell and ability to displace spermine from intracellular sites, thus making spermine available to fulfill the polyamine function(s) essential for growth. These results indicate that the naturally occurring polyamines spermidine or spermine are essential for continued cell growth and cannot be replaced by analogues containing only primary amino groups.     

Regulation of activities of NK cells and CD4 expression in T cells by human HNP-1, -2, and -3

Human neutrophil defensin alpha (HNP) is a group of cationic peptides of diverse physiological roles. Recent studies revealed the nature of HNPs as the dominant HLA-DR binding peptides on malignant cancer cells, which may block the major histocompatibility complex for antigen presentation. Here we show that HNPs may inhibit T cells by downregulating CD4 expression, a molecule of critical importance for T cell's interaction with the target cell. HNPs also inhibited tumor-cell-lysis activities of NK cells by downregulating CD16-CD56 expression. More importantly, HNPs were markedly elevated in 14 cancer tissues out of 15 self-paired human colorectal cancers and their adjacent noncancerous tissues. The subset compositions of HNPs extracted from cancer tissues and neutrophils were identical. Immunohistochemical studies indicated that HNPs mainly distributed in the infiltrated neutrophils in the interstitium. The elevated HNPs in cancer tissues may create a microenvironment unfavorable for adaptive immune reaction, implicating the cancer evasion.     

Regulation of uridine uptake by serum and insulin in density-inhibited 3T3 cells

Stimulation of nucleoside uptake in quiescent 3T3 cells by insulin and serum is preceded by a substantial lag phase. Our findings point to the length of the lag phase as a major target for regulation. The length of such phase varies markedly with the concentration of insulin (10^?9-10^?6 M) or serum (0.5–10%) but it is not eliminated by high, saturating levels of the activating agents. Further, variations in the temperature at which the stimulation process occurs 24–39°C), addition of compounds like prostaglandin E₁ (1–5 μg/ml) or theophylline (0.4 mM) and differences in the age of the cultures primarily affect the length of the lag time while the final uptake rates achieved are remarkably constant. Analysis of the temporal order of the events in the lag phase reveals that there is a discrete temperature-sensitive period located in the early and middle part of the lag, while the prostaglandin E₁-sensitive step(s) appear to be toward the end of the lag. The transition from the basal to the stimulated rate of uptake is abrupt. Indeed, the kinetics of activation does not fit a simple exponential law but a high power of an exponential, suggesting that the switching mechanism involves cooperative steps. Since the transition is abrupt, the nucleoside uptake system exists largely in two alternative states either switched off or on. The regulation of the lag period is by the control of the time at which this switch occurs. On the basis of the data presented here, we propose a working hypothesis of uptake stimulation.     

The role of polyamine depletion and accumulation of decarboxylated S-adenosylmethionine in the inhibition of growth of SV-3T3 cells treated with alpha-difluoromethylornithine.

The effects of alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, on cell growth rate, polyamine content and the content of decarboxylated S-adenosylmethionine in SV-3T3 transformed mouse fibroblasts were studied. DL-alpha-Difluoromethylornithine at 1 mM or higher concentrations decreased the growth rate by over 90% after 2 or more days of exposure, but the cells remained viable, although quiescent for at least 9 days. Addition of 10 microM-spermidine or -spermine or 50 microM-putrescine at any time throughout this period completely reversed the inhibition of growth. Treatment with alpha-difluoromethylornithine decreased putrescine and spermidine contents by more than 98% and that of spermine by 60%, but cells exposed to exogenous polyamines did not require complete replenishment of the polyamine pools to resume growth. In fact, a virtually normal growth rate was obtained in cells lacking putrescine, having 2% of normal spermidine content and 156% of normal spermine. These results suggest that the well-known increase in putrescine and spermidine in cells stimulated for growth is not essential for this to occur and that mammalian cells can utilize spermine as their only polyamine. A substantial reversal of the growth-inhibitory effect of alpha-difluoromethylornithine was produced by a number of polyamines not normally found in mammalian cells, including the spermidine analogues aminopropylcadaverine and sym-homospermidine, which were partially converted into their respective spermine analogues by addition of an aminopropyl group within the cell. The spermine analogue sym-norspermine was also effective, but the maximal growth rate produced by these unphysiological polyamines was only 60-70% of that produced by the normal polyamines. These results indicate that spermidine and spermine have the optimal length for activation of the cellular processes critically dependent on polyamines and should help in identifying these processes. Exposure to alpha-difluoromethylornithine leads to an enormous rise in the concentration of decarboxylated S-adenosylmethionine, which reached a peak at 530-fold after 3 days of exposure and steadily declined to 140-fold after 11 days. This increase was abolished by addition of exogenous polyamines, which rapidly decreased the activity of S-adenosylmethionine decarboxylase. The increase in decarboxylated S-adenosylmethionine is unlikely to be solely responsible for the decrease to the same extent by spermine, sym-norspermidine and sym-homospermidine, which produce 97%, 16% and 60% of the control growth rate, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of proline 3-hydroxylation and prolyl 3-hydroxylase and 4-hydroxylase activities in transformed cells.

Prolyl 3-hydroxylase activity and the extent of collagen proline 3-hydroxylation were studied in six transformed and three control human cell lines. In the transformed cell lines, the enzyme activity was markedly high in two, similar to that in control cells in two and significantly low in two. The extent of proline 3-hydroxylation was markedly high in cell lines with high enzyme activity, but it was also significantly high in some transformed cell lines with enzyme activities similar to those in the controls. The results thus suggest that, in addition to the amount of enzyme activity present, the rate of collagen synthesis also affects the extent of proline 3-hydroxylation in the newly synthesized collagen. The effect of acute cell transformation on prolyl 3-hydroxylase and 4-hydroxylase activities was studied by infecting chick-embryo fibroblasts with Rous sarcoma virus mutant NY68, temperature-sensitive for transformation. At the permissive temperature prolyl 3-hydroxylase activity showed a more rapid increase and decrease than did prolyl 4-hydroxylase activity, the maximal activity for both enzymes being about 2.5 times that in the control chick fibroblasts. When the transformed cells were shifted to the non-permissive temperature the decays in the elevated enzyme activities were similar, suggesting identical half-lives.     

Studies of non-metabolizable polyamines that support growth of SV-3T3 cells depleted of natural polyamines by exposure to alpha-difluoromethylornithine.

A number of synthetic polyamine derivatives that included five achiral gem-dimethylspermidines and two analogous tetramethylated spermines were tested for their abilities to serve as substrates for enzymes metabolizing polyamines and for their capacities to substitute for the natural polyamines in cell growth. It was found that none of the compounds were effective substrates for spermine synthase, and only one, namely 8,8-dimethylspermidine, was a substrate for spermidine/spermine N1-acetyltransferase. However, all of the spermidine derivatives and 1,1,12,12-tetramethylspermine were able to support the growth of SV-3T3 cells in which endogenous polyamine synthesis was prevented by the addition of alpha-difluoromethylornithine. These results suggest that either spermidine or spermine can support cell growth without the need for metabolic interconversion. In contrast with the result with 1,1,12,12-tetramethylspermine, 3,3,10,10-tetramethylspermine did not restore growth of polyamine-depleted SV-3T3 cells. Comparison of the properties of these derivatives may prove valuable in understanding the physiological role of polyamines.