Isolation of aldehyde oxidase (EC 1.2.3.1) from potato extracts by preparative page

A method is proposed for the isolation (and purification) of enzymes, with retention of their activity, from solutions or gels of preparative PAGE runs. It is based on the inclusion of Sephadex G-25 as a supporting medium for a collector buffer in otherwise normal disc-PAGE gels. The collector buffer has a lower pH and higher concentration than the stacking gel buffer. This makes the proteins concentrate in a very narrow, slowly moving band in the Sephadex on electrophoresis, and makes their recovery easy. The method is illustrated by the isolation of aldehyde oxidase from potato extracts (which was unsuccessful by classical methods), and of one isoenzyme from commercial lipoxygenase after preparative PAGE. Recovery of chicken egg albumin after PAGE was over 90%.     

Induction of alpha type DNA polymerases in human cytomegalovirus-infected WI-38 cells.

Productive infection of WI-38 cells with human cytomegalovirus (HCMV) induced the increase in the activity of DNA polymerases as well as the synthesis of viral and cellular DNA. Sedimentation analyses in sucrose gradients of high ionic strength showed that the HCMV infection caused marked increase in the activity of alpha-type polymerases (resolved into alpha1, 8 S, and alpha 2, 6 S, in the present experiments), while the infection little affected the level of beta-type polymerase (about 3.5 S) activity in both the nuclei and cytoplasm. Such increase in alpha-type polymerases was also observed when DNA synthesis in WI-38 cells was enhanced by SV40 infection or by an increased concentration of serum in medium. Phosphonacetate, which selectively blocked the synthesis of HCMV DNA, did not significantly affect the HCMV-mediated induction of DNA polymerases. However, phosphonoacetate added in the reaction mixture for DNA polymerase assay inhibited the activity of the HCMV-induced polyperase alpha, but not of the polymerases alpha2 and beta. These results support the idea that alpha-type polymerases are involved in the replicative synthesis of cellular and viral DNA.     

Alkaline phosphatase and an acid arylamidase as marker enzymes for normal and transformed WI-38 cells

A survey of eleven enzyme activity levels in normal and SV40 transformed (VA-13) WI-38 cells revealed that the transformed cell enzymes differed by a quantitative and qualitative change of alkaline phosphatase and a quantitative loss of an arylamidase. Alkaline phosphatase activity was found to be elevated in the transformed cells at confluency but not in log phase cultures. This elevated activity was heat stable, L-homoarginine resistant and L-phenylalanine sensitive and is probably the term placental isoenzyme. In nontransformed WI-38 cells, the alkaline phosphatase was heat labile, L-homoarginine sensitive and L-phenylalanine resistant and so is probably the liver isoenzyme. While the arylamidase activity from both normal and transformed WI-38 cells had identical pH optima and Km values, the activity was approximately 20 times higher in confluent WI-38 cells than in confluent VA-13 cells. Cytochemical staining techniques for both activities are described that permit identification of fluorescent product within the cells, analysis of activity levels, and separation of cells with high and low activities. Mixtures of WI-38 cells and VA-13 cells separated by flow cytometry on the basis of arylamidase activity were subsequently evaluated for alkaline phosphatase isoenzyme and found to have been simultaneously separated into heat labile and heat stable samples.     

Prostaglandin synthetase activity and hormone responsiveness in normal and SV40 transformed WI-38 fibroblasts.

Prostaglandin (PG) synthetase activity and selective hormone responsiveness were examined in normal and SV40 transformed WI-38 fibroblasts (VA13-2RA). The transformed VA13-2RA cells have significantly reduced rates of PGE1, PGE2, PGF1alpha and PGF2alpha synthesis as compared to the normal WI-38 fibroblast. The transformed cell in contrast to the normal cell hyperresponds to stimulation by L-epinephrine (10 muM) and PGE1 (8.5 muM) but is unresponsive to bradykinin (BK) as measured by the accumulation of intracellular cyclic AMP. Indomethacin treatment does not significantly alter the PGE1 and L-epinephrine (EPI) responsiveness of the normal WI-38 fibroblast, however it abolishes the BK response in these cells. These results provide further evidence for the dependency of cell activation by bradykinin on the PG synthetase system. No experimental data was found to support the role of PGs as negative regulators of PGE1 and EPI responsiveness in the WI-38 fibroblast. Using the VA13-2RA cells, limited attempts to recover PG synthetase activity comparable to that found in normal WI-38 cells were unsuccessful. The VA13-2RA cell and its normal counterpart represent models for investigating the role of PGs in cell function and the mechanism of BK activation and its effect on cell metabolism.     

A mathematical model of human thymidine kinase 2 activity

The mitochondrial enzyme thymidine kinase 2 (TK2) phosphorylates deoxythymidine (dT) and deoxycytidine (dC) to form dTMP and dCMP, which in cells rapidly become the negative-feedback end-products dTTP and dCTP. TK2 kinetic activity exhibits Hill coefficients of ～0.5 (apparent negative cooperativity) for dT and ～1 for dC. We present a mathematical model of TK2 activity that is applicable if TK2 exists as two monomer forms in equilibrium.     

Lithium mimics dexamethasone in stimulating DNA synthesis by WI-38 cells

Lithium interferes with the responses of neural and secretory cells to calcium-mobilizing agonists by blocking the generation of phospholipase C-dependent second messengers. However, the mechanism by which lithium stimulates the proliferation of other cells in response to agonists that do not activate phospholipase C remains obscure. We investigated the pathways that mediate the mitogenic action of lithium on WI-38 cells in a defined, serum-free medium. Lithium, like dexamethasone (Dex), potentiated DNA synthesis in response to the combination of insulin+epidermal growth factor (EGF) (+50%), but not in response to either growth factor alone or with Dex. As in the case of Dex, lithium could be added as late as 8 h following stimulation of quiescent cells by insulin+EGF without loss of potentiating activity. While DNA synthesis in control cultures was essentially complete by 24 h, lithium and Dex stimulated "late" DNA synthesis (24-30 h) 10-fold and 5-fold, respectively. The potentiating activity of Dex, but not that of lithium, was blocked by the specific glucocorticoid receptor antagonist, RU486. Both lithium and Dex stimulated log-phase growth, but only Dex increased saturation density. These data indicate that both lithium and Dex recruit into the cell cycle a subpopulation of cells with a longer mean prereplicative phase (G1). The effect of lithium on DNA synthesis in WI-38 cells may be mediated by the glucocorticoid response pathway at some point distal to activation of the glucocorticoid receptor, or by an independent mechanism that can be switched on late in G1.     

Microneutralization Test for Determination of Rhinovirus and Coxsackievirus A Antibody in Human Diploid Cells

A method for determination of serum-neutralizing antibody titers to rhinovirus type 16 and coxsackievirus type A-4 in human diploid cells (WI-38) grown in Microtiter plates is described. A good correlation was observed when comparing neutralization tests in WI-38 and other cells. The WI-38 Microtiter method is relatively simple and economical. It is suitable for those laboratories which are required to conduct large-scale serological evaluation of antibodies for rhinoviruses and coxsackie A viruses. This system also can be used for cytomegalovirus and varicella-zoster virus, which grow relatively slowly.     

Changes in enzymic activity during cultivation of human cells in vitro

The composition of chromatin, its template activity and the activity of certain chromatin-associated enzymes, including DNA polymerase (DP) and soluble RNase, DNase, DP and seryl tRNA synthetase, were examined in early and late passage of WI-38 cells and of WI-38VA13 cells.No significant changes in soluble RNase, DNase, seryl tRNA synthetase or soluble and chromatin-associated DP were found with increasing passage of WI-38 cells. The activity of seryl tRNA synthetase and DP in WI38VA13 cells was, however, significantly higher than WI-38 cells in all passages. A decline in RNA synthesizing activity of chromatin, an increase in the proportion of RNA and histone in chromatin, as well as an increase in the activities of ‘chromatin-associated enzymes’ (RNase, DNase, protease, nucleoside triphosphatase, DPN pyrophosphorylase) were noted in WI-38 cells with increasing passages. Although RNA synthesizing activity of chromatin from WI38VA13 cells was lower than that from WI-38 cells, the former also were much lower in ‘chromatin-associated enzymes’. An increase of chromatin-associated enzymes responsible for RNA, DNA and protein degradation in WI-38 cells in successive passages, and a much lower activity of these enzymes in WI-38VA13 cells (which have an indefinite doubling potential in vitro) suggests that an elevation in the activity of these enzymes, which would seriously interfere with the chromatin function, could result in ‘aging’ of WI-38 cells.     

Changes in membrane transport function in G0 and G1 cells

Confluent quiescent monolayers of aneuploid and euploid cells in culture can be stimulated to proliferate by appropriate nutritional changes. In confluent monolayers of WI-38 human diploid fibroblasts the uptake of cycloleucine is increased three hours after these cells are stimulated to proliferate by a change of medium plus 10% serum. No changes in the uptake of cycloleucine are observed in logarithmically-growing WI-38 cells exposed to fresh medium plus 10% serum, or in WI-38 confluent monolayers in which the conditioned medium has been replaced by fresh medium with 0.3% serum (a change that does not cause stimulation of cellular proliferation in WI-38 cells). In 3T6 cells in the stationary phase stimulated to proliferate by nutritional changes, there is a prompt increase in the uptake of cycloleucine, within one hour after stimulation of cell proliferation. Similar results were obtained with stationary 2RA cells which are SV-40 transformed WI-38 fibroblasts. In addition, chromatin template activity which is known to increase in the early stages after stimulation of confluent WI-38 cells, was unchanged in confluent 3T6 or 2RA cells stimulated to proliferate. These results show that at least two of the very early biochemical events occurring in response to stimulation of cell proliferation are different in WI-38 diploid cells and in aneuploid 2RA or 3T6 cells. It is proposed that WI-38 cells in the stationary phase are arrested in the G₀ phase of the cell cycle, while 2RA and 3T6 cells are arrested in the G₁ phase.     

Characterization of an Epstein-Barr virus-induced thymidine kinase.

Previous work from our laboratory suggested that the selective inhibition of Epstein-Barr virus (EBV) replication by 1-beta-D-arabinofuranosylthymine in human lymphoid cell lines involved the induction of a new thymidine kinase (TK) able to phosphorylate the thymidine analog. We further characterized this enzyme induced in various EBV-positive cell lines after viral genome activation with a combination of sodium butyrate and 12-O-tetradecanoylphorbol-13-acetate. The following results confirmed the existence of an EBV-specific deoxypyrimidine kinase: induction of EBV-related TK was connected with the appearance of viral early antigens in EBV-carrying cells; unexpected behaviors of the enzyme activity upon different fractionating treatments led to the conclusion that EBV-induced TK was extracted as a complex molecular form, larger than other known cellular or viral isozymes; enzymatic properties distinguished EBV-induced TK from host lymphoid cell isozymes but made it resemble other herpesvirus-specific deoxypyrimidine kinases, i.e., by partial inhibition by dTTP or ammonium sulfate, insensitiveness to dCTP, and nonstringent specificity for normal TK substrates. Genetic evidence is required to definitively ensure that EBV-specific TK actually is virus coded in EBV-transformed human lymphoid cells.     

Effect of vitamin A on epithelial morphogenesis in vitro

Quiescent confluent monolayers of WI-38 human diploid fibroblasts and of 3T6 mouse fibroblasts were stimulated to proliferate by nutritional changes. WI-38 cells had a stringent requirement for serum factor(s) but 3T6 did not require serum in order to proliferate again. In both cell lines there was an early increase in the synthesis of non-histone chromosomal proteins shortly after stimulation of cellular proliferation and this increase was linearly correlated to the number of cells entering the S phase several hours later. Only WI-38 diploid fibroblasts, however, showed an early increase in chromatin template activity 1 h after stimulation of cellular proliferation, while chromatin template activity in 3T6 cells remained unchanged. It is suggested that the activation of gene function represents a critical step for the passage of WI-38 cells in the G0 resting phase to the G1 phase of the cell cycle. It is also suggested that 3T6 cells are unable to enter or stay in a G0 phase but can be arrested predominantly in the G1 phase by nutritional deficit, probably amino acid starvation.     

In vitro study of the modification by bleomycin of thymidine phosphorylation activity in lectin-stimulated normal human lymphocytes

We have investigated the effect of bleomycin (BLM) on thymidine phosphorylation in lectin-stimulated normal human lymphocytes. BLM reduces thymidine phosphorylation by decreasing the activity of thymidine kinase (TK). Accordingly, polyacrylamide gel electrophoresis (PAGE) of extracts of cells incubated for 48, 72 and 96 h showed here that this activity dropped 48, 65 and 67% respectively. The electrophoretic profiles of TK activity were similar but different in amplitude. These effects of the BLM were confirmed firstly by direct measurement of TK activity, secondly by amount of 3H-thymidine incorporation in the cultures before cell lysis. Both the measurement of TK activity and 3H-thymidine incorporation were correlated.     

Inhibition of cyclic nucleotide phosphodiesterase during exposure to WI-38 cells to prostaglandin E1.

Short term incubation of WI-38 cultures with 5.7 micron prostaglandin E1 (PGE1) caused cyclic AMP phosphodiesterase activity in fibroblast homogenates to fall by 25 to 35% as compared to controls. The PGE1-induced decline in phosphodiesterase activity coincided with a rapid increase in intracellular cyclic AMP levels in response to the hormone and was rapidly reversed by washing the cultures free of the prostaglandin before homogenizing the cells. The effect of PGE1 on WI-38 phosphodiesterase activity was localized to the enzyme form(s) present in 27,000 times g supernatant fractions of cell homogenates. These data suggest that the pattern of cyclic AMP accumulation in WI-38 fibroblasts exposed to PGE1 may be related, at least in part, to decreased phosphodiesterase activity during hormone stimulation.     

Dissimilar cyclic nucleotide phosphodiesterase activities in subcellular fractions from normal and SV40-transformed WI-38 fibroblasts.

Broken cell preparations of WI-38 and SV40-transformed WI-38 (VA13) fibroblasts were used to compare the cyclic nucleotide phosphodiesterase activities of the two cell strains. The bulk of the cAMP or cGMP phosphodiesterase activity of WI-38 and VA13 homogenates was found in the 100,000 x g fibroblast supernatant fractions. WI-38 and VA13 soluble phosphodiesterase activities showed anomalous kinetic behavior with either cAMP or cGMP as the substrate. At low substrate concentrations, e.g., 0.1 muM, WI-38 supernatant fractions hydrolyzed cGMP much more rapidly than cAMP. At high substrate concentrations, e.g., 100muM, the same enzyme preparations degraded cAMP more than twice as fast as cGMP. In contrast, VA13 soluble phosphodiesterase activity catalyzed the hydrolysis of a wide range of cAMP and cGMP concentrations at similar rates. Phosphodiesterase activity in WI-38 supernatant fractions was generally more sensitive than that of the comparable VA13 enzyme activity to inhibition by MIX and papaverine. The cAMP phosphodiesterase activity of both WI-38 and VA13 supernatant preparations was decreased by cGMP in a concentration-dependent manner. cAMP was an effective inhibitor of cGMP hydrolysis by VA13 soluble phosphodiesterase activity. Yet, the cGMP phosphodiesterase activity of WI-38 supernatant fractions was only slightly reduced in the presence of cAMP. DEAE-cellulose chromatography of WI-38 and VA13 supernatant preparations revealed two major peaks of phosphodiesterase activity for each cell type. WI-38 peak I showed much greater activity with 1muM cGMP than with 1muM cAMP and appeared to be composed of two different phosphodiesterase activities. WI-38 peak Ia included phosphodiesterase activity which could be stimulated by boiled, dialyzed fibroblast homogenates while WI-38 peak Ib coincided with column fractions which contained most of the cyclic GMP hydrolytic activity. VA13 peak I phosphodiesterase activity was eluted from DEAE cellulose columns at the same ionic strength as WI-38 peak Ia and hydrolyzed these two substrates at nearly identical rates. This enzyme activity was also increased in the presence of boiled, dialyzed fibroblast preparations. Peak II phosphodiesterase activities from both WI-38 and VA13 fibroblasts were relatively specific for cAMP as the substrate. Phosphodiesterase activity with the properties of WI-38 peak Ib was not isolated from VA13 supernatant fractions. These results suggested that the dissimilar patterns of cAMP accumulation in WI-38 and VA13 cultures may be at least partially related to different phosphodiesterase activities in the normal and the transformed fibroblasts.     

Differential effects of phorbol ester on the in vitro invasiveness of malignant and non-malignant human fibroblast cells

The effect of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) on cell invasion was studied using an in vitro assay for cell invasion through a reconstituted basement membrane matrix (Matrigel). TPA inhibited the invasiveness of malignant human fibrosarcoma HT1080 cells. In contrast, WI-38 lung fibroblasts, which show a very low invasive capacity, were stimulated (3-fold) to invade Matrigel after exposure to TPA for 48 hours. The inhibitory or stimulatory effects of TPA on cell invasion were correlated with a decrease or an increase in cell motility and collagenase IV activity, respectively. Synthetic diacylglycerols partially mimicked the inhibitory action of TPA on HT1080 cells but failed to stimulate WI-38 cell invasion. Immunoblots demonstrated that in both cell lines the alpha and beta isoforms of protein kinase C were equally down-regulated after a 5 hour exposure to TPA despite the basal low level of protein kinase C polypeptide in the malignant cells. Thus, whereas in WI-38 cells induction of an invasive behavior could be observed in the absence of protein kinase C, in the malignant cells disappearance of the kinase was associated with a non-invasive phenotype.     

Differences in oxygen-dependent regulation of enzymes between tumor and normal cell systems in culture

Metabolic studies in tumor cells have indicated that bioenergetic regulatory mechanisms geared to acute changes in oxygen availability are abnormal. In the present studies we have examined bioenergetic adaptations to chronic oxygen depletion in culture maintained tumor cells in comparison to normal cell lines. Activities of two key glycolytic enzymes (pyruvate kinase (PyKI) and phosphofructokinase (PFK)) were measured in two tumor cell lines (fibrosarcoma (FS) and Hela) and two normal cell lines (rat lung fibroblasts (RLF) and WI-38) maintained in culture for up to 96 hours under aerobic (PO2 approximately 140) and hypoxic PO2 approximately 15) conditions. Exposure to low O2 tensions for 96 hours resulted in significant increases in PyKi and PFK in both RLF and WI-38, ut did not alter activities of these enzymes in either FS or HeLa cell systems. Activities of two enzymes involved in O2 metabolism (cytochrome oxidase (CyOx) and superoxide dismutase (SOD) were also measured in the two tumor cell lines and in RLF. chronic hypoxia significantly decreased the activities of CyOx and SOD in RLF cell systems but did not alter the activities of these enzymes in the tumor cells. In these studies, the tumor-derived cell lines do not demonstrate specific enzymatic responses to sustained oxygen depletion in vitro noted in normal cell systems, suggesting significant abnormalities in regulatory mechanisms geared to chronic changes in molecular O2.     

The role of mitochondrial dNTP levels in cells with reduced TK2 activity

Both the nuclear and mitochondrial DNA (mtDNA) depend on separate balanced pools of dNTPs for correct function of DNA replication and repair of DNA damage. Import of dNTPs from the cytosolic compartment to the mitochondria has been suggested to have the potential of rectifying a mitochondrial dNTP imbalance. Reduced TK2 activity has been demonstrated to result in mitochondrial dNTP imbalance and consequently mutations of mtDNA in non-dividing cells. In this study, the consequences of a reduced thymidine kinase 2 (TK2) activity were measured in proliferating HeLa cells, on both whole-cell as well as mitochondrial dNTP levels. With the exception of increased mitochondrial dCTP level no significant difference was found in cells with reduced TK2 activity. Our results suggest that import of cytosolic dNTPs in mitochondria of proliferating cells can compensate a TK2 induced imbalance of the mitochondrial dNTP pool.     

The Role of Mitochondrial dNTP Levels in Cells with Reduced TK2 Activity

Both the nuclear and mitochondrial DNA (mtDNA) depend on separate balanced pools of dNTPs for correct function of DNA replication and repair of DNA damage. Import of dNTPs from the cytosolic compartment to the mitochondria has been suggested to have the potential of rectifying a mitochondrial dNTP imbalance. Reduced TK2 activity has been demonstrated to result in mitochondrial dNTP imbalance and consequently mutations of mtDNA in non-dividing cells. In this study, the consequences of a reduced thymidine kinase 2 (TK2) activity were measured in proliferating HeLa cells, on both whole-cell as well as mitochondrial dNTP levels. With the exception of increased mitochondrial dCTP level no significant difference was found in cells with reduced TK2 activity. Our results suggest that import of cytosolic dNTPs in mitochondria of proliferating cells can compensate a TK2 induced imbalance of the mitochondrial dNTP pool.     

Effect of cyclic AMP on chromatin-bound protein kinases in WI-38 fibroblasts stimulated to proliferate.

When resting confluent monolayers of WI-38 fibroblasts are stimulated to proliferate by serum, DNA synthesis begins to increase between 15-18 h after stimulation. Chromatin-bound protein kinase activity increases in stimulated cells within 1 h after the nutritional change, concomitant with an increase in the template activity of nuclear chromatin. Addition of dibutyryl 3' : 5'-cyclic adenosine monophosphate (dibutyryl cyclic) AMP to the stimulating medium inhibits the entrance of cells into S phase, but only if dibutyryl cyclic AMP (5-10(-4) M) is added before the onset of DNA synthesis. The increases in chromatin template activity and in the chromatin-bound kinase activity are not inhibited by dibutyryl cyclic AMP in the early hours after stimulation, but are completely inhibited after the 5th hour from the nutritional change. This seems to indicate that in stimulated WI-38 cells, dibutyryl cyclic AMP exerts its inhibitory action somewhere between 5 and 12 h after stimulation. A number of protein kinase activities were extracted from chromatin with 0.3 M NaCl and partially resolved on a phosphocellulose column. Two distinct peaks of protein kinase activity appeared to be markedly increased in WI-38 cells 6 h after serum stimulation. Both peaks of increased activity were inhibited by dibutyryl cyclic AMP in vivo. Adenosine, sodium butyrate and adenosine 5'-monophosphate (AMP) do not inhibit the increase in DNA synthesis nor the increase in protein kinase activity. The results suggest that stimulation of cell proliferation in confluent monolayers of WI-38 cells causes an increase (or the new appearance) of certain chromatin-bound protein kinases, and that this increase is inhibited by cyclic AMP in vivo.     

epsilon-(gamma-Glutamyl)lysine isopeptide bonds in normal and virus transformed human fibroblasts.

Normal human lung cells (WI-38) possessed 20–40 times more ε-(γ-glutamyl)lysine isopeptide bonds than Simian virus transformed counterparts, WI-38 VA13A and WI-38 VA13-2RA. Normal cells arrested in an essentially nonmitotic state had more isopeptide bonds than proliferating cells. Isopeptide content paralleled the transglutaminase activity of these cells. The results suggest that isopeptide crosslinks contribute to a cellular architecture conducive to a nonproliferating state.