Studies on the nature of protease-induced growth stimulation in normal and transformed BHK cells.

In the presence of growth-limiting serum concentrations trypsin displays mitogenic activity on actively-growing but not quiescent BHK cells. These results suggest that BHK cells arrested in G1 (G0) are not sensitive to protease-induced growth stimulation. Previous work strongly suggested that the trypsin active-site is not directly involved in its mitogenic activity on BHK cells. Additional studies on denatured trypsin fragments further indicate that the molecular conformation and size of native trypsin may not be absolutely required for mitogenic activity. Cellular multiplication induced by the addition of fresh serum to quiescent BHK cultures is not inhibited by high concentrations of soybean trypsin inhibitor. Similar to our previous findings with trypsin, it has been further observed that plasmin is not sufficient to initiate the growth of BHK cells in soft agar. Trypsin also fails to enhance the growth of a thermosensitive polyoma-transformed BHK line in soft agar at the restrictive temperature. Finally, the growth of transformed BHK cells in soft agar does not display a requirement for plasminogen and is not inhibited by soybean trypsin inhibitor. These studies argue against the involvement of plasmin or other exogenous trypsin-like enzymes in the growth and transformation of BHK cells.     

Attachment of fibroblasts to a polyanionic surface promoted by adenosine and prostaglandin E1

Dextran sulphate, which inhibits the growth of normal fibroblasts, marginally reduces their rate of initial attachment to polystyrene dishes. Transformed cells grow readily in dextran sulphate but their attachment is markedly inhibited. Adenosine and prostaglandin E₁ promote the attachment of transformed cells in the presence and absence of dextran sulphate. On dishes pretreated with serum and dextran sulphate, concentrations of both adenosine and PGE₁ as low as 10⁻⁸ M are active. Other purine derivatives and prostaglandins are inactive. The effect is discussed in relation to the properties common to these two substances in other cell systems.     

Purine and pyrimidine modification of growth and surface properties of baby hamster kidney cells (BHK21/C13)

Hypoxanthine or adenosine and thymidine added to soft agar medium stimulated the growth of non-transformed BHK21/C13 cells. Increases in concanavalin A agglutinability and synthesis of a plasminogen activator also occurred. Thymidine was required for colony formation in agar but not for increased lectin agglutinability or activator activity.     

The control of cell proliferation by preformed purines: a genetic study. I. Isolation and preliminary characterization of Chinese hamster lines with single or multiple defects in purine "salvage" pathways.

Sublines with single or multiple defects in purine "salvage" enzymes were isolated from the Chinese hamster fibroblastic line GMA32 through single or successive one-step selections for resistance to purine analogs. They were examined for their ability to incorporate purine bases and nucleosides into macromolecules, for their sensitivity to growth inhibitory purines, and for their rescue by exogenous purines from deprivation imposed by metabolic inhibitors of endogenous synthesis. The results show that a deficiency of either adenosine kinase (EC 2.7.1.20), adenine phosphoribosyltransferase (EC 2.4.2.7) or hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) abolishes the ability of adenine to cause cell death by interfering with pyrimidine synthesis; on the other hand, the pyrimidine starvation caused by adenosine is fully prevented only by a deficiency of adenosine kinase.     

The use of agarose in the determination of anchorage-independent growth.

At the present time, growth in agar suspension is one of the best in vitro correlates of tumorigenicity. Growth in agarose, however, has not been evaluated extensively as an in vitro criterion for tumorigenicity. In the present study we have tested 19 cell lines, including six mouse-human hybrids, for growth in agarose and agar in the presence and absence of exogenous hypoxanthine. None of the six nontumorigenic cell lines grew in agar or agarose. Ten of the 13 tumorigenic cell lines grew in both agar and agarose with about equal efficiency. The remaining three tumorigenic cell lines grew well in agarose but poorly or not at all in agar. Hypoxanthine did not stimulate the growth in agar or agarose of any of the cell lines except BHK. We conclude that growth in agarose may be a more sensitive marker for tumorigenicity than growth in agar and that BHK is exceptional in its sensitivity to supplemental purines.     

Purine Uptake and Utilization by the Avian Malaria Parasite Plasmodium lophurae*

SYNOPSIS. Plasmodium lophurae cannot carry out extensive de novo purine biosynthesis, and depends upon the host erythrocyte for a supply of preformed purines. Exogenous purines taken up by the parasitized erythrocyte may constitute a major source of preformed purines for parasite nucleotide biosynthesis. The uptake of exogenous radioactive purine compounds and their incorporation into nucleic acids by duck erythrocytes parasitized with P. lophurae, uninfected erythrocytes, and erythrocyte-free parasites were studied. P. lophurae was found to have a remarkable ability, both intracellularly and extracellularly, to take up and utilize certain exogenous purines such as adenosine, inosine, and hypoxanthine. Incorporation studies indicated that this species has a functional purine salvage pathway by which inosine, hypoxanthine, and adenosine can be converted to both adenine and guanine nucleotides.     

The use of agarose in the determination of anchorage-independent growth

Summary At the present time, growth in agar suspension is one of the best in vitro correlates of tumorigenicity. Growth in agarose, however, has not been evaluated extensively as an in vitro criterion for tumorigenicity. In the present study we have tested 19 cell lines, including six mouse-human hybrids, for growth in agarose and agar in the presence and absence of exogenous hypoxanthine. None of the six nontumorigenic cell lines grew in agar or agarose. Ten of the 13 tumorigenic cell lines grew in both agar and agarose with about equal efficiency. The remaining three tumorigenic cell lines grew well in agarose but poorly or not at all in agar. Hypoxanthine did not stimulate the growth in agar or agarose of any of the cell lines except BHK. We conclude that growth in agarose may be a more sensitive marker for tumorigenicity than growth in agar and that BHK is exceptional in its sensitivity to supplemental purines. This research was supported by DHEW NCI Contract No. NO-CP-2-3234 and by USPHS Medical Scientist Training Grant GM 02042-07.     

Evaluation of cellular purine transport and metabolism in the Caco-2 cell using comprehensive high-performance liquid chromatography method for analysis of purines

ABSTRACT

Using Caco-2 cells and our previously developed high-performance liquid chromatography method for quantification of purine bases, nucleosides, and nucleotides, we evaluated cellular purine transport and uptake. The analytes were separated using YMC-Triart C18 column with gradient elution. We used Caco-2 cells as intestinal model cells and monitored purine transport across a monolayer for 2 h. The degree of change of purine concentrations in the permeate was very slight; however, it was possible to simultaneously determine these parameters for all purines because of our method's high sensitivity. In the present study, the purine bases (adenine, guanine, hypoxanthine, and xanthine) showed a relatively high permeability as compared with the nucleosides (adenosine, guanosine, inosine, and xanthosine). Increased concentration of metabolites in the permeate was also observed following the addition of purines. In a cell uptake assay, both the cell culture medium (extracellular) and the cells extracted from Caco-2 with acetonitrile:water (7:3) (intracellular) were measured. The additional nucleoside did not increase significantly within the cells. On the other hand, we observed that nucleotide, such as ATP, increased in the cell in a time-dependent manner following the addition of nucleoside. The additional nucleosides were considered to be rather recycled via the salvage pathway than metabolized to purine bases and/or uric acid in the cell. Such differences might have affected the increase in the serum uric acid levels depending on purine form.     

Regulation of cyclic nucleotide phosphodiesterase forms by serum and insulin in cultured fibroblasts.

A rapid reduction of cyclic nucleotide phosphodiesterase activity occurs after the replating of confluent cultures of BHK 21 c/13 fibroblasts into fresh medium. This reduction in activity depends on the density to which the cultures are reseeded and the concentration of serum in the medium. Enzyme activity in BHK cells is restored after 24 to 48 hours if cells are diluted into medium containing 10% fetal calf serum or 0.5% fetal calf serum supplemented with insulin (10(-6)M), but not into 0.5% serum alone. The restoration in enzyme activity is blocked by cycloheximide or Actinomycin D. When BHK cells become quiescent by maintanance in 0.5% serum conditions for 48 hours, a rapid (15--60 minutes) increase in cyclic AMP phosphodiesterase activity occurs when 10% serum is added to the cultures. Enzyme activity is increased even further after 24 to 48 hours in the 10% serum. Cycloheximide or Actinomycin D do not affect the rapid increase in enzyme activity in response to serum, but completely inhibit the long term increase. In contrast to serum, insulin (10(-8) to 10(-6)M) has no short term effect, but does increase enzyme activity after 24 to 48 hours to levels comparable to those seen with addition of 10% serum. As is the case with serum, this long term effect of insulin on enzyme activity is prevented by inhibitors of protein and RNA synthesis. Kinetic analyses of cyclic AMP phosphodiesterase activity in homogenates of quiescent BHK cells indicate the presence of only high Km (congruent to 20 muM) enzyme activity. Addition of serum or insulin to quiescent cells results in the appearance of apparent low Km enzyme activity in homogenates. Sucrose gradient analysis of BHK cells displays two forms of cyclic AMP phosphodiesterase enzyme activity: a 3--4 S form and 5--6 S form. In quiescent cells, the 5--6 S form greatly predominates relative to the 3--4 S form. Addition of serum to quiescent cells results in a rapid appearance of increased 3--4 S form enzyme activity. Insulin also increases the activity of this higher affinity 3--4 S enzyme form after 24 to 48 hours in culture. The functional significance of short and long term regulation of cyclic nucleotide phosphodiesterase(s) in cells is discussed.     

Purine metabolism in Leishmania donovani and Leishmania braziliensis

We have studied purine metabolism in the culture forms of Leishmania donovani and Leishmania braziliensis. These organisms are incapable of synthesizing purines de novo from glycine, serine, or formate and require an exogenous purine for growth. This requirement is better satisfied by adenosine or hypoxanthine than by guanosine. Bothe adenine and inosine are converted to a common intermediate, hypoxanthine, before transformation to nucleotides. This is due to the activity of an adenine aminohydrolase (EC 3.5.4.2), a rather unusual finding in a eukaryotic cell. There is a preferential synthesis of adenine nucleotides, even when guanine or xanthine are used as precursors.The pathways of purine nucleotide interconversions in these Leishmania resemble those found in mammalian cells except for the absence of de novo purine biosynthesis and the presence of an adenine-deaminating activity.     

Cytochalasin B releases a major surface-associated glycoprotein, fibronectin, from cultured fibroblasts

BHK21 cells cultured in minimal essential medium (Eagle) supplemented with 10% dialyzed fetal calf serum did not grow as they did in whole serum containing medium. Logarithmic growth was, however, initiated after a lag period, the length of which was dependent upon the cell density: medium volume ratio. The quiescent cells conditioned the medium during this lag period, and growth stimulation was apparently due to the release of serine into the medium. Cells cultured in 10% dialyzed serum plus the low molecular weight fraction of serum (serum dialysate), grew with kinetics similar to cells cultured in serum containing medium. When serum dialysate was chromatographed on Bio-gel P-2 the growth promoting activity eluted with the amino acids. Each of the non-essential amino acids was tested for its ability to stimulate the growth of cells in 10% dialyzed serum. Serine was capable of stimulating cell growth to the same extent as 10% serum dialysate and its concentration optimum was similar to its concentration in 10% serum dialysate. The remaining non-essential amino acids were either slightly stimulatory or had no effect on cell growth. Shifting a logarithmically growing population of cells to serine-free medium resulted in the accumulation of 95% of the cells in the G1 phase of the cell cycle within 24 h. Escape from the G1 block could occur if serine was added to the medium or if the cells were allowed to condition the medium. Entry of cells into S phase after the addition of 0.05 μmoles/ml of serine followed a 4–6 h lag and 80% of the cells were synthesizing DNA 12 h after shift-up.     

Purine requirements for the expression of Cape buffalo serum trypanocidal activity

Cape buffalo serum contains xanthine oxidase which generates trypanocidal H₂O₂ during the catabolism of hypoxanthine and xanthine. The present studies show that xanthine oxidase-dependent trypanocidal activity in Cape buffalo serum was also elicited by purine nucleotides, nucleosides, and bases even though xanthine oxidase did not catabolize those purines. The paradox was explained in part, by the presence in serum of purine nucleoside phosphorylase and adenosine deaminase, that, together with xanthine oxidase, catabolized adenosine, inosine, hypoxanthine, and xanthine to uric acid yielding trypanocidal H₂O₂. In addition, purine catabolism by trypanosomes provided substrates for serum xanthine oxidase and was implicated in the triggering of xanthine oxidase-dependent trypanocidal activity by purines that were not directly catabolized to uric acid in Cape buffalo serum, namely guanosine, guanine, adenine monophosphate, guanosine diphosphate, adenosine 3′:5-cyclic monophosphate, and 1-methylinosine. The concentrations of guanosine and guanine that elicited xanthine oxidase-dependent trypanocidal activity were 30–270-fold lower than those of other purines requiring trypanosome-processing which suggests differential processing by the parasites.     

Efficiency of purine utilization by Helicobacter pylori: roles for adenosine deaminase and a NupC homolog

The ability to synthesize and salvage purines is crucial for colonization by a variety of human bacterial pathogens. Helicobacter pylori colonizes the gastric epithelium of humans, yet its specific purine requirements are poorly understood, and the transport mechanisms underlying purine uptake remain unknown. Using a fully defined synthetic growth medium, we determined that H. pylori 26695 possesses a complete salvage pathway that allows for growth on any biological purine nucleobase or nucleoside with the exception of xanthosine. Doubling times in this medium varied between 7 and 14 hours depending on the purine source, with hypoxanthine, inosine and adenosine representing the purines utilized most efficiently for growth. The ability to grow on adenine or adenosine was studied using enzyme assays, revealing deamination of adenosine but not adenine by H. pylori 26695 cell lysates. Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines. This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides. Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens. In this study we define a more comprehensive model for purine acquisition and salvage in H. pylori that includes purine uptake by a NupC homolog and catabolism of adenosine via adenosine deaminase.     

Metabolism of Exogenous Purine Bases and Nucleosides by Salmonella typhimurium

Purine-requiring mutants of Salmonella typhimurium LT2 containing additional mutations in either adenosine deaminase or purine nucleoside phosphorylase have been constructed. From studies of the ability of these mutants to utilize different purine compounds as the sole source of purines, the following conclusions may be drawn. (i) S. typhimurium does not contain physiologically significant amounts of adenine deaminase and adenosine kinase activities. (ii) The presence of inosine and guanosine kinase activities in vivo was established, although the former activity appears to be of minor significance for inosine metabolism. (iii) The utilization of exogenous purine deoxyribonucleosides is entirely dependent on a functional purine nucleoside phosphorylase. (iv) The pathway by which exogenous adenine is converted to guanine nucleotides in the presence of histidine requires a functional purine nucleoside phosphorylase. Evidence is presented that this pathway involves the conversion of adenine to adenosine, followed by deamination to inosine and subsequent phosphorolysis to hypoxanthine. Hypoxanthine is then converted to inosine monophosphate by inosine monophosphate pyrophosphorylase. The rate-limiting step in this pathway is the synthesis of adenosine from adenine due to lack of endogenous ribose-l-phosphate.     

Helicobacter pylori Salvages Purines from Extracellular Host Cell DNA Utilizing the Outer Membrane-Associated Nuclease NucT

Helicobacter pylori is a bacterial pathogen that establishes life-long infections in humans, and its presence in the gastric epithelium is strongly associated with gastritis, peptic ulcer disease, and gastric cancer. Having evolved in this specific gastric niche for hundreds of thousands of years, this microbe has become dependent on its human host. Bioinformatic analysis reveals that H. pylori has lost several genes involved in the de novo synthesis of purine nucleotides, and without this pathway present, H. pylori must salvage purines from its environment in order to grow. While the presence and abundance of free purines in various mammalian tissues has been loosely quantified, the concentration of purines present within the gastric mucosa remains unknown. There is evidence, however, that a significant amount of extracellular DNA is present in the human gastric mucosal layer as a result of epithelial cell turnover, and this DNA has the potential to serve as an adequate purine source for gastric purine auxotrophs. In this study, we characterize the ability of H. pylori to grow utilizing only DNA as a purine source. We show that this ability is independent of the ComB DNA uptake system, and that H. pylori utilization of DNA as a purine source is largely influenced by the presence of an outer membrane-associated nuclease (NucT). A ΔnucT mutant exhibits significantly reduced extracellular nuclease activity and is deficient in growth when DNA is provided as the sole purine source in laboratory growth media. These growth defects are also evident when this nuclease mutant is grown in the presence of AGS cells or in purine-free tissue culture medium that has been conditioned by AGS cells in the absence of fetal bovine serum. Taken together, these results indicate that the salvage of purines from exogenous host cell DNA plays an important role in allowing H. pylori to meet its purine requirements for growth.     

Growth limitation of BHK-21 cells and its relation to folate metabolism.

The role of folate metabolism in growth control in monolayer and suspension cell cultures was studied in three related cell lines: BHK-21, polyoma-transformed BHK-21 (PyBHK), and an aminopterin-resistant derivative of BHK-21 (A5). BHK-21 cells had extremely low levels of dihydrofolate reductase, PyBHK had higher levels, and A5 had extremely high levels. Hypoxanthine and thymidine together, but not individually, induced BHK-21 to grow in agar, and stimulated its growth in agarose and monolayer culture. PyBHK and A5 grew spontaneously in agar, and hypoxanthine plus thymidine had little or no effect on their growth either in suspension or in monolayer cultures. We found that exogenous folinic acid, a derivative of folate metabolism that bypasses the function of dihydrofolate reductase, mimicked the growth-stimulatory effects of exogenous hypoxanthine plus thymidine BHK-21. We conclude that the growth limitation of BHK-21 in suspension culture is due, in part, to a deficiency of dihydrofolate reductase. This enzyme deficiency limits nucleoside synthesis and can be overcome by supplying end products of this pathway.     

Phosphohydrolases on the cell surface of BHK cells: loss during long term culture.

Low passage BHK 21/13 cells contain two cell surface enzymes, a nucleotide pyrophosphatase and a monophosphoester hydrolase, which together hydrolyze exogenous UDP-galactose to free galactose. During serial passage, BHK cells successively lose both enzymes. Concomitant with the loss of these enzymatic activities, changes in cell morphology, as well as in the serum requirement for the initiation of DNA synthesis, were observed. Clonal sublines of BHK cells were isolated, which differed qualitatively in their ability to hydrolyze UDP-galactose. Clonal BHK sublines, which exhibited both enzymatic activities on their cell surface, resembled low passage BHK cells in morphology and serum requirement for the initiation of DNA synthesis. Sublines not containing these enzymes resembled BHK cells of high passage cultures. The ability of intact BHK cells to hydrolyze exogenous nucleotide sugars may serve as an indicator for the progression of BHK cells from a normal to a more transformed state.     

Extracellular purine and pyrimidine catabolism in cell culture

The presence of purines and pyrimidines bases, nucleosides, and nucleotides in the culture medium has shown to differently affect the growth of a Chinese hamster ovary (CHO) cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP; Carvalhal et al., Biotech Prog. 2003;19:69-83). CHO, BHK, as well as Sf9 cell growth was clearly reduced in the presence of purines but was not affected by pyrimidines at the concentrations tested. The knowledge about the mechanisms by which nucleotides exert their effect when present outside the cells remains very incomplete. The catabolism of both extracellular purines and pyrimidines was followed during the culture of CHO cells. Purines/pyrimidines nucleotides added at a concentration of 1 mM to the culture medium decreased to negligible concentrations in the first 2 days. Purine and pyrimidine catabolism originated only purinic and pyrimidic end-products, respectively. The comparison between AMP catabolism in serum-free cultures (CHO cells expressing Factor VII and Sf9 cells) and in cultures containing serum (CHO cells expressing SEAP and BHK cells expressing Factor VII) showed that AMP extracellular catabolism is mediated by both cells and enzymes present in the serum. This work shows that the quantification of purines and pyrimidines in the culture medium is essential in animal cell culture optimization. When using AMP addition as a chemical cell growth strategy for recombinant protein production improvement, AMP extracellular concentration monitoring allows the optimization of the multiple AMP addition strategy for a prolonged cell culture duration with high specific productivity.     

Purine metabolism in microplasmodia of Physarum polycephalum.

The uptake and utilization of purine nucleosides and purines in microplasmodia of Physarum polycephalum were investigated. The results revealed a unique pattern, namely that exogenous purine nucleosides are readily taken up and metabolised, while free purine bases are hardly taken up. The pathways of incorporation have been elucidated in studies with whole cells and with cell-free extracts. The ribonucleosides (adenosine, inosine and guanosine) can be converted into ribonucleotides in two ways; either directly catalysed by a kinase or by a phosphorolytic cleavage to the free base (adenine, hypoxanthine and guanine respectively) which can then be activated by a purine phosphoribosyltransferase. Apparently the purine phosphoribosyltransferases do not react with exogenous purine bases. The deoxyribonucleosides (deoxyadenosine, deoxyinosine and deoxyguanosine) are also phosphorolysed by purine nucleoside phosphorylase to adenine, hypoxanthine and guanine respectively. A portion of deoxyadenosine is directly phosphorylated to dAMP. It appears that only a minor part of the soluble nucleotide pool can be synthesised from exogenous supplied nucleosides and that none of the deoxyribonucleosides specifically label DNA. There is no catabolism of the purine moiety. In agreement with the above findings, we have found that analoguees of purine nucleosides are more toxic than their corresponding purine base analogues.     

Growth induction by serum or polyoma virus inhibits the aggregation of trypsinised suspensions of BHK21 tissue culture fibroblasts

BHK21 hamster tissue culture fibroblasts, when brought into suspension with trypsin, aggregate spontaneously in serum-free medium. The amount of aggregation appears to depend on the proportion of quiescent (growth inhibited) cells which were present in the culture. It is greatest in cells from cultures whose growth is inhibited, either by high cell density or by low serum concentration. When serum is added to low serum cultures, growth is induced and S phase and mitosis follow at 14 and 20 h respectively. It has now been found that aggregation ceases less than 2 h after the addition of serum. Growth can also be induced in such cultures by infection with high multiplicities of polyoma virus. Infected cells enter S phase about 28 h after infection. It has been found that aggregation ceases between 12 and 20 h after infection. Thus in both these cases loss of aggregation precedes S phase by about 12 h. It is concluded that the absence of aggregation in suspensions derived from polyoma virus-transformed lines of BHK21 cells is a consequence of their resistance to density-dependent inhibition of growth. Failure to undergo spontaneous aggregation appears to be an indicator of an early surface change associated with the induction of growth.