Mitogenic enhancement of purine base phosphoribosylation in Swiss mouse 3T3 cells

Increased uptake of guanosine and the purine bases, adenine, hypoxanthine, and guanine, occurs within minutes after addition of fresh serum or purified growth-promoting agents to density-arrested cultures of Swiss 3T3, Swiss 3T6, and tertiary mouse embryo fibroblasts. Enhancement of uptake is maximal by about 50 min, is potentiated by combinations of growth promoters, and involves a process distinguishable from that of the enhanced uridine uptake on the basis of time course, pattern of growth-factor responsiveness and the failure of uridine to inhibit purine uptake. Enhanced purine uptake in 3T3 cells results from stimulation of the phosphoribosylation of purine bases and consequent trapping of nucleotide derivatives rather than from increased rates of membrane transport of the purine compounds. Increased availability of 5-phosphoribosyl 1-pyrophosphate (PRPP), identifiable within 30 min of serum addition, provides a likely explanation for increased purine base phosphoribosylation. Mitogenic stimulation of purine uptake is unaffected by cycloheximide but is markedly reduced in sodium-free medium. Enhanced purine uptake does not clearly depend upon changes in concentrations of effectors of intracellular PRPP synthesis. Nevertheless, when the possibility of direct mitogenic activation of PRPP synthase was studied, no differences were found in activities of this enzyme in extracts from stimulated and unstimulated 3T3 cells and mouse embryo fibroblasts.     

Extracellular purine nucleosides stimulate cell division and morphogenesis: Pathological and physiological implications

Extracellular purine nucleosides and nucleotides are ubiquitous, phylogenetically ancient, intercellular signals. Purines are released from hypoxic, damaged or dying cells. Purine nucleosides and nucleotides are potent mitogens for several types of cells such as fibroblasts, endothelial cells and neuroglia. They also induce other cell types to differentiate. For example, they act synergistically with nerve growth factor to stimulate neurite outgrowth from a pheochromocytoma cell line (PC12). We propose that after injury to tissues, including the central nervous system, purine nucleosides and nucleotides interact synergistically with other growth factors. They stimulate proliferation and morphological changes in the various cell types involved in the wound healing response. In the central nervous system this response includes glial proliferation, capillary endothelial cell proliferation, and sprouting of nerve axons. Since many actions of extracellular purines are mediated through specific cell surface receptors, this hypothesis has broad pharmacological implications.     

Sialidase activity of oncogenic cells transformed by herpes simplex virus

Sialidase in normal nononcogenic hamster embryo fibroblasts was not active toward added disialo- and trisialoganglioside, but all transformed lines of hamster embryo fibroblasts studied had sialidase active toward added ganglioside substrate. The levels of exogenous sialidase activity suggested a parallel trend in the amount of this activity and the degree of oncogenicity of the transformed cells. Exogenous sialidase activity in a weakly oncogenic cell line was found to increase after passage of the cells into hamsters and isolation and cell culture of the resulting tumors which gave rise to a cell line of relatively high oncogenicity.     

Human tumor cells in culture stimulate glycosaminoglycan synthesis by human skin fibroblasts

The human tumor cell lines, MM-96, FME, HCT-8, HT-29, MCF-7 and T-47D, in culture produced a factor or factors able to stimulate glycosaminoglycan (GAG) synthesis in human skin fibroblasts (HF). Conditioned growth media from the melanoma MM-96 and the colon carcinoma HT-29 produced a 10- and 8-fold stimulation of HF GAG synthesis, respectively, with an even larger stimulation of hyaluronic acid. Conditioned media from the melanoma FME and the breast carcinomas MCF-7 and T-47D stimulated GAG synthesis 2-fold, whereas media from the colon carcinoma HCT-8 gave a variable response often with no effect on GAG levels. Conditioned media from HF cultures had no effect on tumor cell GAG synthesis. Coculture of tumor cells and HF also resulted in increased GAG synthesis, and the degree of stimulation was similar to that with the conditioned media. Tumor cell-conditioned media were also effective in stimulating GAG synthesis by porcine smooth muscle cells and by chick embryo fibroblasts in culture, although the increase in GAG synthesis was much less than with HF cultures. These findings support the concept that the stromal desmoplasia characteristic of many growing and invasive tumors in vivo arises by tumor cell modulation of GAG synthesis by surrounding normal connective tissue cells.     

Identification and characterization of trypanocides by functional expression of an adenosine transporter from Trypanosoma brucei in yeast

The causative agents of sleeping sickness, Trypanosoma brucei rhodesiense and T. brucei gambiense, do not synthesize purines de novo but salvage purine bases and nucleosides from their hosts. We used yeast as an expression system for functional characterization of the trypanosomal adenosine transporter TbAT1. A selection of purine analogs and flavonoids were tested for their ability to interfere with adenosine transport, with the aims of identifying (a) trypanocidal TbAT1 substrates, and (b) inhibitors of trypanosomal purine transport. Cordycepin (3'-deoxyadenosine) was a TbAT1 substrate of high activity against T. brucei rhodesiense (IC50 0.2 nM). Inhibitors of mammalian nucleoside transport were not active, while the flavonol silibinin was a potent, noncompetitive inhibitor of TbAT1-mediated adenosine transport in yeast. Silibinin also inhibited melarsen-induced lysis of bloodstream form trypanosomes. IC50 values to T. brucei rhodesiense and to human carcinoma cells were 0.6 and 140 microM, respectively, indicating a good selectivity towards the parasites. Further studies are necessary to elucidate the effects of flavonoids on trypanosomal purine transport and their potential as trypanocides.     

A study of dose-response relationships of allpurinol in the presence of low or high purine turnover

Summary Effects of allopurinol (125–500 mg/m² body surface) were studied in normal subjects during periods of 18 days both during a purine-free, isoenergetic liquid formula diet and additional intake of ribonucleic acid, 4 g/day. Plasma uric acid and renal excretion of uric acid, oxypurines (hypoxanthine plus xanthine) and orotic acid were measured and total purine excretion calculated. Effects of allopurinol were evaluated by comparison of the results obtained in the steady state during diet alone (average of days 7–10) with those during allopurinol administration (days 16–18).During the purine-free diet, plasma uric acid was lowered more than urinary uric acid by allopurinol on doses of 250–500 mg/m² (44%–54% of control values on 500 mg/m²), demonstrating an increase in renal clearance. At the same dose, the uric acid lowering effect of allopurinol was more pronounced with than without purine loads (plasma 41%, urine 32% of control on 500 mg/m² during purine intake), while renal uric acid clearance was decreased. The more pronounced reduction of uric acid excretion during purine administration was balanced to the greater part by a more pronounced increased in oxypurine excretion. Total purine excretion was reduced by about 20% during the purine-free diet irrespective of dose. The size of this purine deficit was doubled, but was also independent of dose during addition of purines. Orotic acid excretion increased with dose during allopurinol treatment and was reduced by addition of purines.With respect to uric acid lowering effects, these results are in accordance with findings in patients overproducing uric acid endogenously and suggest that the uric acid lowering effect of allopurinol is enhanced with increasing concentrations of purine bases, presumably due to the tight binding of oxipurinol to xanthine oxidase. The small uricosuric effect of allopurinol seen during ingestion of the purine-free diet possibly is attributable to drug-induced orotic aciduria. The increase in size during purine intake of the purine deficit may result from reduced absorption of dietary purines during allopurinol treatment. Apparently, maximum effects of allopurinol on endogenous synthesis and/or absorption of purines from the gut are exerted by low doses.Abbreviations HPRT Hypoxanthine phosphoribosyl transferase - PRPP Phosphoribosyl pyrophosphate - RNA Ribonucleic acid This study was supported in part by grants of the Deutsche Forschungsgemeinschaft and Deutsche Wellcome GmbHDedicated to Prof. Nepomuk Zöllner on the occasion of his 65th birthday     

Changes in cell-surface sialic acid content during chick embryo development

The amount of cell-surface sialic acid found in the material released by trypsin from chick fibroblasts rose markedly from day 8 to day 16 of embryo development. The rise seemed to result from two processes: increased sialylation of N-linked carbohydrate chains, and enhancement of the amount of O-linked structures. Eight-day cells were more quickly detached from the substrate than 16-day cells, since in 8-day fibroblasts detachment of half the adherent cells only required 10 min, compared to 20 min in 16-day fibroblasts. Re-adhesion to the substrate was also faster for the younger fibroblasts, and 40% of the 8-day trypsin-treated cells re-adhered within 30 min compared to about 3 h for the same proportion of 16-day cells. The age-dependent differences in the amount of cell-surface sialic acid do not account for the differences in the adhesive capacity of embryo cells. The fact that neuraminidase did not affect cell detachment or re-adhesion indicates that cell-surface sialic acid does not play an important part in the adhesive capacity of embryo fibroblasts.     

De novo purine synthesis, purine salvage, and DNA synthesis in normal and Lesch-Nyhan fibroblasts infected with Mycoplasma pneumoniae.

The effects of Mycoplasma pneumoniae on host cell metabolism were studied by using two types of host cells, MRC-5 human lung fibroblasts, a normal cell line, and Lesch-Nyhan fibroblasts, a cell line deficient in hypoxanthine-guanine phosphoribosyl transferase (EC 2.4.2.8). The susceptibilities of the two cell types were determined by infecting the cells with M. pneumoniae at different multiplicities of infection (MOI). Our data indicate that the Lesch-Nyhan cells were four times more susceptible to damage by M. pneumoniae than the MRC-5 cells. The effects of different MOIs (10 and 50) on de novo purine synthesis. DNA synthesis, and the development of a cytopathic effect were determined. In both cell types, the higher MOI inhibited de novo purine synthesis to a greater extent than the lower MOI. This correlated closely with the cytopathic effect which developed in the monolayers (i.e., the more the inhibition of de novo purine synthesis, the greater the cytopathic effect which developed). In the Lesch-Nyhan cells, DNA synthesis was completely inhibited by the high MOI, whereas in the MRC-5 cells, DNA synthesis was stimulated by the high MOI. In the MRC-5 cells infected with M. pneumoniae, purine salvage activity increased, as indicated by an increase in adenosine deaminase (EC 3.5.4.4) activity. These data indicate that M. pneumoniae alters host cell metabolism, particularly the nucleic acid metabolic pathways. This may explain in part the mechanism of pathogenesis of M. pneumoniae infection.     

Trophic effects of purines in neurons and glial cells

In addition to their well known roles within cells, purine nucleotides such as adenosine 5' triphosphate (ATP) and guanosine 5' triphosphate (GTP), nucleosides such as adenosine and guanosine and bases, such as adenine and guanine and their metabolic products xanthine and hypoxanthine are released into the extracellular space where they act as intercellular signaling molecules. In the nervous system they mediate both immediate effects, such as neurotransmission, and trophic effects which induce changes in cell metabolism, structure and function and therefore have a longer time course. Some trophic effects of purines are mediated via purinergic cell surface receptors, whereas others require uptake of purines by the target cells. Purine nucleosides and nucleotides, especially guanosine, ATP and GTP stimulate incorporation of [3H]thymidine into DNA of astrocytes and microglia and concomitant mitosis in vitro. High concentrations of adenosine also induce apoptosis, through both activation of cell-surface A3 receptors and through a mechanism requiring uptake into the cells. Extracellular purines also stimulate the synthesis and release of protein trophic factors by astrocytes, including bFGF (basic fibroblast growth factor), nerve growth factor (NGF), neurotrophin-3, ciliary neurotrophic factor and S-100beta protein. In vivo infusion into brain of adenosine analogs stimulates reactive gliosis. Purine nucleosides and nucleotides also stimulate the differentiation and process outgrowth from various neurons including primary cultures of hippocampal neurons and pheochromocytoma cells. A tonic release of ATP from neurons, its hydrolysis by ecto-nucleotidases and subsequent re-uptake by axons appears crucial for normal axonal growth. Guanosine and GTP, through apparently different mechanisms, are also potent stimulators of axonal growth in vitro. In vivo the extracellular concentration of purines depends on a balance between the release of purines from cells and their re-uptake and extracellular metabolism. Purine nucleosides and nucleotides are released from neurons by exocytosis and from both neurons and glia by non-exocytotic mechanisms. Nucleosides are principally released through the equilibratory nucleoside transmembrane transporters whereas nucleotides may be transported through the ATP binding cassette family of proteins, including the multidrug resistance protein. The extracellular purine nucleotides are rapidly metabolized by ectonucleotidases. Adenosine is deaminated by adenosine deaminase (ADA) and guanosine is converted to guanine and deaminated by guanase. Nucleosides are also removed from the extracellular space into neurons and glia by transporter systems. Large quantities of purines, particularly guanosine and, to a lesser extent adenosine, are released extracellularly following ischemia or trauma. Thus purines are likely to exert trophic effects in vivo following trauma. The extracellular purine nucleotide GTP enhances the tonic release of adenine nucleotides, whereas the nucleoside guanosine stimulates tonic release of adenosine and its metabolic products. The trophic effects of guanosine and GTP may depend on this process. Guanosine is likely to be an important trophic effector in vivo because high concentrations remain extracellularly for up to a week after focal brain injury. Purine derivatives are now in clinical trials in humans as memory-enhancing agents in Alzheimer's disease. Two of these, propentofylline and AIT-082, are trophic effectors in animals, increasing production of neurotrophic factors in brain and spinal cord. Likely more clinical uses for purine derivatives will be found; purines interact at the level of signal-transduction pathways with other transmitters, for example, glutamate. They can beneficially modify the actions of these other transmitters.     

Purine metabolism in the intact sporozoites and merozoites of Eimeria tenella

Intact Eimeria tenella sporozoites and merozoites did not incorporate radiolabeled formate or glycine into their purine nucleotides suggesting a lack of de novo purine synthesis. However, [U-14C]glucose was incorporated into the cellular purine and pyrimidine nucleotide pools of both forms probably via conversion to radiolabeled ribose-1-phosphate and/or 5'-phosphoribosyl-1-alpha-pyrophosphate and the resulting action of various purine and pyrimidine salvage enzymes. Both forms of the parasite salvaged radiolabeled purine bases and nucleosides in a similar fashion. These purines were incorporated into ribonucleotides and into RNA and DNA. Adenine and inosine were transformed to hypoxanthine. Adenosine was converted to both inosine and hypoxanthine. Hypoxanthine and xanthine were not oxidized to uric acid but were metabolized to nucleotides. Guanosine was cleaved to guanine; guanine was deaminated to xanthine. The results demonstrate the presence of several purine salvage pathways. Purine phosphoribosylating and nucleoside phosphorylating activities as well as purine nucleoside cleaving and adenosine, adenine and guanine deaminating activities were evident. The metabolic evidence suggests the enzymes required to convert the newly formed nucleoside monophosphates to ATP and GTP were present also.     

In vitro differentiation of mouse embryonic stem cells: enrichment of endodermal cells in the embryoid body

Embryonic stem (ES) cells have the potential to differentiate into all three germ layers, providing new perspectives not only for embryonic development but also for the application in cell replacement therapies. Even though the formation of an embryoid body (EB) in a suspension culture has been the most popular method to differentiate ES cells into a wide range of cells, not much is known about the characteristics of EB cells. To this end, we investigated the process of EB formation in the suspension culture of ES cells at weekly intervals for up to 6 weeks. We observed that the central apoptotic area is most active in the first week of EB formation and that the cell adhesion molecules, except beta-catenin, are highly expressed throughout the examination period. The sequential expression of endodermal genes in EBs during the 6-week culture correlated closely with that of normal embryo development. The outer surface of EBs stained positive for alpha-fetoprotein and GATA-4. When isolated from the 2-week-old EB by trypsin treatment, these endodermal lineage cells matured in vitro into hepatocytes upon stimulation with various hepatotrophic factors. In conclusion, our results demonstrate that endodermal cells can be retrieved from EBs and matured into specific cell types, opening new therapeutic usage of these in vitro differentiated cells in the cell replacement therapy of various diseases.     

Variations among species and cell types in the effects of caffeine on mutagen-induced cytotoxicity and postreplication repair of DNA

The influence of caffeine on cytotoxicity and postreplication repair of DNA was examined following exposure of several cell types to physical and chemical agents known to damage DNA. The cell types used in this study were normal human fibroblasts (HS-WP), human xeroderma pigmentosum fibroblasts (SGL), Chinese hamster V79 cells, mouse BALB/c-3T3 cells, and secondary Syrian hamster embryo cells. The DNA damaging agents were ultraviolet light (UV), N-2-acetoxy-fluorenylacetamide (AFAA), nitroquinoline-N-oxide (NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Induction of cytotoxicity in Chinese hamster V79 cells due to ultraviolet light or AFAA exposure was enhanced by caffeine at a concentration of 1.0 mM in the culture medium, but not at 0.2 or 0.05 mM. Caffeine also inhibited postreplication repair in these cells at the same concentrations. In contrast, postreplication repair was not affected by caffeine at concentrations up to 1.0 mM in normal human fibroblasts (HS-WP), human xeroderma pigmentosum fibroblasts (SGL), secondary Syrian hamster embryo cells, and mouse BALB/c-3T3 cells following treatment with ultraviolet light, AFAA, NQO, or MNNG. Cytotoxicity in BALB/c-3T3 cells following exposure to ultraviolet light or AFAA was enhanced in the presence of caffeine at 1.0 or 0.2 mM, although these concentrations of caffeine had no effect on postreplication repair in these cells. The inhibitory effect of caffeine on postreplication repair was found only in Chinese hamster V79 cells among the five cell types used in this study. Both normal and xeroderma pigmentosum human cells repaired mutagen-induced DNA damage equally well in the absence or presence of caffeine at concentrations of 1.0 mM or less.     

Induction of matrix metalloproteinase 9 expression in breast carcinoma cells by a soluble factor from fibroblasts

Tumor-stromal interactions appear to play an important role in the induction of metalloproteinase expression in malignant tumors. We describe a tissue culture system in which expression of MMP-9 (gelatinase B or the 92 kDa type IV collagenase/gelatinase) was induced by co-cultivation of fibroblasts with breast cancer cell lines. While neither the breast cancer cells nor the normal rat embryo fibroblasts made MMP-9 alone in culture, human MMP-9 was made in the co-cultures. The MMP-9 was secreted in a latent form. The induction occurred at least in part through increases in the MMP-9 mRNA levels in the breast cancer cells. These increases did not appear to require protein synthesis. Conditioned medium from the fibroblasts could duplicate the induction of MMP-9 in the breast cancer cell lines. The active factor in the medium was inactivated by heat or by trypsin suggesting that it was a protein. This protein was in the size range of 30–100 kDa. Thus, fibroblasts could secrete a factor which was able to regulate the expression of MMP-9 in breast cancer cells.     

ATP, beta-gamma-methylene-ATP, and adenosine inhibit non-cholinergic non-adrenergic transmission in rat urinary bladder

ATP and adenosine caused a dose-dependent and reversible inhibition of the atropine-resistant contraction response to transmural nerve stimulation in the rat urinary bladder. Both purines also inhibited contraction responses to acetylcholine and direct muscle stimulation, indicating a postjunctional effect on the transmission. It seems as ATP per se inhibits the excitatory transmission, because the stable ATP-analogue beta-gamma-methylene-ATP was inhibitory as well, and because exogenous adenosine deaminase annulled the inhibition by adenosine but not that by ATP or beta-gamma-methylene-ATP. Blockade of purine inactivation enhanced the inhibitory action of ATP and adenosine, and by itself inhibited the transmission. These results are consistent with the possibility that endogenous purines may modulate non-cholinergic non-adrenergic excitatory transmission in the rat urinary bladder.     

Production,purification and characterization of rat interferon

Summary Priming with heterologous mouse interferon, increased production of an antiviral substance induced in rat diploid fibroblasts by Newcastle disease virus. This substance was characterized as an acid stable interferon. This rat interferon exhibited marked cross-species antiviral activity when tested in mouse L929B cells, guinea pig embryo fibroblasts, human fibroblasts and bovine cells but was not active on chick embryo cells.     

ATP, β-γ-methylene-ATP,and adenosine inhibit non-cholinergic non-adrenergic transmission in rat urinary bladder

ATP and adenosine caused a dose-dependent and reversible inhibition of the atropine-resistant contraction response to transmural nerve stimulation in the rat urinary bladder. Both purines also inhibited contraction responses to acetylcholine and direct muscle stimulation, indicating a postjunctional effect on the transmission. It seems as ATP per se inhibits the excitatory transmission, because the stable ATP-analogue β-γ-methylene-ATP was inhibitory as well, and because exogenous adenosine deaminase annulled the inhibition by adenosine but not that by ATP or β-γ-methylene-ATP. Blockade of purine in-activation enhanced the inhibitory action of ATP and adenosine, and by itself inhibited the transmission. These results are consistent with the possibility that endogenous purines may modulate non-cholinergic non-adrenergic excitatory transmission in the rat urinary bladder.     

The induction of aneuploidy by alkylated purines: effects on early and late cell cycle events

It has been shown that alkylated bases induce aneuploidy inmammalian cells in culture. The mechanism of action is not clear,however, data with 6-dimethyl amino purine (6DMAP) suggest thatthis analogue might act by affecting the cytoskeleton and proteinkinases involved in cell cycle regulation (cdc2/p34). The aimof this work was to study the effect of O⁶methylguanine (O⁶meG),O⁶ethylguanine (O⁶etG) and 6DMAP on DNA synthesis induced bygrowth factors in two cell lines, 3T3 and CHEF/18 fibroblasts,which respond in opposite ways to substances affecting the cytoskeleton,colchicine and cholera toxin: DNA synthesis initiation is stimulatedin 3T3 cells and inhibited in CHEF/18 cells by such compounds.Our results indicate that O⁶meG and O⁶etG behave like choleratoxin, in as much as they inhibit DNA synthesis induced by epidermalgrowth factor plus insulin in CHEF/18 cells, and stimulate itin 3T3 cells. 6DMAP behaves differently and inhibits DNA synthesisin both cell lines. The inhibition (or stimulation) was greaterwhen alkylated bases were added before S phase started, suggestingthat these compounds might affect early events of the cell cycle.In CHEF/18 cells the three alkylated bases were able to induceaberrant metaphases and ana-telophases with different efficiency(70–100%). The effect was not dependent on the G₁–Sblock and it was reversible even after cell commitment to DNAsynthesis. ⁴To whom correspondence should be addressed     

Prostaglandin production and cellular aging

Prostaglandin (PG) production by human embryo lung fibroblasts (HELF) is stimulated by a number of effectors including angiotensin, thrombin, bradykinin and ascorbic acid. The types of prostaglandins produced are to a degree effector-dependent. For example, angiotensin stimulates mainly PGE₂ synthesis, thrombin stimulates production of both PGE₂ and prostacyclin while bradykinin and ascorbic acid stimulate production of PGE₂, PGF_2α, prostacyclin and thromboxane A₂. Upon senescence in culture, prostacyclin (PGI₂) production by HELF drops dramatically in response to ascorbic or arachidonic acids. An overall drop in prostaglandin synthesis is observed with bradykinin stimulation. Prostaglandin production is also related to senescence in human skin fibroblasts (HSF). These cells produce predominantly PGI₂. Prostacyclin production in response to bradykinin drops in HSF as they are obtained from individuals of increasing chronologic age. Thus our results indicate changes in prostaglandin production upon senescence, a dependency of these changes on the given stimulus and a correlation between in vivo and in culture aging with respect to prostaglandin production.     

Purine metabolism in Trypanosoma cruzi

Culture forms of Trypanosoma cruzi are incapable of synthesizing purines de novo from formate, glycine, or serine and require an exogenous purine for growth. Adenine, hypoxanthine, guanine, xanthine and their respective ribonucleosides are equal in their abilities to support growth. Radiolabeled purine bases, with the exception of guanine, are stable and are converted to their respective ribonucleotides directly by phosphoribosyltransferase activity. Guanine is both converted to its ribonucleotide and deaminated to xanthine. Purine nucleosides are not hydrolysed to any extent but are converted to their respective ribonucleotides. This conversion may involve a rate-limiting ribonucleoside cleaving activity or a purine nucleoside kinase or phosphotransferase activity. The apparent order of salvage efficiency for the bases and their respective ribonucleosides is adenine > hypoxanthine > guanine > xanthine.     

Mutations of ATIC and ADSL affect purinosome assembly in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency

The purinosome is a multienzyme complex composed by the enzymes active in de novo purine synthesis (DNPS) that cells transiently assemble in their cytosol upon depletion or increased demand of purines. The process of purinosome formation has thus far been demonstrated and studied only in human epithelial cervical cancer cells (HeLa) and human liver carcinoma cells (C3A) transiently expressing recombinant fluorescently labeled DNPS proteins. Using parallel immunolabeling of various DNPS enzymes and confocal fluorescent microscopy, we proved purinosome assembly in HeLa, human hepatocellular liver carcinoma cell line (HepG2), sarcoma osteogenic cells (Saos-2), human embryonic kidney cells (HEK293), human skin fibroblasts (SF) and primary human keratinocytes (KC) cultured in purine-depleted media. Using the identical approach, we proved in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency that various mutations of ATIC and ADSL destabilize to various degrees of purinosome assembly and found that the ability to form purinosomes correlates with clinical phenotypes of individual ADSL patients. Our results thus shown that the assembly of functional purinosomes is fully dependent on the presence of structurally unaffected ATIC and ADSL complexes and presumably also on the presence of all the other DNPS proteins. The results also corroborate the hypothesis that the phenotypic severity of ADSL deficiency is mainly determined by structural stability and residual catalytic capacity of the corresponding mutant ADSL protein complexes, as this is prerequisite for the formation and stability of the purinosome and at least partial channeling of succinylaminoimidazolecarboxamide riboside-ADSL enzyme substrates-through the DNPS pathway.