Effect of 5'-difluoromethylthioadenosine, an inhibitor of methylthioadenosine phosphorylase, on proliferation of cultured cells

5'-Methylthioadenosine (MTA) is formed from decarboxylated S-adenosylmethionine during biosynthesis of polyamines. This nucleoside is cleaved by methylthioadenosine phosphorylase (MTA Pase) to adenine and 5-methylthioribose-I-phosphate in mammalian cells. 5'-Difluoromethylthioadenosine (DFMTA), a synthetic analog of MTA, was not a substrate for MTA Pase, but was a strong competitive inhibitor of the enzyme (Ki = 0.48 microM). DFMTA caused marked accumulation of labeled MTA formed from [35S]methionine in Raji cells, which contain MTA Pase, but not in CCRF-CEM cells, which do not contain this enzyme, suggesting that it also inhibits the enzyme in intact cells. DFMTA inhibited the growth of a variety of cultured cells and its cytostatic effect was roughly proportional to the MTA Pase activity of the cells. MTA also depressed the growth of cultured cells but, in contrast with DFMTA, its inhibitory effect was greater in MTA Pase-deficient cells (CCRF-CEM) than MTA Pase-containing cells (Raji). Inhibition of growth of Raji cells by DFMTA was partially reversed by exogenous adenine, a reaction product of MTA Pase. These results suggest that the utilization of adenine formed from MTA was important for proliferation of cells containing MTA Pase under the culture conditions employed, and that DFMTA inhibited cell growth by inhibiting MTA Pase activity.     

In vivo metabolism of 5'-methylthioadenosine in lemna

Evidence is presented that Lemna converts 5′-methylthioadenosine (MTA) to methionine. The methylthio moiety and four of the ribose carbons of the nucleoside contribute the methylthio and the four-carbon moieties of methionine. Plants grown in the presence of inhibitors which block methionine biosynthesis convert MTA to methionine at a rate sufficient to sustain normal growth (at least 4.4 nanomoles per colony per doubling with a molar yield of at least 65%). The pathway for conversion is shown to be constitutive in plants grown in standard medium and to function at a rate sufficient to dispose of MTA arising as a result of polyamine synthesis, and to explain the observed rate (1.4 nanomoles per colony per doubling) of preferential recycling of methionine sulfur (Giovanelli, Mudd, Datko 1981 Biochem Biophys Res Commun 100: 831-839). Rapid entry of methionine methyl into S-adenosylmethionine and phosphorylcholine was observed for plants grown in standard medium. Adenine generated during this cycle is efficiently salvaged into ADP and ATP.

Conversion of MTA to methionine completes the steps in methionine thiomethyl recycling (Giovanelli, Mudd, Datko 1981 Biochem Biophys Res Commun 100: 831-839) in which the sulfur of methionine is retained while the four-carbon moiety is not. The findings further show that the four-carbon moiety of methionine can be derived via the ribose moiety of MTA in addition to the established route from O-phosphohomoserine via transsulfuration. Previous observations (Giovanelli, Mudd, Datko 1980 Biochemistry of Plants pp 453-505) can now be interpreted as establishing that exogenous methionine down-regulates its own net synthesis via the transsulfuration pathway.

     

The metabolism of 5'-methylthioadenosine and 5-methylthioribose 1-phosphate in Saccharomyces cerevisiae

Cordycepin sensitive mutants of Saccharomyces cerevisiae, which are permeable to 5'-deoxy-5'-methylthioadenosine (MTA), were used to study the fate of the methylthioribose carbons of this purine nucleoside. Evidence is presented for the recycling of the methylthio group and part of the ribose portion of MTA in a biosynthetic pathway which leads to the synthesis of methionine. The main pathway involves the phosphorylytic cleavage of MTA by MTA phosphorylase yielding 5-methylthioribose 1-phosphate and adenine as products. Loss of the phosphate group of 5-methylthioribose 1-phosphate, concurrent with the rearrangement of the ribose carbons, leads to the synthesis of 2-keto-4-methylthiobutyric acid. In the final step of the sequence, 2-keto-4-methylthiobutyric acid is converted to methionine via transamination. Several compounds not directly associated with the biosynthesis of methionine were also isolated. These compounds, which may arise through the degradation of intermediates in the pathway, were: 5'-methylthioinosine, a deaminated catabolite of MTA; 5-methylthioribose, a result of the phosphorylysis of 5-methylthioribose 1-phosphate, and 3-methylthiopropionaldehyde, 3-methylthiopropionic acid and 2-hydroxy-4-methylthiobutyric acid, all arising from the catabolism of 2-keto-4-methylthiobutyric acid.     

Selective Inhibition of Nerve Growth Factor-Stimulated Protein Kinases by K-252a and 5''-S-Methyladenosine in PC12 Cells

K-252a, a protein kinase inhibitor isolated from the culture broth of Nocardiopsis sp., inhibits the nerve growth factor (NGF)-stimulated phosphorylation of microtubule-associated protein 2 (MAP2) and Kemptide (synthetic Leu-Arg-Arg-Ala-Ser-Leu-Gly) by blocking the activation of two independent kinases in PC12 cells: MAP2/pp250 kinase and Kemptide kinase. The NGF-stimulated activation of these kinases is inhibited in a dose-dependent manner following treatment of the cells with K-252a. Although these kinases also are activated by epidermal growth factor (EGF) and 12-O-tetradecanoyl-phorbol 13-acetate, K-252a has no inhibitory effect when these agents are used. Half-maximal inhibition of the activation of both kinases was observed at 10-30 nM K-252a. K-252a was shown to directly inhibit the activity of MAP2/pp250 kinase and Kemptide kinase when added to the phosphorylation reaction mixture in vitro; however, half-maximal inhibition under these conditions was observed at greater than or equal to 50 nM K-252a. These data suggest that K-252a exerts its effects at a step early in the cascade of events following NGF binding. The effects of K-252a are similar to those reported for 5'-S-methyladenosine (MTA) and other methyltransferase inhibitors. Treatment of PC12 cells with MTA inhibited NGF-, but not EGF-mediated activation of MAP2/pp250-kinase (Ki greater than 500 microM). MTA, when added to the phosphorylation reaction mixture in vitro, directly inhibited kinase activity (Ki = 50 microM), suggesting that the effects of MTA may be the result of its action on protein kinases rather than methyltransferases.     

Growth and metastases of human lung cancer are inhibited in mouse xenografts by a transition state analogue of 5'-methylthioadenosine phosphorylase

The S-adenosylmethionine (AdoMet) salvage enzyme 5'-methylthioadenosine phosphorylase (MTAP) has been implicated as both a cancer target and a tumor suppressor. We tested these hypotheses in mouse xenografts of human lung cancers. AdoMet recycling from 5'-methylthioadenosine (MTA) was blocked by inhibition of MTAP with methylthio-DADMe-Immucillin-A (MTDIA), an orally available, nontoxic, picomolar transition state analogue. Blood, urine, and tumor levels of MTA increased in response to MTDIA treatment. MTDIA treatment inhibited A549 (human non-small cell lung carcinoma) and H358 (human bronchioloalveolar non-small cell lung carcinoma cells) xenograft tumor growth in immunodeficient Rag2(-/-)γC(-/-) and NCr-nu mice. Systemic MTA accumulation is implicated as the tumor-suppressive metabolite because MTDIA is effective for in vivo treatment of A549 MTAP(-/-) and H358 MTAP(+/+) tumors. Tumors from treated mice showed increased MTA and decreased polyamines but little alteration in AdoMet, methionine, or adenine levels. Gene expression profiles of A549 tumors from treated and untreated mice revealed only modest alterations with 62 up-regulated and 63 down-regulated mRNAs (≥ 3-fold). MTDIA antitumor activity in xenografts supports MTAP as a target for lung cancer therapy.     

Action of Sulfanilamide on Ochromonas malhamensis

SYNOPSIS. Sulfanilamide inhibited the growth of O. malhamensis. Sulfanilamide growth inhibition was reversed competitively by PABA and by very high concentrations of folic acid. Folic acid at low concentrations, however, accentuated sulfa inhibition of growth. Vitamin B₁₂, methionine, p -aminobenzoylglutamic acid and pteroic add were effective to some extent as antagonists of sulfa. A marked reduction in the folate synthesis was accompanied by sulfa growth inhibition. This was restored on growth restoration by PABA, folic acid, vitamin B₁₂ and methionine. The reduction in folate synthesis held for all the folate fractions except one derivative—a formyl poly-glutamate. Sulfanilamide-inhibited cells had a considerable activity for in vitro synthesis of folate activity from precursors. ∼75% activity being retained at the 90% growth inhibition level. There was no change in chlorophyll, RNA and DNA contents as a result of sulfa growth inhibition.     

Conversion of 5-S-ethyl-5-thio-D-ribose to ethionine in Klebsiella pneumoniae. Basis for the selective toxicity of 5-S-ethyl-5-thio-D-ribose

5-S-Ethyl-5-thio-D-ribose (ethylthioribose) exhibits antiprotozoal activity against Plasmodium falciparum, Giardia lamblia, and Ochromonas malhamensis, but is nontoxic to cultured human and murine bone marrow cells (Riscoe, M. K., Ferro, A. J., and Fitchen, J. H. (1988) Antimicrob. Agents Chemother. 32, 1904-1906). We propose the following mechanism to account for the observed selective toxicity of ethylthioribose. 1) The cytocidal action of ethylthioribose against protozoa is a result of its conversion to ethionine, a well-known cytotoxic agent. 2) This transformation occurs through the pathway which normally converts 5-S-methyl-5-thio-D-ribose (methylthioribose) to methionine. 3) Conversion of ethylthioribose to ethionine cannot occur in mammalian cells since these cells cannot phosphorylate methylthioribose (ethylthioribose), a first step in the pathway to methionine (ethionine). To test this hypothesis, [5-3H]ethylthioribose has been synthesized and its metabolism by cell-free extracts of Klebsiella pneumoniae and rat liver was examined. The pathway by which methylthioribose is converted to methionine in K. pneumoniae is well characterized. When supplemented with ATP and L-glutamine, the bacterial extract efficiently converted [5-3H]ethylthioribose to [3H]ethionine. By contrast, ethionine was not produced upon incubation of [5-3H]ethylthioribose, ATP, and L-glutamine with rat liver homogenate. The mammalian cell extract lacks a kinase activity capable of converting ethylthioribose to 1-phospho-5-S-ethyl-5-thio-alpha-D-ribofuranoside, an obligate intermediate in the biosynthesis of ethionine from ethylthioribose in K. pneumoniae. These results support our hypothesis and provide a basis for understanding the apparently selective toxicity of ethylthioribose.     

The role of 5'-methylthioadenosine on rat calvaria cell differentiation.

The role of 5'-methylthioadenosine (MTA), formed during the process of polyamine biosynthesis, on differentiation of osteoprogenitor cells was assessed by its effects on alkaline phosphatase (ALP) activity, bone nodule formation and osteopontin contents of cultured rat calvaria (RC) cells. These three markers were stimulated by exogenous MTA and were depressed by 5'-difluoromethylthioadenosine (DFMTA), a synthetic inhibitor of MTA phosphorylase, which cleaves MTA to adenine and 5-methylthioribose-1-phosphate. 5-Methylthioribose and 2-keto-4-methylthiobutyrate, metabolites of 5-methylthioribose-1-phosphate, had no effects on ALP activity and bone nodule formation in the presence or absence of DFMTA. On the other hand, adenine enhanced ALP activity, bone nodule formation and osteopontin contents in mineralized nodules and also partially reversed DFMTA-induced inhibition of these three markers. MTA, its metabolites and DFMTA did not affect the growth of RC cells under these culture conditions. These results suggest that adenine formed from MTA is important in the differentiation of RC cells.     

Functional Identification of APIP as Human mtnB,a Key Enzyme in the Methionine Salvage Pathway

The methionine salvage pathway is widely distributed among some eubacteria, yeast, plants and animals and recycles the sulfur-containing metabolite 5-methylthioadenosine (MTA) to methionine. In eukaryotic cells, the methionine salvage pathway takes place in the cytosol and usually involves six enzymatic activities: MTA phosphorylase (MTAP, EC 2.4.2.28), 5′-methylthioribose-1-phosphate isomerase (mtnA, EC 5.3.1.23), 5′-methylthioribulose-1-phosphate dehydratase (mtnB, EC: 4.2.1.109), 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase (mtnC, EC 3.1.3.77), aci-reductone dioxygenase (mtnD, EC 1.13.11.54) and 4-methylthio-2-oxo-butanoate (MTOB) transaminase (EC 2.6.1.-). The aim of this study was to complete the available information on the methionine salvage pathway in human by identifying the enzyme responsible for the dehydratase step. Using a bioinformatics approach, we propose that a protein called APIP could perform this role. The involvement of this protein in the methionine salvage pathway was investigated directly in HeLa cells by transient and stable short hairpin RNA interference. We show that APIP depletion specifically impaired the capacity of cells to grow in media where methionine is replaced by MTA. Using a Shigella mutant auxotroph for methionine, we confirm that the knockdown of APIP specifically affects the recycling of methionine. We also show that mutation of three potential phosphorylation sites does not affect APIP activity whereas mutation of the potential zinc binding site completely abrogates it. Finally, we show that the N-terminal region of APIP that is missing in the short isoform is required for activity. Together, these results confirm the involvement of APIP in the methionine salvage pathway, which plays a key role in many biological functions like cancer, apoptosis, microbial proliferation and inflammation.     

Biosynthesis of methionine in mouse cells cultured in vitro

In the mouse cell-lines cultured in vitro, viz. L-cells and mouse embryo fibroblasts, the methylation of homocysteine to methionine is carried out by vitamin B12-dependent 5-methyltetrahydrofolate:L-homocysteine methyltransferase only. In these cells grown in the standard Eagle medium, the activity of another methyltransferase, which utilizes betaine as the methyl donor, was not detected. The high activity of the vitamin B12-dependent methionine synthetase is typical for mouse cells from the logarithmic phase of growth. In L-cells 60%, and in the mouse fibroblasts 30% of the enzyme exist in the holo-form; the ratio between the holo- and apoenzyme activity remains stable in cells from logarithmic and stationary cultures. The level of the activity of methionine synthetase strongly depends on the presence of vitamin B12, folate and methionine in the culture medium and is greater after prolonged contact of the cells with these agents.     

Euryhaline Chrysomonads: Nutrition and Toxigenesis in Prymnesium parvum, with Notes on Isochrysis galbana and Monochrysis lutheri

SYNOPSIS. The nutritional requirements of 3 isolates of Prymnesium parvum (2 Israeli, 1 Scottish) included vitamin B₁₂ and thiamine. For comparison, 2 other brackish chrysomonads were studied: Monochrysis lutheri isolated by Droop in Scotland and Isochrysis galbana purified by McLaughlin from a culture obtained from the Plymouth laboratory.
The isolates of Prymnesium parvum and Isochrysis galbana had a molecular B₁₂ specificity like Ochromonas malhamensis : no response to Factor B, pseudovitamin B₁₂, Factor A or Factor H. M. lutheri , in contrast, responded to pseudovitamin B₁₂, Factor H, and Factor A.
Thiamine was essential; 1.0 μg.% allowed full growth of P. parvum. The NaCl concentration for good growth was 0.3–5.0%; growth was possible from 6–12%. Dark growth was not achieved.
Ammonia, as suggested from its use in suppressing outbreaks of P. parvum , was sharply inhibitory, less so at high concentrations of NaCl or at acid pH.
Nitrate, ammonia, arginine, asparagine, methionine, histidine, alanine, glycine, serine, proline, leucine, isoleucine, tyrosine, aspartic and glutamic acids, acetylurea, and creatine served as nitrogen sources in both acid and alkaline media.
The phosphate requirement of P. parvum and M. lutheri and Isochrysis galbana was satisfied by inorganic phosphate, commercial glycerophosphate, yeast adenylic acid, cytidylic acid, monoethyl phosphate, and riboflavin monophosphate.
Laboratory cultures in defined media of the isolates of P. parvum were toxigenic to Lebistes and Gambusia. Culture fluids from alkaline media were more toxic than those from acid media, as previously noted in Israel.
Culture media suitable for production of large quantities of these organisms were developed.     

5'-Methylthioadenosine nucleosidase and 5-methylthioribose kinase activities in relation to stress-induced ethylene biosynthesis

The activities of 5'-methylthioadenosine (MTA) nucleosidase (EC 2.2.2.28) and 5-methylthioribose (MTR) kinase (EC 2.7.1.100) were related to changes in ethylene biosynthesis in tomato ( Lycopersicon esculentum Mill. cv. Rutgers) and cucumber ( Cucumis sativus Mill. cv. Poinsett 76) fruit following wounding and chemically induced stresses. Stress ethylene formation in wounded tomato and cucumber tissue continued to increase after wounding, reached its peak by 3h, and then declined. The activities of MTA nucleosidase and MTR kinase increased parallel to stress ethylene in both tissues. At peak ethylene formation, MTA and MTR kinase activities were 2- to 4-fold higher in wounded than in intact tissue. Wounded, mature-green tomato tissue treated with specific inhibitors of MTA nucleosidase and MTR kinase showed a significant reduction in the activities of these enzymes, which was concomitant with a decline in stress ethylene biosynthesis. When mature-green tomato discs were infiltrated with [¹⁴CH₃] MTA and wounded, radioactive MTR and methionine were formed. Incubation of mature-green tomato discs with Cu²⁺ and Li⁺ in the presence of kinetin increased ethylene biosynthesis. MTA nucleosidase activity was higher than that of the control in the presence of Cu²⁺ but not in the presence of Li⁺, while MTR kinase activity was lower than that of the control in both Cu²⁺ and Li⁺ treatments. Data indicate that MTA nucleosidase and MTR kinase are required for wound-induced ethylene biosynthesis but not for chemical stress-induced ethylene by Cu²⁺ or Li⁺ treatments.     

Femtomolar transition state analogue inhibitors of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Escherichia coli

Escherichia coli 5'-methylthioadenosine/S-adenosyl-homocysteine nucleosidase (MTAN) hydrolyzes its substrates to form adenine and 5-methylthioribose (MTR) or S-ribosylhomocysteine (SRH). 5'-Methylthioadenosine (MTA) is a by-product of polyamine synthesis and SRH is a precursor to the biosynthesis of one or more quorum sensing autoinducer molecules. MTAN is therefore involved in quorum sensing, recycling MTA from the polyamine pathway via adenine phosphoribosyltransferase and recycling MTR to methionine. Hydrolysis of MTA by E. coli MTAN involves a highly dissociative transition state with ribooxacarbenium ion character. Iminoribitol mimics of MTA at the transition state of MTAN were synthesized and tested as inhibitors. 5'-Methylthio-Immucillin-A (MT-ImmA) is a slow-onset tight-binding inhibitor giving a dissociation constant (K(i)(*)) of 77 pm. Substitution of the methylthio group with a p-Cl-phenylthio group gives a more powerful inhibitor with a dissociation constant of 2 pm. DADMe-Immucillins are better inhibitors of E. coli MTAN, since they are more closely related to the highly dissociative nature of the transition state. MT-DADMe-Immucillin-A binds with a K(i)(*) value of 2 pm. Replacing the 5'-methyl group with other hydrophobic groups gave 17 transition state analogue inhibitors with dissociation constants from 10(-12) to 10(-14) m. The most powerful inhibitor was 5'-p-Cl-phenylthio-DADMe-Immucillin-A (pClPhT-DADMe-ImmA) with a K(i)(*) value of 47 fm (47 x 10(-15) m). These are among the most powerful non-covalent inhibitors reported for any enzyme, binding 9-91 million times tighter than the MTA and SAH substrates, respectively. The inhibitory potential of these transition state analogue inhibitors supports a transition state structure closely resembling a fully dissociated ribooxacarbenium ion. Powerful inhibitors of MTAN are candidates to disrupt key bacterial pathways including methylation, polyamine synthesis, methionine salvage, and quorum sensing. The accompanying article reports crystal structures of MTAN with these analogues.     

Occurrence of 5''-Deoxy-5''-Methylthioadenosine Phosphorylase in the Mammalian CNS: Distribution and Kinetic Studies on the Rat Brain Enzyme

5'-Deoxy-5'-methylthioadenosine (MTA) phosphorylase catalyzes the cleavage of MTA, a secondary product of polyamine biosynthesis, to 5-methylthioribose-1-phosphate and adenine. The occurrence and the general properties of the enzyme were studied in mammalian brain with the following results. (1) Cerebral tissues contained levels of MTA phosphorylase that were comparable to those occurring in other mammalian tissues. (2) Interspecies differences in the enzyme distribution were quite limited, with the highest specific activity values observed in pig brain. Moreover, the enzyme seemed to be generally more concentrated in the cerebellar fractions. (3) Rat brain MTA phosphorylase was highly localized in the cellular soluble fraction. In the first days of rat life, its specific activity in the whole brain was observed to decline significantly from a value of 17.6 units/mg at 1-5 days of age to 13.7 units/mg at 6-10 days of age, remaining then fairly constant up to maturity. (4) Kinetic studies performed with the soluble enzyme extracted from rat brain showed: a pH optimum of 7.4; a Km value for MTA of about 10 microM; an inhibitory effect of the MTA analog 5'-deoxy-5'-isobutylthioadenosine; and a remarkable resistance of the enzyme to heat treatment.     

Variations in the Growth Response of Four Different Vitamin B12 Assay Microorganisms to the Same Tissue and Standard Preparations

Four microbiological assay procedures (Escherichia coli, Lactobacillus leichmanni, Euglena gracilis, and Ochromonas malhamensis) for the vitamin B(12) content of rat liver and kidney tissue were compared. Significant differences in the growth response for portions of the same sample based on the same standard preparations were noted. The liver values with E. coli were strikingly higher than those obtained with the other microorganisms, about which there was rather general agreement. With kidney tissue, E. coli again gave higher values, but not as significantly high as those obtained with L. leichmannii and E. gracilis. O. malhamensis values were significantly lower. These differences were greater than were expected and could not be explained by the differences of specificity and sensitivity of the assay organisms. Several methods of sample preparation, the possible presence of an alkali-heat-stable factor, different methods of sterilization of the sample plus medium, and different chromatographic procedures were explored in an attempt to find explanations for the variations in growth response, with the thought that these various methods would clarify in what form or forms the free and bound vitamin B(12) might exist in the samples. No clear-cut explanation was found.     

A Potential Pathway for Galactose Metabolism in Cucumis sativus L., A Stachyose Transporting Species

The ribose moiety of 5′-methylthioadenosine (MTA) is metabolized to form the four-carbon unit (2-aminobutyrate) of methionine in tomato tissue (Lycopersicon esculentum Mill., cv. Pik Red). When [U-¹⁴C-adenosine] MTA was administered to tomato tissue slices, label was recovered in 5-methylthioribose (MTR), methionine, 1-aminocyclopropane-1-carboxylic acid (ACC), C₂H₄ and other unidentified compounds. However, when [U-¹⁴C-ribose]MTR was administered, radioactivities were recovered in methionine, ACC and C₂H₄, but not MTA. This suggests that C₂H₄ formed in tomato pericarp tissue may be derived from the ribose portion of MTA via MTR, methionine and ACC. The conversion of MTR to methionine is not inhibited by aminoethoxyvinylglycine (AVG), but is O₂ dependent. These data present a new salvage pathway for methionine biosynthesis which may be important in relation to polyamine and ethylene biosynthesis in tomato tissue.     

Lipopolysaccharide-induced NF-kappa B activation in mouse 70Z/3 pre-B lymphocytes is inhibited by mevinolin and 5''-methylthioadenosine: roles of protein isoprenylation and carboxyl methylation reactions.

We show that both the lipopolysaccharide (LPS)-induced activation of NF-kappa DNA binding and kappa gene expression are blocked by treating murine pre-B lymphocyte 70Z/3 cells with 5'-methylthioadenosine (MTA), an inhibitor of several S-adenosylmethionine-dependent methylation reactions. We further show that the LPS-induced incorporation of radioactivity from [methyl-3H]methionine into methyl ester-like linkages on a group of membrane polypeptides is also inhibited by MTA treatment, suggesting the involvement of protein methylation reactions in the LPS signal transduction pathway. We also find that NF-kappa B and kappa gene activation in LPS-treated 70Z/3 cells is blocked by mevinolin, an inhibitor that prevents protein isoprenylation. Interestingly, mevinolin-treated cells also exhibited a marked reduction in the methylation of membrane proteins. Neither MTA nor mevinolin significantly inhibited NF-kappa B activation by phorbol myristate acetate, suggesting that these agents act early in signal transduction. These results provide the first evidence that carboxyl methylated and/or isoprenylated proteins play an essential role in the LPS-signaling pathway.     

The transfer of 5'-methylthioadenosine from B82 cells through the medium to CHW-1102 cells

The Chinese hamster cell line. CHW-1102, which is deficient in hypoxanthine guanine phosphoribosyl transferase (HGPRT+), incorporated a [3H]purine metabolite(s) from medium in which B82 cells, but not V79, A9 and BHK cells, had been grown for 24 h with [3H]hypoxanthine. A thin-layer chromatographic comparison of the medium revealed a large radioactive peak that was unique to the B82 medium and co-chromatographed with methylthioadenosine (MTA), but not with most other common purine bases and nucleosides. The addition of either MTA, adenine, or adenosine to B82 medium reduced the amount of radioactive material incorporated by CHW-1102 cells. Methylglyoxal bis(guanylhydrazone) inhibited the production of the [3H]metabolite(s) that were incorporated from B82 medium by CHW-1102 cells. Little MTA phosphorylase activity was detected in the mouse L cell lines, L929, B82, and A9, but activity was present in CHW-1102 cells. These results suggest that one of the metabolites in B82 medium is [3H]MTA, and this is taken up and cleaved by CHW-1102 cells to yield [3H]adenine, which is incorporated into nucleic acids. This accounts for the majority of contact-independent metabolite transfer (CIMT). In cocultures some interactions between B82 and CHW-1102 cells were positive for contact-dependent metabolite transfer (CDMT) or metabolic cooperation.