Purine Biosynthesis Enzyme Research Articles

Reconstruction of Purine Metabolism in Bacillus subtilis to Obtain the Strain Producer of AICAR: A New Drug with a Wide Range of Therapeutic Applications

AICAR is a natural compound, an analogue and precursor of adenosine. As activator of AMP-activated protein kinase (AMPK), AICAR has a broad therapeutic potential, since it normalizes the carbohydrate and lipid metabolism and inhibits the proliferation of tumor cells. The synthesis of AICAR inBacillus subtiliscells is controlled by the enzymes of purine biosynthesis; their genes constituting purine operon (pur-operon). Reconstruction of purine metabolism inB. subtiliswas performed to achieve overproduction of AICAR. For this purpose, the genepurH, which encodes formyltransferase/IMP-cyclohydrolase, an enzyme that controls the conversion of AICAR to IMP, was removed from theB. subtilisgenome, ensuring the accumulation of AICAR. An insertion inactivating the genepurRthat encodes the negative transcriptional regulator of the purine biosynthesis operon was introduced into theB.subtilischromosome in order to boost the production of AICAR; the transcription attenuator located in the leader sequence ofpur-operon was deleted. Furthermore, the expression integrative vector carrying a strong promoter of therpsFgene encoding the ribosomal protein S6 was designed. The heterologousEscherichia coligenepurFencoding the first enzyme of the biosynthesis of purines with impaired allosteric regulation, as well as the modifiedE.coligeneprsresponsible for the synthesis of the precursor of purines - phosphoribosyl pyrophosphate (PRPP) - was cloned into this vector under the control of therpsFgene promoter. The modifiedpurFandprsgenes were inserted into the chromosome of theB. subtilisstrain.B. subtilisstrain obtained by these genetic manipulations accumulates 11-13 g/L of AICAR in the culture fluid.

Purine biosynthesis and the case of the “missing” genes

This work is a survey of known and suspected purine biosynthesis genes in sequenced archaea. We searched existing sequence databases for genes for the 11 steps (17 distinct genes) of de novo purine biosynthesis in 65 sequenced archaea, and carefully inspected each hit for the presence of active site residues and other residues known or suspected to be required for function. We found a number of problems with existing annotations, such as missing active site residues, fusions, and incorrect initiation sites. There is also evidence that the pathway has been shaped by horizontal gene transfer, duplication, and gene loss. Two groups of archaea lack a gene normally required for purine biosynthesis, suggesting that there are at least two more variants in this pathway to discover. We propose the existence of a gene encoding a GAR transformylase enzyme that is structurally unrelated to either known GAR transformylase, expected to be present in seven of the 65 archaea studied. A second proposed gene encodes a novel IMP cyclohydrolase function and is expected to be present in the Crenarchaea and Pyrococci.Functional characterization of some purine biosynthesis enzymes will also be reported.

Gcn4p-mediated transcriptional repression of ribosomal protein genes under amino-acid starvation

Gcn4p is a well-characterized bZIP transcription factor that activates more than 500 genes encoding amino acids and purine biosynthesis enzymes, and many stress-response genes under various stress conditions. Under these stresses, it had been shown that transcriptions of ribosomal protein (RP) genes were decreased. However, the detailed mechanism of this downregulation has not been elucidated. In this study, we present a novel mechanistic model for a repressive role of Gcn4p on RP transcription, especially under amino-acid starvation. It was found that Gcn4p bound directly to Rap1p, which in turn inhibited Esa1p-Rap1p binding. The inhibition of Esa1p recruitment to RP promoters ultimately reduced the level of histone H4 acetylation and RP transcription. These data revealed that Gcn4p has simultaneous dual roles as a repressor for RP genes as well as an activator for amino-acid biosynthesis genes. Moreover, our results showed evidence of a novel link between general control of amino-acid biosynthesis and ribosome biogenesis mediated by Gcn4p at an early stage of adaptation to amino-acid starvation.

Pemetrexed Indirectly Activates the Metabolic Kinase AMPK in Human Carcinomas

The chemotherapeutic drug pemetrexed, an inhibitor of thymidylate synthase, has an important secondary target in human leukemic cells, aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate-dependent enzyme of purine biosynthesis. The purine intermediate aminoimidazolecarboxamide ribonucleotide (ZMP), which accumulates behind this block, transmits an inhibitory signal to the mTORC1 complex via activation of the cellular energy sensor AMP-activated kinase (AMPK). Given that the PI3K-AKT-mTOR pathway is frequently deregulated during carcinogenesis, we asked whether the indirect activation of AMPK by pemetrexed offers an effective therapeutic strategy for carcinomas with defects in this pathway. Activation of AMPK by ZMP in pemetrexed-treated colon and lung carcinoma cells and the downstream consequences of this activation were strikingly more robust than previously seen in leukemic cells. Genetic experiments demonstrated the intermediacy of AICART inhibition and the centrality of AMPK activation in these effects. Whereas AMPK activation resulted in marked inhibition of mTORC1, other targets of AMPK were phosphorylated that were not mTORC1-dependent. Whereas AMPK activation is thought to require AMPKα T172 phosphorylation, pemetrexed also activated AMPK in carcinoma cells null for LKB1, the predominant AMPKα T172 kinase whose deficiency is common in lung adenocarcinomas. Like rapamycin analogs, pemetrexed relieved feedback suppression of PI3K and AKT, but the prolonged accumulation of unphosphorylated 4E-BP1, a tight-binding inhibitor of cap-dependent translation, was seen following AMPK activation. Our findings indicate that AMPK activation by pemetrexed inhibits mTORC1-dependent and -independent processes that control translation and lipid metabolism, identifying pemetrexed as a targeted therapeutic agent for this pathway that differs significantly from rapamycin analogs.

Mechanism of Action of Escherichia coli Phosphoribosylaminoimidazolesuccinocarboxamide Synthetase

The conversion of ATP, L-aspartate, and 5-aminoimidazole-4-carboxyribonucleotide (CAIR) to 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide (SAICAR), ADP, and phosphate by phosphoribosylaminoimidazolesuccinocarboxamide synthetase (SAICAR synthetase) represents the eighth step of de novo purine nucleotide biosynthesis. SAICAR synthetase and other enzymes of purine biosynthesis are targets of natural products that impair cell growth. Prior to this study, no kinetic mechanism was known for any SAICAR synthetase. Here, a rapid equilibrium random ter-ter kinetic mechanism is established for the synthetase from Escherichia coli by initial velocity kinetics and patterns of linear inhibition by IMP, adenosine 5'-(beta,gamma-imido)triphosphate (AMP-PNP), and maleate. Substrates exhibit mutual binding antagonism, with the strongest antagonism between CAIR and either ATP or L-aspartate. CAIR binds to the free enzyme up to 200-fold more tightly than to the ternary enzyme-ATP-aspartate complex, but the latter complex may be the dominant form of SAICAR synthetase in vivo. IMP is a competitive inhibitor with respect to CAIR, suggesting the possibility of a hydrogen bond interaction between the 4-carboxyl and 5-amino groups of enzyme-bound CAIR. Of several aspartate analogues tested (hadacidin, l-malate, succinate, fumarate, and maleate), maleate was by far the best inhibitor, competitive with respect to L-aspartate. Inhibition by IMP and maleate is consistent with a chemical mechanism for SAICAR synthetase that parallels that of adenylosuccinate synthetase.

Anthranilate synthase can generate sufficient phosphoribosyl amine for thiamine synthesis in Salmonella enterica.

In bacteria, the biosynthetic pathway for the hydroxymethyl pyrimidine moiety of thiamine shares metabolic intermediates with purine biosynthesis. The two pathways branch after the compound aminoimidazole ribotide. Past work has shown that the first common metabolite, phosphoribosyl amine (PRA), can be generated in the absence of the first enzyme in purine biosynthesis, PurF. PurF-independent PRA synthesis is dependent on both strain background and growth conditions. Standard genetic approaches have not identified a gene product singly responsible for PurF-independent PRA formation. This result has led to the hypothesis that multiple enzymes contribute to PRA synthesis, possibly as the result of side products from their dedicated reaction. A mutation that was able to restore PRA synthesis in a purF gnd mutant strain was identified and found to map in the gene coding for the TrpD subunit of the anthranilate synthase (AS)-phosphoribosyl transferase (PRT) complex. Genetic analyses indicated that wild-type AS-PRT was able to generate PRA in vivo and that the P362L mutant of TrpD facilitated this synthesis. In vitro activity assays showed that the mutant AS was able to generate PRA from ammonia and phosphoribosyl pyrophosphate. This work identifies a new reaction catalyzed by AS-PRT and considers it in the context of cellular thiamine synthesis and metabolic flexibility.

Chimeric yeast prions with unstable inheritance

The Saccharomyces cerevisiae [PSI] factor, a cytoplasmic omnipotent nonsense suppressor, is a conformationally changed (prion) form of translation termination factor eRF3 (Sup35p). Induction and maintenance of the [PSI] factor depend on the prionizing peptide located in the N domain of Sup35p. The N domain of Sup35p was fused with phosphoribosylaminoimidazole carboxylase (Ade2p), a purine biosynthesis enzyme, and the hybrid protein (NM-Sup35p::Ade2p) was tested for induction of the [PSI] factor. Transformation with a centromeric plasmid carrying the gene for NM-Sup35p::Ade2p induced a [PSI]-like factor in yeast cells, which was evident from efficient nonsense suppression. The suppressory effect depended on the presence of the prionizing peptide both in the hybrid protein and in Sup35p synthesized from the chromosomal gene, as well as on the presence of the prion-like [PIN] factor in the cell.

Repression of GCN4 mRNA Translation by Nitrogen Starvation in Saccharomyces cerevisiae

Saccharomyces cerevisiae activates a regulatory network called "general control" that provides the cell with sufficient amounts of protein precursors during amino acid starvation. We investigated how starvation for nitrogen affects the general control regulatory system, because amino acid biosynthesis is part of nitrogen metabolism. Amino acid limitation results in the synthesis of the central transcription factor Gcn4p, which binds to specific DNA-binding motif sequences called Gcn4-protein-responsive elements (GCREs) that are present in the promoter regions of its target genes. Nitrogen starvation increases GCN4 transcription but efficiently represses expression of both a synthetic GCRE6::lacZ reporter gene and the natural amino acid biosynthetic gene ARO4. Repression of Gcn4p-regulated transcription by nitrogen starvation is independent of the ammonium sensing systems that include Mep2p and Gpa2p or Ure2p and Gln3p but depends on the four upstream open reading frames in the GCN4 mRNA leader sequence. Efficient translation of GCN4 mRNA is completely blocked by nitrogen starvation, even when cells are simultaneously starved for amino acids and eukaryotic initiation factor-2 alpha is fully phosphorylated by Gcn2p. Our data suggest that nitrogen starvation regulates translation of GCN4 by a novel mechanism that involves the four upstream open reading frames but that still acts independently of eukaryotic initiation factor-2 alpha phosphorylation by Gcn2p.

Crystal structure of a bifunctional transformylase and cyclohydrolase enzyme in purine biosynthesis.

ATIC, the product of the purH gene, is a 64 kDa bifunctional enzyme that possesses the final two activities in de novo purine biosynthesis, AICAR transformylase and IMP cyclohydrolase. The crystal structure of avian ATIC has been determined to 1.75 A resolution by the MAD method using a Se-methionine modified enzyme. ATIC forms an intertwined dimer with an extensive interface of approximately 5,000 A(2) per monomer. Each monomer is composed of two novel, separate functional domains. The N-terminal domain (up to residue 199) is responsible for the IMPCH activity, whereas the AICAR Tfase activity resides in the C-terminal domain (200-593). The active sites of the IMPCH and AICAR Tfase domains are approximately 50 A apart, with no structural evidence of a tunnel connecting the two active sites. The crystal structure of ATIC provides a framework to probe both catalytic mechanisms and to design specific inhibitors for use in cancer chemotherapy and inflammation.

The purine biosynthesis enzyme PRAT detected in proenzyme and mature forms during development of Drosophila melanogaster

Glutamine phosphoribosylpyrophosphate amidotransferase (PRAT; EC 2.4.2.14) is the first and rate-limiting enzyme of de novo purine biosynthesis. PRAT expression in Drosophila development was examined to determine if it is correlated with cell proliferation and/or nutritional isolation. An antiserum, raised against the 16 carboxyl-terminal amino acids of PRAT, detects two proteins corresponding to a 60 kDa proenzyme and 55 kDa mature enzyme, consistent with a 53 amino acid propeptide predicted from the gene sequence. Mature enzyme is maternally expressed, and proenzyme appears in embryos at 2–8 h, corresponding to the interval during which zygotic transcription is initiated. Upon hatching of first instar larvae, proenzyme levels are reduced and remain low relative to mature enzyme. Adult females have higher levels of both proteins relative to males, consistent with maternal expression. Maternal expression reflects a requirement for the enzyme during embryogenesis, while reduction in expression following hatching reflects a switch to an exogenous source of purines. Prat mRNA levels follow a similar overall pattern in the same developmental stages examined for the protein. Discovery of a second gene encoding PRAT with 78% amino acid identity leads to the possibility that the antiserum raised against the carboxyl-terminus detects two enzymes.

Towards understanding the first genome sequence of a crenarchaeon by genome annotation using clusters of orthologous groups of proteins (COGs).

Standard archival sequence databases have not been designed as tools for genome annotation and are far from being optimal for this purpose. We used the database of Clusters of Orthologous Groups of proteins (COGs) to reannotate the genomes of two archaea, Aeropyrum pernix, the first member of the Crenarchaea to be sequenced, and Pyrococcus abyssi. A. pernix and P. abyssi proteins were assigned to COGs using the COGNITOR program; the results were verified on a case-by-case basis and augmented by additional database searches using the PSI-BLAST and TBLASTN programs. Functions were predicted for over 300 proteins from A. pernix, which could not be assigned a function using conventional methods with a conservative sequence similarity threshold, an approximately 50% increase compared to the original annotation. A. pernix shares most of the conserved core of proteins that were previously identified in the Euryarchaeota. Cluster analysis or distance matrix tree construction based on the co-occurrence of genomes in COGs showed that A. pernix forms a distinct group within the archaea, although grouping with the two species of Pyrococci, indicative of similar repertoires of conserved genes, was observed. No indication of a specific relationship between Crenarchaeota and eukaryotes was obtained in these analyses. Several proteins that are conserved in Euryarchaeota and most bacteria are unexpectedly missing in A. pernix, including the entire set of de novo purine biosynthesis enzymes, the GTPase FtsZ (a key component of the bacterial and euryarchaeal cell-division machinery), and the tRNA-specific pseudouridine synthase, previously considered universal. A. pernix is represented in 48 COGs that do not contain any euryarchaeal members. Many of these proteins are TCA cycle and electron transport chain enzymes, reflecting the aerobic lifestyle of A. pernix. Special-purpose databases organized on the basis of phylogenetic analysis and carefully curated with respect to known and predicted protein functions provide for a significant improvement in genome annotation. A differential genome display approach helps in a systematic investigation of common and distinct features of gene repertoires and in some cases reveals unexpected connections that may be indicative of functional similarities between phylogenetically distant organisms and of lateral gene exchange.

Methotrexate inhibits the first committed step of purine biosynthesis in mitogen-stimulated human T-lymphocytes: a metabolic basis for efficacy in rheumatoid arthritis?

The immunosuppressive and anti-inflammatory effects of low-dose methotrexate (MTX) have been related directly to inhibition of folate-dependent enzymes by polyglutamated derivatives, or indirectly to adenosine release and/or apoptosis and clonal deletion of activated peripheral blood lymphocytes in S-phase. In this study of phytohaemagglutinin-stimulated primary human T-lymphocytes we show that MTX (20 nM to 20 microM) was cytostatic not cytotoxic, halting proliferation at G(1). This stasis of blastogenesis was associated with an inhibition of purine ribonucleotide synthesis but a stimulation of pyrimidine biosynthesis, the normal mitogen-induced expansion of ATP and GTP pools over 72 h being restricted to concentrations of unstimulated T-cells, whereas the increment in UTP pools exceeded that of controls. Decreased incorporation of H(14)CO(3) or [(14)C]glycine into purine ribonucleotides, with no radiolabel accumulation in any de novo synthetic intermediate but enhanced H(14)CO(3) incorporation into UTP, supported these MTX-related effects. Exaggerated [(14)C]hypoxanthine salvage (which normalized the purine and UTP pools) confirmed the increased availability of 5-phosphoribosyl-1-pyrophosphate (PP-ribose-P) as the molecular mechanism underlying these disparate changes. These results provide the first substantive evidence that the immunosuppressive effects of low-dose MTX in primary blasting human T-lymphocytes relate not to the inhibition of the two folate-dependent enzymes of purine biosynthesis but to inhibition of the first enzyme, amidophosphoribosyltransferase, thereby elevating PP-ribose-P and stimulating UTP synthesis. Varying cell types or incubation conditions employed by other workers, especially malignant/activated cells with high basal metabolic rates, might mask the effects noted in primary human T-lymphocytes. The findings imply the involvement of low-dose MTX in the inhibition of T-lymphocyte proliferation and proliferation-dependent processes in rheumatoid arthritis.

A diverse superfamily of enzymes with ATP‐dependent carboxylate—amine/thiol ligase activity

The recently developed PSI-BLAST method for sequence database search and methods for motif analysis were used to define and expand a superfamily of enzymes with an unusual nucleotide-binding fold, referred to as palmate, or ATP-grasp fold. In addition to D-alanine-D-alanine ligase, glutathione synthetase, biotin carboxylase, and carbamoyl phosphate synthetase, enzymes with known three-dimensional structures, the ATP-grasp domain is predicted in the ribosomal protein S6 modification enzyme (RimK), urea amidolyase, tubulin-tyrosine ligase, and three enzymes of purine biosynthesis. All these enzymes possess ATP-dependent carboxylate-amine ligase activity, and their catalytic mechanisms are likely to include acylphosphate intermediates. The ATP-grasp superfamily also includes succinate-CoA ligase (both ADP-forming and GDP-forming variants), malate-CoA ligase, and ATP-citrate lyase, enzymes with a carboxylate-thiol ligase activity, and several uncharacterized proteins. These findings significantly extend the variety of the substrates of ATP-grasp enzymes and the range of biochemical pathways in which they are involved, and demonstrate the complementarity between structural comparison and powerful methods for sequence analysis.

Regulation of signal transduction

1. 1. A systematic study is reported on the control of 1-phosphatidylinositol 4-kinase (PI kinase) and PI 4-phosphate 5-kinase (PIP kinase), enzymes of the phosphatidylinositol phosphorylation pathway which leads to the production of second messengers, IP 3 and DAG. In liver of normal male, adult, fed Wistar rats the steady state activity of PI kinase was 0.5 ± 0.01 and that of PIP kinase was 0.046 ± 0.003 nmol/hr/mg protein. The concentration of IP 3 was 1.8 ± 0.1 pmol/mg protein. 2. 2. That the two kinases have short half-lives was observed in starvation, where in the rat liver or bone marrow activities rapidly decreased and on refeeding were restored in a day. Injection to rats of the protein synthetic inhibitor, cycloheximide, yielded t 1 2 = 8 min for the two enzymes in bone marrow and t 1 2 = 80 min in liver. 3. 3. Linkage of the signal transduction enzymes with proliferation was shown by the high activities as compared to liver of these enzymes in rat organs of high cell renewal capacity, e.g., thymus, bone marrow, spleen and testes. 4. 4. Linkage with malignant proliferation was indicated by the observation that in rat hepatomas the enzyme activities increased 5- to 9-fold and were highest in rapidly growing hepatoma 3924A (29- and 45-fold). 5. 5. In human primary ovarian carcinoma PI and PIP kinase activities were elevated 4.4 and 2.9-fold, respectively, and in OVCAR-5 cells, 32- and 11-fold, respectively. Similar increases were observed in MDA-MB-435 human breast carcinoma cells in comparison with normal breast parenchymal cells. 6. 6. The linkage of signal transduction enzyme activities with malignant proliferation was also observed in experiments when human breast carcinoma cells were plated in flasks and expressed their proliferative capacity in the log phase. PI and PIP kinase activities steadily and coordinately increased to a peak 11-fold rise in mid-log phase. In late log and plateau phases the kinase activities gradually declined to the starting level. Similar observations were made for the two enzymes in human ovarian carcinoma OVCAR-5 cells and in rat hepatoma 3924A cells in tissue culture. 7. 7. In animals injected with cycloheximide the bone marrow PI and PIP kinase activities exhibited t 1 2 = 0.12 hr , the shortest decay rate in comparison with 8 enzymes of purine and pyrimidine biosynthesis with t 1 2 = = 0.6 to 4.3 hr . 8. 8. Injection of tiazofurin decreased PI and PIP kinase activities in the bone marrow with t 1 2 = 82 and 78 min , respectively. IP 3 concentration decreased with a t 1 2 = 23 min . 9. 9. Injection of quercetin decreased PI kinase activity in the bone marrow with t 1 2 = 17 min ; however, PIP kinase activity only decreased to 70% and activity leveled off at this value for 24 hr. The IP 3 concentration followed the decline in PI kinase activity with t 1 2 = 40 min . 10. 10. Addition of quercetin to OVCAR-5 cells in culture showed that different concentrations of this drug have little effect on PIP kinase activity; however, PI kinase activity decreased in a dose-dependent fashion with IC 50 = 44 μ m. The IP 3 concentration sharply declined with an IC 50 of 46 μ m and correlated best with the decrease in PI kinase activity. 11. 11. Quercetin inhibited the proliferation of OVCAR cells with an IC 50 of 63 μ m and of human breast carcinoma cells with IC 50 = 55 μ m. The decline of PI kinase activity preceded the inhibition of cell proliferation whereas PIP kinase activity was inhibited only to a minor extent. 12. 12. In vivo studies showed that in the bone marrow IP 3 concentration could be depleted by only 40–60% by injection of cycloheximide, tiazofurin or quercetin. Therefore, we postulated that there might be a salvage pathway that could replenish or maintain IP 3 concentration in a homeostatic adaptation. This hypothesis is strengthened by reports in the literature indicating that IP 3 may be generated by degradation of IP 6 and observations that addition of IP 6 to cancer cells yields IP 3 provide support for this hypothesis for a salvage pathway for IP 3 production.

Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes.

Glycinamide ribonucleotide (GAR) synthetase, GAR transformylase and aminoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 10-step de novo purine biosynthetic pathway. From a cDNA library of Arabidopsis thaliana, cDNAs encoding the above three enzymes were cloned by functional complementation of corresponding Escherichia coli mutants. Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transformylase or AIR synthetase. Comparisons of the three Arabidopsis purine biosynthetic enzymes with corresponding enzymes/polypeptide-fragments from procaryotic and eucaryotic sources indicate a high degree of conserved homology at the amino acid level, in particular with procaryotic enzymes. Assays from extracts of E. coli expressing the complementing clones verified the specific enzymatic activity of Arabidopsis GAR synthetase and GAR transformylase. Sequence analysis, as well as Northern blot analysis indicate that Arabidopsis has single and monofunctional enzymes. In this respect the organization of these three plant purine biosynthesis genes is fundamentally different from the multifunctional purine biosynthesis enzymes characteristic of other eucaryotes and instead resembles the one gene, one enzyme relationship found in procaryotes.

Structure of the allosteric regulatory enzyme of purine biosynthesis.

Multi-wavelength anomalous diffraction (MAD) has been used to determine the structure of the regulatory enzyme of de novo synthesis of purine nucleotides, glutamine 5-phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase, from Bacillus subtilis. This allosteric enzyme, a 200-kilodalton tetramer, is subject to end product regulation by purine nucleotides. The metalloenzyme from B. subtilis is a paradigm for the higher eukaryotic enzymes, which have been refractory to isolation in stable form. The two folding domains of the polypeptide are correlated with functional domains for glutamine binding and for transfer of ammonia to the substrate PRPP. Eight molecules of the feedback inhibitor adenosine monophosphate (AMP) are bound to the tetrameric enzyme in two types of binding sites: the PRPP catalytic site of each subunit and an unusual regulatory site that is immediately adjacent to each active site but is between subunits. An oxygen-sensitive [4Fe-4S] cluster in each subunit is proposed to regulate protein turnover in vivo and is distant from the catalytic site. Oxygen sensitivity of the cluster is diminished by AMP, which blocks a channel through the protein to the cluster. The structure is representative of both glutamine amidotransferases and phosphoribosyltransferases.

Regulation of deoxycytidine kinase activity and inhibition by DFDC

1. (1) Deoxycytidine kinase activity increased in a transformation- and progression-linked fashion in rat hepatomas of different proliferation rates (1). The activity also increased and was growth rate-linked in a series of tissue culture cell lines of human and animal tumors. 2. (2) Deoxycytidine kinase activity was stringently linked with expression of the neoplastic proliferative program as it sharply increased in log phase in tissue culture cells of hepatoma 3924A and several human carcinoma strains. 3. (3) Deoxycytidine kinase is subject to nutritional and hormonal regulation. On starvation the activity in liver decreased and on refeeding it returned to normal. Steroid hormone increased liver enzymic activity. Deoxycytidine kinase is substrate-inducible, since deoxycytidine injections in rat led to a 2- to 3-fold increase in hepatic enzyme activity. 4. (4) Actinomycin or cycloheximide treatment blocked the increase in liver deoxycytidine kinase activity induced by steroid or deoxycytidine treatment. Therefore, it is assumed that the rise in deoxycytidine kinase activity requires new RNA and protein synthesis. 5. (5) Cycloheximide treatment of rats carrying hepatomas yielded a t 1 2 = 3.4 hr in the tumor for deoxycytidine kinase activity which was the shortest among the examined enzymes of purine and pyrimidine biosynthesis. 6. (6) Actinomycin treatment of rats carrying hepatomas yielded a t 1 2 of 5.8 hr for deoxycytidine kinase activity in the tumor which was one of the shortest in the examined enzymes of purine and pyrimidine biosynthesis. 7. (7) Difluorodeoxycytidine (DFDC) is a competitive inhibitor ( K i = 7 − 28 μ m ) of deoxycytidine kinase from rat hepatoma and from human pancreatic carcinoma and ovarian carcinoma cells in culture.

Purine biosynthetic genes are required for cadmium tolerance in Schizosaccharomyces pombe.

Phytochelatins (PCs) are metal-chelating peptides produced in plants and some fungi in response to heavy metal exposure. A Cd-sensitive mutant of the fission yeast Schizosaccharomyces pombe, defective in production of a PC-Cd-sulfide complex essential for metal tolerance, was found to harbor mutations in specific genes of the purine biosynthetic pathway. Genetic analysis of the link between metal complex accumulation and purine biosynthesis enzymes revealed that genetic lesions blocking two segments of the pathway, before and after the IMP branchpoint, are required to produce the Cd-sensitive phenotype. The biochemical functions of these two segments of the pathway are similar, and a model based on the alternate use of a sulfur analog substrate is presented. The novel participation of purine biosynthesis enzymes in the conversion of the PC-Cd complex to the PC-Cd-sulfide complex in the fission yeast raises an intriguing possibility that these same enzymes might have a role in sulfur metabolism in the fission yeast S. pombe, and perhaps in other biological systems.

Purine biosynthetic genes are required for cadmium tolerance in Schizosaccharomyces pombe.

Phytochelatins (PCs) are metal-chelating peptides produced in plants and some fungi in response to heavy metal exposure. A Cd-sensitive mutant of the fission yeast Schizosaccharomyces pombe, defective in production of a PC-Cd-sulfide complex essential for metal tolerance, was found to harbor mutations in specific genes of the purine biosynthetic pathway. Genetic analysis of the link between metal complex accumulation and purine biosynthesis enzymes revealed that genetic lesions blocking two segments of the pathway, before and after the IMP branchpoint, are required to produce the Cd-sensitive phenotype. The biochemical functions of these two segments of the pathway are similar, and a model based on the alternate use of a sulfur analog substrate is presented. The novel participation of purine biosynthesis enzymes in the conversion of the PC-Cd complex to the PC-Cd-sulfide complex in the fission yeast raises an intriguing possibility that these same enzymes might have a role in sulfur metabolism in the fission yeast S. pombe, and perhaps in other biological systems.

Novel chemotherapeutic agents in clinical development

The development of new chemotherapeutic agents for cancer treatment is pursued with the hope of finding compounds with novel chemical characteristics, unique mechanisms of action, and with improved therapeutic indexes. Seven novel agents at different stages of clinical development have been selected for review. D1694 is a thymidylate synthase inhibitor that has intriguing preclinical activity. The anthrapyrazoles consist of three analogues that are undergoing phase II testing at present. One of these agents, CI-941, has been reported to have significant clinical activity in breast cancer. Lometrexol is an inhibitor of glycinamide ribonuclide transformylase, a critical enzyme in purine biosynthesis, that is undergoing phase II testing. Taxotere, a semisynthetic analogue of taxol that stabilizes microtubules, is currently undergoing phase I testing. Gemcitabine, a fluorinated analogue of deoxycytidine that can inhibit ribonucleotide reductase and be incorporated into DNA, is undergoing phase II testing. BMY-25067 is a mitomycin C analogue that is less myelosuppressive and more active than mitomycin C in preclinical models. Topotecan, a topoisomerase I inhibitor, has been shown to cause myelosuppression as the dose-limiting toxicity in phase I testing. Although each of these agents have some unique and novel characteristics that merit their clinical testing, these agents' long-range clinical role will depend on their efficacy in randomized phase III comparative trials.