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Chembiochem : a European Journal of... Apr 2020N-Nitroso-containing natural products are bioactive metabolites with antibacterial and anticancer properties. In particular, compounds containing the diazeniumdiolate...
N-Nitroso-containing natural products are bioactive metabolites with antibacterial and anticancer properties. In particular, compounds containing the diazeniumdiolate (N-nitrosohydroxylamine) group display a wide range of bioactivities ranging from cytotoxicity to metal chelation. Despite the importance of this structural motif, knowledge of its biosynthesis is limited. Herein we describe the discovery of a biosynthetic gene cluster in Streptomyces alanosinicus ATCC 15710 responsible for producing the diazeniumdiolate natural product l-alanosine. Gene disruption and stable isotope feeding experiments identified essential biosynthetic genes and revealed the source of the N-nitroso group. Additional biochemical characterization of the biosynthetic enzymes revealed that the non-proteinogenic amino acid l-2,3-diaminopropionic acid (l-Dap) is synthesized and loaded onto a free-standing peptidyl carrier protein (PCP) domain in l-alanosine biosynthesis, which we propose may be a mechanism of handling unstable intermediates generated en route to the diazeniumdiolate. These discoveries will facilitate efforts to determine the biochemistry of diazeniumdiolate formation.
Topics: Alanine; Azo Compounds; Bacterial Proteins; Biosynthetic Pathways; Molecular Structure; Multigene Family; Streptomyces
PubMed: 31643127
DOI: 10.1002/cbic.201900565 -
Oncotarget Mar 2023Homozygous deletion of upregulates synthesis of purine (DNSP) and increases the proliferation of neoplastic cells. This increases the sensitivity of breast cancer...
INTRODUCTION
Homozygous deletion of upregulates synthesis of purine (DNSP) and increases the proliferation of neoplastic cells. This increases the sensitivity of breast cancer cells to DNSP inhibitors such as methotrexate, L-alanosine and pemetrexed.
MATERIALS AND METHODS
7,301 cases of MBC underwent hybrid-capture based comprehensive genomic profiling (CGP). Tumor mutational burden (TMB) was determined on up to 1.1 Mb of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. Tumor cell PD-L1 expression was determined by IHC (Dako 22C3).
RESULTS
208 (2.84%) of MBC featured loss. loss patients were younger ( = 0.002) and were more frequently ER- (30% vs. 50%; < 0.0001), triple negative (TNBC) (47% vs. 27%; < 0.0001) and less frequently HER2+ (2% vs. 8%; = 0.0001) than intact MBC. Lobular histology and mutations were more frequent in intact (14%) than loss MBC ( < 0.0001). (100%) and (97%) loss (9p21 co-deletion) were significantly associated with loss ( < 0.0001). Likely associated with the increased TNBC cases, BRCA1 mutation was also more frequent in loss MBC (10% vs. 4%; < 0.0001). As for immune checkpoint inhibitors biomarkers, higher TMB >20 mut/Mb levels in the intact MBC ( < 0.0001) and higher PD-L1 low expression (1-49% TPS) in the loss ( = 0.002) were observed.
CONCLUSIONS
loss in MBC has distinct clinical features with genomic alterations (GA) affecting both targeted and immunotherapies. Further efforts are necessary to identify alternative means of targeting PRMT5 and MTA2 in -ve cancers to benefit from the high-MTA environment of -deficient cancers.
Topics: Humans; B7-H1 Antigen; Homozygote; Triple Negative Breast Neoplasms; Sequence Deletion; Purine-Nucleoside Phosphorylase; Genomics; Histone Deacetylases; Repressor Proteins; Protein-Arginine N-Methyltransferases
PubMed: 36913304
DOI: 10.18632/oncotarget.28376 -
Biomedicines Mar 2022Glioblastoma (GBM) is a lethal brain cancer exhibiting high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work shows that...
Glioblastoma (GBM) is a lethal brain cancer exhibiting high levels of drug resistance, a feature partially imparted by tumor cell stemness. Recent work shows that homozygous deletion, a genetic alteration occurring in about half of all GBMs, promotes stemness in GBM cells. Exploiting MTAP loss-conferred deficiency in purine salvage, we demonstrate that purine blockade via treatment with L-Alanosine (ALA), an inhibitor of de novo purine synthesis, attenuates stemness of -deficient GBM cells. This ALA-induced reduction in stemness is mediated in part by compromised mitochondrial function, highlighted by ALA-induced elimination of mitochondrial spare respiratory capacity. Notably, these effects of ALA are apparent even when the treatment was transient and with a low dose. Finally, in agreement with diminished stemness and compromised mitochondrial function, we show that ALA sensitizes GBM cells to temozolomide (TMZ) in vitro and in an orthotopic GBM model. Collectively, these results identify purine supply as an essential component in maintaining mitochondrial function in GBM cells and highlight a critical role of mitochondrial function in sustaining GBM stemness. We propose that purine synthesis inhibition can be beneficial in combination with the standard of care for -deficient GBMs, and that it may be feasible to achieve this benefit without inflicting major toxicity.
PubMed: 35453502
DOI: 10.3390/biomedicines10040751 -
Oncotarget Nov 2014Myxofibrosarcomas are genetically complex and involve recurrently deleted chromosome 9p, for which we characterized the pathogenically relevant target(s) using genomic...
Myxofibrosarcomas are genetically complex and involve recurrently deleted chromosome 9p, for which we characterized the pathogenically relevant target(s) using genomic profiling. In 12 of the 15 samples, we detected complete or partial losses of 9p. The only aggressiveness-associated, differentially lost region was 9p21.3, spanning the potential inactivated methylthioadenosine phosphorylase (MTAP) that exhibited homozygous (4/15) or hemizygous (3/15) deletions. In independent samples, MTAP gene status was assessed using quantitative- and methylation-specific PCR assays, and immunoexpression was evaluated. We applied MTAP reexpression or knockdown to elucidate the functional roles of MTAP and the therapeutic potential of L-alanosine in MTAP-preserved and MTAP-deficient myxofibrosarcoma cell lines and xenografts. MTAP protein deficiency (37%) was associated with MTAP gene inactivation (P < 0.001) by homozygous deletion or promoter methylation, and independently portended unfavorable metastasis-free survival (P = 0.0318) and disease-specific survival (P = 0.014). Among the MTAP-deficient cases, the homozygous deletion of MTAP predicted adverse outcome. In MTAP-deficient cells, MTAP reexpression inhibited cell migration and invasion, proliferation, and anchorage-independent colony formation and downregulated cyclin D1. This approach also attenuated the tube-forming abilities of human umbilical venous endothelial cells, attributable to the transcriptional repression of MMP-9, and abrogated the susceptibility to L-alanosine. The inhibiting effects of MTAP expression on tumor growth, angiogenesis, and the induction of apoptosis by L-alanosine were validated using MTAP-reexpressing xenografts and reverted using RNA interference in MTAP-preserved cells. In conclusion, homozygous deletion primarily accounts for the adverse prognostic impact of MTAP deficiency and confers the biological aggressiveness and susceptibility to L-alanosine in myxofibrosarcomas.
Topics: Alanine; Animals; Apoptosis; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Down-Regulation; Female; Fibrosarcoma; Heterografts; Human Umbilical Vein Endothelial Cells; Humans; Male; Mice; Mice, SCID; Middle Aged; Molecular Targeted Therapy; Purine-Nucleoside Phosphorylase; Survival Analysis
PubMed: 25426549
DOI: 10.18632/oncotarget.2552 -
Blood Feb 2006The deficiency of methylthioadenosine phosphorylase (MTAP) in T-cell acute lymphoblastic leukemia (T-ALL) and other cancers, while constitutively expressed in normal...
EFA (9-beta-D-erythrofuranosyladenine) is an effective salvage agent for methylthioadenosine phosphorylase-selective therapy of T-cell acute lymphoblastic leukemia with L-alanosine.
The deficiency of methylthioadenosine phosphorylase (MTAP) in T-cell acute lymphoblastic leukemia (T-ALL) and other cancers, while constitutively expressed in normal cells, allows for selective therapy using L-alanosine, an inhibitor of de novo AMP synthesis. We demonstrate that MTAP- T-ALL cells obtained at relapse are as sensitive to L-alanosine toxicity as diagnosis samples. The therapeutic index of L-alanosine can be increased by the use of a MTAP substrate, which protects MTAP+ normal cells. Since MTAP substrates MTA and 5'deoxyadenosine are prone to toxicities associated with adenosine, we synthesized and evaluated a potentially nontoxic MTAP substrate, 9-beta-D-erythrofuranosyladenine (EFA). The cytotoxicity of EFA to hematopoietic progenitors erythroid burst-forming units (BFU-Es) and granulocyte-macrophage colony-forming units (CFU-GMs) was at least 26- to 41-fold less than that of MTA. In addition, EFA selectively rescued MTAP+ MOLT-4 cells from L-alanosine toxicity at 25 microM with negligible toxicity even at 100 microM. As for MTA, significant, albeit incomplete, rescue was achieved at 12.5 microM, but higher concentrations were toxic. EFA at 20 microM or less rescued primary MTAP+ T-ALL cells and normal lymphocytes from L-alanosine toxicity. Collectively, these data indicate that EFA is an effective agent for salvaging MTAP+ cells from L-alanosine toxicity and is superior to MTA due to lower cytotoxicity.
Topics: Adenine; Alanine; Antibiotics, Antineoplastic; Cell Line, Tumor; Deoxyadenosines; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Enzyme Inhibitors; Erythroid Precursor Cells; Furans; Granulocyte Precursor Cells; Humans; Leukemia-Lymphoma, Adult T-Cell; Purine-Nucleoside Phosphorylase; Salvage Therapy; Thionucleosides
PubMed: 16234352
DOI: 10.1182/blood-2005-06-2430 -
Japanese Journal of Cancer Research :... Jun 1989L-Alanosine, an analog of L-aspartic acid, was investigated as one of a series of chemical compounds that may have inhibitory effects on the repair of potentially lethal...
L-Alanosine, an analog of L-aspartic acid, was investigated as one of a series of chemical compounds that may have inhibitory effects on the repair of potentially lethal damage caused by radiation using an in vivo murine fibrosarcoma (Meth-A tumor) in BALB/cBy male mice. The combined treatment of single administration of L-alanosine (600 mg/kg) and single dose of X-irradiation (20 Gy) on Meth-A tumors produced 62% tumor control, while the radiation alone resulted in less than 5% tumor control. The potentiating effect by L-alanosine was higher when the drug was administered 8 h prior to X-irradiation. The dose modification factor of the drug is estimated to be 1.4 for Meth-A tumor. The increased tumor control rates with combined alanosine and radiation were highly dependent upon the time and sequence of the combined treatment. The reason for reduced efficacy at treatment times of less than 8 h prior to X-irradiations appears to be related in part to the modulation of the body temperature by L-alanosine when combined with Ketamine, an anesthetic agent.
Topics: Alanine; Animals; Antibiotics, Antineoplastic; Body Temperature; Combined Modality Therapy; Dose-Response Relationship, Drug; Fibrosarcoma; Ketamine; Male; Methylcholanthrene; Mice; Mice, Inbred BALB C; Radiation-Sensitizing Agents
PubMed: 2503478
DOI: 10.1111/j.1349-7006.1989.tb01680.x -
The Journal of Biological Chemistry Feb 1994Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the initial step in the conversion of IMP to AMP. Two isoforms of this enzyme have been observed in vertebrates. A... (Comparative Study)
Comparative Study
Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the initial step in the conversion of IMP to AMP. Two isoforms of this enzyme have been observed in vertebrates. A muscle isozyme is highly abundant in cardiac and skeletal muscle tissue and is thought to play a role in muscle energy metabolism. The non-muscle isozyme, which is present at low levels in most tissues, likely functions in de novo AMP biosynthesis. The analysis of the non-muscle isozyme has been hampered by its low abundance and instability during purification. In this study a genetic selection scheme was used to generate a murine T-lymphoma cell line which was at least 100-fold enriched for the non-muscle isozyme, as a result of amplification of the non-muscle synthetase gene. This cell line made possible the purification of the non-muscle isozyme, and the subsequent isolation of isozyme-specific peptides. Based on peptide sequence information a degenerate oligonucleotide probe was designed and used to screen a mouse kidney cDNA library. A 1.5-kilobase cDNA encoding the non-muscle isozyme was cloned and found to contain an open reading frame of 1368 base pairs encoding 456 amino acids. Gene transfer experiments showed that the cDNA encoded a 50-kDa protein, the size expected for mammalian synthetases, that correlated with the presence of high levels of synthetase activity. The deduced amino acid sequence of the mouse non-muscle synthetase is approximately 75% identical to the previously reported mouse muscle synthetase. Southern blot analysis of mouse genomic DNA with the isozyme-specific cDNA probes revealed that the synthetase isozymes are encoded by separate genes. The non-muscle gene is expressed in most tissues but is virtually undetectable in striated muscle tissues. Three different transcripts (1.7, 2.8, and 3.4 kilobases) are detected for the non-muscle isozyme which show a similar tissue distribution. The availability of a cDNA for the non-muscle isozyme of adenylosuccinate synthetase will facilitate further comparative analyses with the previously cloned muscle isozyme.
Topics: Adenylosuccinate Synthase; Alanine; Amino Acid Sequence; Base Sequence; Cloning, Molecular; Consensus Sequence; DNA Primers; Drug Resistance; Gene Amplification; Genes; Humans; Isoenzymes; Liver; Molecular Sequence Data; Muscles; Sequence Alignment; Sequence Homology, Amino Acid; Tissue Distribution
PubMed: 8308018
DOI: No ID Found -
Journal of Virology Dec 1981Human-mouse somatic cell hybrids were made between adenine phosphoribosyltransferase-deficient mouse L cells and a strain of human primary fibroblasts and selected in...
Human-mouse somatic cell hybrids were made between adenine phosphoribosyltransferase-deficient mouse L cells and a strain of human primary fibroblasts and selected in medium containing alanosine and adenine (J. A. Tischfield and F. H. Ruddle, Proc. Natl. Acad. Sci. U.S.A. 71:45-49, 1974). These hybrids were tested for the generation of defective interfering (DI) particles of vesicular stomatitis virus to determine whether or not a host gene controls the induction of DI particles. None of the seven independently arising hybrid clones tested generated detectable DI particles during 13 successive undiluted passages. In addition, the parental human cells also failed to generate DI particles. In contrast, the parental mouse cells generated a detectable level of DI particles during continuous passage. Thus, failure to generate DI particles appears to act in a dominant fashion in these hybrids. Human chromosome 16 and adenine phosphoribosyltransferase were present, as a direct consequence of the selection system, in all of the hybrid clones that failed to generate DI particles. It was the only human chromosome observed in the cells of every hybrid clone. This was verified by both isozyme and karyotype analyses. After hybrids were back-selected (with 2,6-diaminopurine) for loss of human adenine phosphoribosyltransferase and chromosome 16, they gained the ability to generate DI particles. Replication of DI particles already present in virus stocks, however, was normal in all of the hybrid clones and the parental human cells. This suggests that the induction, but not the replication, of DI particles is affected by the human genome and that a factor on human chromosome 16 seems to selectively suppress the mouse cell's ability to generate DI particles in the hybrids. These results support the idea that the induction of DI particles is controlled in part by host cell function(s), as suggested previously (C. Y. Kang and R. Allen, J. Virol. 25:202-206, 1978).
Topics: Animals; Chromosomes, Human, 16-18; Defective Viruses; Humans; Hybrid Cells; Mice; Models, Biological; Vesicular stomatitis Indiana virus; Viral Interference; Virus Activation; Virus Replication
PubMed: 6275129
DOI: 10.1128/JVI.40.3.946-952.1981 -
American Journal of Physiology. Cell... Oct 2000Intracellular ATP depletion is a hallmark event in ischemic injury. It has been extensively characterized in models of chemical anoxia in vitro. In contrast, the fate of...
Intracellular ATP depletion is a hallmark event in ischemic injury. It has been extensively characterized in models of chemical anoxia in vitro. In contrast, the fate of GTP during ischemia remains unknown. We used LLC-PK proximal tubular cells to measure GTP and ATP changes during anoxia. In 45 min, antimycin A decreased ATP and GTP to 8% and 2% of controls, respectively. Ischemia in vivo resulted in comparable reductions in GTP and ATP. After 2 h of recovery, GTP levels in LLC-PK cells increased to 65% while ATP increased to 29%. We also investigated steady-state models of selective ATP or GTP depletion. Combinations of antimycin A and mycophenolic acid selectively reduced GTP to 51% or 25% of control. Similarly, alanosine selectively reduced ATP to 61% or 26% of control. Selective GTP depletion resulted in significant apoptosis. Selective ATP depletion caused mostly necrosis. These models of ATP or GTP depletion can prove useful in dissecting the relative contribution of the two nucleotides to the ischemic phenotype.
Topics: Adenosine Triphosphate; Alanine; Animals; Antibiotics, Antineoplastic; Antimycin A; Apoptosis; Cell Hypoxia; Cells, Cultured; Deoxyglucose; Enzyme Inhibitors; Guanosine; Guanosine Triphosphate; Ischemia; Kidney Cortex; Kidney Tubules, Proximal; LLC-PK1 Cells; Male; Models, Biological; Mycophenolic Acid; Necrosis; Oxidative Phosphorylation; Rats; Rats, Sprague-Dawley; Swine
PubMed: 11003607
DOI: 10.1152/ajpcell.2000.279.4.C1270 -
Cancer Biology & Therapy Sep 2012Methylthioadenosine phosphorylase (MTAP), a key enzyme in the catabolism of 5'-deoxy-5'-methylthioadenosine (MTA), catalyzes the formation of adenine and...
Methylthioadenosine phosphorylase (MTAP), a key enzyme in the catabolism of 5'-deoxy-5'-methylthioadenosine (MTA), catalyzes the formation of adenine and 5-methylthioribose-1-phosphate. MTAP is expressed in all cells throughout the body, but a significant percentage of human tumors have lost MTAP expression, thereby making MTAP-loss a potential therapeutic target. Here, we have tested an MTAP-targeting strategy based on the idea that MTAP-expressing cells can be protected from toxic purine and uracil analogs by addition of MTA, but MTAP-deleted tumor cells cannot. Addition of as little as 10 μM MTA could entirely protect isogenic MTAP (+) , but not MTAP (-) , HT1080 cells from toxicity caused by the chemotherapy agents 6-thioguanine (6TG) or 5-fluorouracil (5FU). Inhibitor studies showed that MTA protection requires functional MTAP activity. Addition of adenine protected both MTAP (+) and MTAP (-) cells from 6TG and 5FU, consistent with the idea that adenine produced from the MTAP reaction competes with 6TG and 5FU for a rate limiting pool of phosphoribosyl-1-pyrophosphate (PRPP), which is required for the conversion of purine and uracil bases into nucleotides. Extracellular MTA can also protect mouse mesothelioma cells from killing by 6-TG or the drug L-alanosine in an MTAP-dependent manner. In addition, MTA can protect non-transformed MTAP (+) mouse embryo fibroblasts from 6TG toxicity. Taken together, our data suggest that the addition of MTA to anti-purine-based chemotherapy may greatly increase the therapeutic index of this class of drugs if used specifically to treat MTAP (-) tumors.
Topics: Adenosine; Animals; Cell Line; Cell Line, Tumor; Disease Models, Animal; Fibrosarcoma; Fluorouracil; Humans; Mesothelioma; Mice; Mice, Inbred C57BL; Neoplasms; Purine-Nucleoside Phosphorylase; Thioguanine; Thionucleosides
PubMed: 22825330
DOI: 10.4161/cbt.21115