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author:("Tang, baiting")
1.  Quantitation of Cellular Metabolic Fluxes of Methionine 
Analytical chemistry  2014;86(3):1583-1591.
Methionine is an essential proteogenic amino acid. In addition, it is a methyl donor for DNA and protein methylation and a propylamine donor for polyamine biosyn-thesis. Both the methyl and propylamine donation pathways involve metabolic cycles, and methods are needed to quantitate these cycles. Here, we describe an analytical approach for quantifying methionine metabolic fluxes that accounts for the mixing of intracellular and extracellular methionine pools. We observe that such mixing prevents isotope tracing experiments from reaching the steady state due to the large size of the media pools and hence precludes the use of standard stationary metabolic flux analysis. Our approach is based on feeding cells with 13C methionine and measuring the isotope-labeling kinetics of both intracellular and extracellular methionine by liquid chromatography−mass spectrometry (LC-MS). We apply this method to quantify methionine metabolism in a human fibrosarcoma cell line and study how methionine salvage pathway enzyme methylthioadenosine phosphorylase (MTAP), frequently deleted in cancer, affects methionine metabolism. We find that both transmethylation and propylamine transfer fluxes amount to roughly 15% of the net methionine uptake, with no major changes due to MTAP deletion. Our method further enables the quantification of flux through the pro-tumorigenic enzyme ornithine decarboxylase, and this flux increases 2-fold following MTAP deletion. The analytical approach used to quantify methionine metabolic fluxes is applicable for other metabolic systems affected by mixing of intracellular and extracellular metabolite pools.
PMCID: PMC4060246  PMID: 24397525
2.  Increasing the therapeutic index of 5-fluorouracil and 6-thioguanine by targeting loss of MTAP in tumor cells 
Cancer Biology & Therapy  2012;13(11):1082-1090.
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.
PMCID: PMC3461815  PMID: 22825330
chemotherapy; mesothelioma; methionine; osteosarcoma; purine
3.  Germline Mutations in Mtap Cooperate with Myc to Accelerate Tumorigenesis in Mice 
PLoS ONE  2013;8(6):e67635.
The gene encoding the methionine salvage pathway methylthioadenosine phosphorylase (MTAP) is a tumor suppressor gene that is frequently inactivated in a wide variety of human cancers. In this study, we have examined if heterozygosity for a null mutation in Mtap (MtaplacZ) could accelerate tumorigenesis development in two different mouse cancer models, Eμ-myc transgenic and Pten+/−.
Mtap Eμ-myc and Mtap Pten mice were generated and tumor-free survival was monitored over time. Tumors were also examined for a variety of histological and protein markers. In addition, microarray analysis was performed on the livers of MtaplacZ/+ and Mtap+/+ mice.
Survival in both models was significantly decreased in MtaplacZ/+ compared to Mtap+/+ mice. In Eµ-myc mice, Mtap mutations accelerated the formation of lymphomas from cells in the early pre-B stage, and these tumors tended to be of higher grade and have higher expression levels of ornithine decarboxylase compared to those observed in control Eµ-myc Mtap+/+ mice. Surprisingly, examination of Mtap status in lymphomas in Eµ-myc MtaplacZ/+ and Eµ-myc Mtap+/+ animals did not reveal significant differences in the frequency of loss of Mtap protein expression, despite having shorter latency times, suggesting that haploinsufficiency of Mtap may be playing a direct role in accelerating tumorigenesis. Consistent with this idea, microarray analysis on liver tissue from age and sex matched Mtap+/+ and MtaplacZ/+ animals found 363 transcripts whose expression changed at least 1.5-fold (P<0.01). Functional categorization of these genes reveals enrichments in several pathways involved in growth control and cancer.
Our findings show that germline inactivation of a single Mtap allele alters gene expression and enhances lymphomagenesis in Eµ-myc mice.
PMCID: PMC3694069  PMID: 23840755
4.  Chemical Genetic Screening for Compounds that Preferentially Inhibit Growth of Methylthioadenosine Phosphorylase (MTAP) Deficient Saccharomyces Cerevisiae 
Methylthioadenosine phosphorylase (MTAP), a key enzyme in the methionine salvage pathway, is inactivated in a variety of human cancers. Since all human tissues express MTAP, it would be of potential interest to identify compounds that selectively inhibit the growth of MTAP deficient cells. To determine if MTAP inactivation could be targeted, we have performed a differential chemical genetic screen in isogenic MTAP+ and MTAP− S. cerevisiae. A low molecular weight compound library containing 30,080 unique compounds was screened for those that selectively inhibit growth of MTAP− yeast using a differential growth assay. One compound, containing a 1,3,4-thiadiazine ring, repeatedly showed a differential dose response, with MTAP− cells exhibiting a four-fold shift in IC50 compared to MTAP+ cells. Several structurally related derivatives of this compound also showed enhanced growth inhibition in MTAP− yeast. These compounds were also examined for growth inhibition of isogenic MTAP+ and MTAP− HT1080 fibrosarcoma cells, and four of the five compounds exhibited evidence of modest, but significant, increased potency in MTAP− cells. In summary, these studies show the feasibility of differential growth screening technology and have identified a novel class of compounds that can preferentially inhibit growth of MTAP− cells.
PMCID: PMC3019245  PMID: 21131597
Methionine Salvage Pathway; Drug screening; Yeast; Genetic-chemical interaction
5.  Methionine-deficient diet induces post-transcriptional down-regulation of Cystathionine β-Synthase 
Elevated plasma total homocysteine (tHcy) is a risk factor for a variety of human diseases. Homocysteine is formed from methionine and has two primary metabolic fates: remethylation to form methionine or commitment to the transulfuration pathway by the action of cystathionine β-synthase (CBS). Here, we have examined the metabolic response in mice of a shift from a methionine-replete (M+) to a methionine-free (M−) diet.
Methods/Principle Findings
We found that shifting three-month old C57BL6 mice to a M− diet caused a transient increase in tHcy, as well as an increase in the tHcy/methionine ratio. Since CBS is a key regulator of tHcy, we examined CBS protein levels and found that within 3 days on methionine-deficient diet, animals had a 50% reduction in the levels of liver CBS protein and enzyme activity. Examination of CBS mRNA and studies of transgenic animals that express CBS from a heterologous promoter indicate that this reduction is occurring post-transcriptionally. Loss of CBS protein was unrelated to intracellular levels of S-adenosylmethionine, a known regulator of CBS activity and stability.
Our results imply that methionine deprivation induces a metabolic state in which methionine is effectively conserved in tissue by shutdown of the transsulfuration pathway via an S-adenosylmethionine-independent mechanism that signals a rapid down-regulation of CBS protein.
PMCID: PMC2956870  PMID: 20036517
Metabolism; Genetics; Amino Acids; Cardiovascular Disease
6.  Mice Heterozygous for Germline Mutations in Methylthioadenosine Phosphorylase (MTAP) Die Prematurely of T-cell Lymphoma 
Cancer research  2009;69(14):5961-5969.
Large homozygous deletions of 9p21 that inactivate CDKN2A, ARF, and MTAP are common in a wide variety of human cancers. The role for CDKN2A and ARF in tumorigenesis is well established, but whether MTAP loss directly affects tumorigenesis is unclear. MTAP encodes the enzyme methylthioadenosine phosphorylase, a key enzyme in the methionine salvage pathway. To determine if loss of MTAP plays a functional role in tumorigenesis, we have created an MTAP-knockout mouse. Mice homozygous for a MTAP null allele (MtaplacZ) have an embryonic lethal phenotype dying around day 8 post-conception. Mtap/MtaplacZ heterozygotes are born at Mendelian frequencies and appear indistinguishable from wild-type mice during the first year of life, but they tend to die prematurely with a median survival of 585 days. Autopsies on these animals reveal that they have greatly enlarged spleens, altered thymic histology, and lymphocytic infiltration of their livers, consistent with lymphoma. Immunohistochemical staining and FACS analysis indicate that these lymphomas are primarily T-cell in origin. Lymphoma infiltrated tissues tend to have reduced levels of Mtap mRNA and MTAP protein, and unaltered levels of methyldeoxycytidine. These studies show that Mtap is a tumor suppressor gene independent of CDKN2A and ARF.
PMCID: PMC2757012  PMID: 19567676
Cancer; Tumor Suppressor Gene; Methionine; Embryonic Lethal
7.  Expression of mutant human cystathionine beta-synthase rescues neonatal lethality but not homocystinuria in a mouse model 
Human molecular genetics  2005;14(15):2201-2208.
Cystathionine β-synthase (CBS) deficiency is a recessive genetic disorder in humans characterized by elevated levels in total plasma homocysteine (tHcy) and frequent thrombosis in humans. The I278T mutation is the most common mutation found in human CBS deficient patients. The T424N mutation was identified as a mutation in human CBS that could restore function to I278T in S. cerevisiae. In this report, we have engineered mice that express human I278T and I278T/T424N proteins from a metallotheinein driven transgene. These transgene-containing mice were then bred to CBS knockout animals (Cbs-) to generate mice that express only human I278T or I278T/T424N protein. Both the I278T and the I278T/T424N transgenes are able to entirely rescue the previously described neonatal mortality phenotype despite the animals having a mean tHcy of 250 μM. The transgenic Cbs-/- animals exhibit facial alopecia, have moderate liver steatosis and are slightly smaller than heterozygous littermates. In contrast to human CBS deficiency, these mice do not exhibit extreme methioninemia. The mutant proteins are stable in the liver, kidney and colon, and liver extracts have only 2-3% of the CBS enzyme activity found in wild type mice. Surprisingly, the I278T/T424N enzyme had exactly the same activity as the I278T enzyme indicating that T424N is unable to suppress I278T in mice. Our results show that elevated tHcy per se is not responsible for the neonatal lethality observed in Cbs-/- animals and suggests that CBS protein may have a function in addition to its role in homocysteine catabolism. These transgenic animals should be useful in the study of homocysteine related human disease.
PMCID: PMC1283068  PMID: 15972722

Results 1-7 (7)