scholarly journals Transcriptome and metabolome analysis of crGART, a novel cell model of de novo purine synthesis deficiency: Alterations in CD36 expression and activity

2020 ◽  
Author(s):  
Randall C Mazzarino ◽  
Veronika Baresova ◽  
Marie Zikánová ◽  
Nathan Duval ◽  
Terry G. Wilkinson ◽  
...  
Keyword(s):  
Cell Line ◽  
De Novo ◽  
Cell Model ◽  
Growth Conditions ◽  
Second Step ◽  
Metabolome Analysis ◽  
Purine Synthesis ◽  
Cd36 Expression ◽  
Cluster Of Differentiation ◽  
Cd36 Gene

ABSTRACTIn humans, GART [phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) / phosphoribosylglycinamide synthetase (EC 6.3.4.13) / phosphoribosylaminoimidazole synthetase (EC 6.3.3.1)] is a trifunctional protein which catalyzes the second, third, and fifth reactions of the ten step de novo purine synthesis (DNPS) pathway. The second step of DNPS is conversion of phosphoribosylamine (5-PRA) to glycineamide ribonucleotide (GAR). 5-PRA is extremely unstable under physiological conditions and is unlikely to accumulate in the absence of GART activity. Recently, a HeLa cell line null mutant for GART was constructed via CRISPR-Cas9 mutagenesis. This cell line, crGART, is an important cellular model of DNPS inactivation that does not accumulate DNPS pathway intermediates. In the current study, we characterized the crGART versus HeLa transcriptomes in purine-supplemented and purine-depleted growth conditions. We observed multiple transcriptome changes and discuss pathways and ontologies particularly relevant to Alzheimer disease and Down syndrome. We also report initial analysis of the Cluster of Differentiation (CD36) gene, which is highly expressed in crGART versus HeLa.

scholarly journals Transcriptome and metabolome analysis of crGART, a novel cell model of de novo purine synthesis deficiency: Alterations in CD36 expression and activity

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0247227
Author(s):  
Randall C. Mazzarino ◽  
Veronika Baresova ◽  
Marie Zikánová ◽  
Nathan Duval ◽  
Terry G. Wilkinson ◽  
...  
Keyword(s):  
Cell Line ◽  
De Novo ◽  
Cell Model ◽  
Growth Conditions ◽  
P Value ◽  
Metabolome Analysis ◽  
Purine Synthesis ◽  
Basal Expression ◽  
Elevated Expression ◽  
Cluster Of Differentiation

In humans, GART [phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) / phosphoribosylglycinamide synthetase (EC 6.3.4.13) / phosphoribosylaminoimidazole synthetase (EC 6.3.3.1)] is a trifunctional protein which catalyzes the second, third, and fifth reactions of the ten step de novo purine synthesis (DNPS) pathway. The second step of DNPS is conversion of phosphoribosylamine (5-PRA) to glycineamide ribonucleotide (GAR). 5-PRA is extremely unstable under physiological conditions and is unlikely to accumulate in the absence of GART activity. Recently, a HeLa cell line null mutant for GART was constructed via CRISPR-Cas9 mutagenesis. This cell line, crGART, is an important cellular model of DNPS inactivation that does not accumulate DNPS pathway intermediates. In the current study, we characterized the crGART versus HeLa transcriptomes in purine-supplemented and purine-depleted growth conditions. We observed multiple transcriptome changes and discuss pathways and ontologies particularly relevant to Alzheimer disease and Down syndrome. We selected the Cluster of Differentiation (CD36) gene for initial analysis based on its elevated expression in crGART versus HeLa as well as its high basal expression, high log2 value, and minimal P-value.

scholarly journals DDRE-28. MECHANISTIC AND THERAPEUTIC LINKS BETWEEN PURINE BIOSYNTHESIS AND DNA DAMAGE IN GLIOBLASTOMA

2021 ◽  
Vol 3 (Supplement_1) ◽  
pp. i12-i12
Author(s):  
Andrew Scott ◽  
Weihua Zhou ◽  
Kari Wilder-Romans ◽  
Jiane Feng ◽  
Zhe Wu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Brain Tumors ◽  
Cell Line ◽  
De Novo ◽  
Radiation Treatment ◽  
Adult Brain ◽  
Purine Biosynthesis ◽  
Purine Synthesis ◽  
Normal Cortex

Abstract Glioblastoma (GBM) is the most common and aggressive adult brain cancer. Radiation therapy (RT) is a critical treatment modality, and development of RT resistance is the predominant cause of recurrence and mortality in GBM patients. Using cell line models as well as patient-derived xenografts and neurospheres in orthotopic brain tumor models, we have identified increased rates and dependence upon de novo purine biosynthesis as a hallmark of GBM RT resistance. More recently, we have discovered that radiation treatment acutely stimulates flux through de novo purine synthesis in cell line and neurosphere models of GBM. This RT-induced increase in de novo purine synthesis is dependent on signaling through the DNA damage response and thus appears to be an adaptive mechanism to supply purines to repair radiation-induced DNA damage. To determine whether this regulatory mechanism also exists in vivo, we have used advanced metabolomic and metabolic tracing techniques with 13C-labeled glucose and 15N-labeled glutamine in mice bearing RT-resistant GBM patient-derived orthotopic brain tumors. We found that that orthotopic GBM PDXs had elevated activity of de novo purine synthesis that increased further after RT, while normal cortex had little activity even after RT. These observations have therapeutic relevance, as targeting this metabolic pathway with the FDA-approved purine biosynthesis inhibitor mycophenolate mofetil (MMF) overcomes GBM radiation resistance in mouse models in vivo. The lack of de novo purine synthesis in normal cortex suggests that targeting this pathway may be tumor specific. Collectively our data suggest that de novo synthesis of purines mediates RT resistance in GBM and that treatment of brain tumors with MMF in combination with RT may be a promising therapeutic strategy in patients.

scholarly journals A new folate antimetabolite, 5,10-dideaza-5,6,7,8-tetrahydrofolate is a potent inhibitor of de novo purine synthesis

1989 ◽  
Vol 264 (1) ◽  
pp. 328-333 ◽  
Author(s):  
G P Beardsley ◽  
B A Moroson ◽  
E C Taylor ◽  
R G Moran
Keyword(s):  
De Novo ◽  
Potent Inhibitor ◽  
Purine Synthesis

scholarly journals Paraburkholderia phymatum Homocitrate Synthase NifV Plays a Key Role for Nitrogenase Activity during Symbiosis with Papilionoids and in Free-Living Growth Conditions

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 952
Author(s):  
Paula Bellés-Sancho ◽  
Martina Lardi ◽  
Yilei Liu ◽  
Sebastian Hug ◽  
Marta Adriana Pinto-Carbó ◽  
...  
Keyword(s):  
Nitrogenase Activity ◽  
Root Nodules ◽  
Growth Conditions ◽  
Lysine Biosynthesis ◽  
Metabolome Analysis ◽  
Plant Host ◽  
Free Living ◽  
Bacterial Enzyme ◽  
Homocitrate Synthase ◽  
Iron Molybdenum Cofactor

Homocitrate is an essential component of the iron-molybdenum cofactor of nitrogenase, the bacterial enzyme that catalyzes the reduction of dinitrogen (N2) to ammonia. In nitrogen-fixing and nodulating alpha-rhizobia, homocitrate is usually provided to bacteroids in root nodules by their plant host. In contrast, non-nodulating free-living diazotrophs encode the homocitrate synthase (NifV) and reduce N2 in nitrogen-limiting free-living conditions. Paraburkholderia phymatum STM815 is a beta-rhizobial strain, which can enter symbiosis with a broad range of legumes, including papilionoids and mimosoids. In contrast to most alpha-rhizobia, which lack nifV, P. phymatum harbors a copy of nifV on its symbiotic plasmid. We show here that P. phymatum nifV is essential for nitrogenase activity both in root nodules of papilionoid plants and in free-living growth conditions. Notably, nifV was dispensable in nodules of Mimosa pudica despite the fact that the gene was highly expressed during symbiosis with all tested papilionoid and mimosoid plants. A metabolome analysis of papilionoid and mimosoid root nodules infected with the P. phymatum wild-type strain revealed that among the approximately 400 measured metabolites, homocitrate and other metabolites involved in lysine biosynthesis and degradation have accumulated in all plant nodules compared to uninfected roots, suggesting an important role of these metabolites during symbiosis.

DNA methylation in 5-aza-2'-deoxycytidine-resistant variants of C3H 10T1/2 C18 cells

1984 ◽  
Vol 4 (10) ◽  
pp. 2098-2102
Author(s):  
E Flatau ◽  
F A Gonzales ◽  
L A Michalowsky ◽  
P A Jones
Keyword(s):  
Cell Line ◽  
Cytosine Methylation ◽  
De Novo ◽  
Lethal Dose ◽  
Cytotoxic Effects ◽  
Drug Treatments ◽  
A Cell ◽  
Mouse Cells ◽  
Low Levels ◽  
Dna Cytosine Methylation

A cell line (T17) was derived from C3H 10T1/2 C18 cells after 17 treatments with increasing concentrations of 5-aza-2'-deoxycytidine. The T17 cell line was very resistant to the cytotoxic effects of 5-aza-2'-deoxycytidine, and the 50% lethal dose for 5-aza-2'-deoxycytidine was ca. 3 microM, which was 30-fold greater than that of the parental C3H 10T1/2 C18 cells. Increased drug resistance was not due to a failure of the T17 cell line to incorporate 5-aza-2'-deoxycytidine into DNA. The cells were also slightly cross-resistant to 5-azacytidine. The percentage of cytosines modified to 5-methylcytosine in T17 cells was 0.7%, a 78% decrease from the level of 3.22% in C3H 10T1/2 C18 cells. The DNA cytosine methylation levels in several clones isolated from the treated lines were on the order of 0.7%, and clones with methylation levels lower than 0.45% were not obtained even after further drug treatments. These highly decreased methylation levels appeared to be unstable, and DNA modification increased as the cells divided in the absence of further drug treatment. The results suggest that it may not be possible to derive mouse cells with vanishingly low levels of 5-methylcytosine and that considerable de novo methylation can occur in cultured lines.

Sign in / Sign up

Export Citation Format

Share Document