Metabolic Characterization of Antifolate Responsiveness and Non-responsiveness in Malignant Pleural Mesothelioma Cells

Antifolates are a class of drugs effective for treating malignant pleural mesothelioma (MPM). The majority of antifolates inhibit enzymes involved in purine and pyrimidine synthesis such as dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and glycinamide ribonucleotide formyltransferase (GART). In order to select the most suitable patients for effective therapy with drugs targeting specific metabolic pathways, there is a need for better predictive metabolic biomarkers. Antifolates can alter global metabolic pathways in MPM cells, yet the metabolic profile of treated cells has not yet been clearly elucidated. Here we found that MPM cell lines could be categorized into two groups according to their sensitivity or resistance to pemetrexed treatment. We show that pemetrexed susceptibility could be reversed and DNA synthesis rescued in drug-treated cells by the exogenous addition of the nucleotide precursors hypoxanthine and thymidine (HT). We observed that the expression of pemetrexed-targeted enzymes in resistant MPM cells was quantitatively lower than that seen in pemetrexed-sensitive cells. Metabolomic analysis revealed that glycine and choline, which are involved in one-carbon metabolism, were altered after drug treatment in pemetrexed-sensitive but not resistant MPM cells. The addition of HT upregulated the concentration of inosine monophosphate (IMP) in pemetrexed-sensitive MPM cells, indicating that the nucleic acid biosynthesis pathway is important for predicting the efficacy of pemetrexed in MPM cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, and points to potential biomarkers for informing clinical decisions regarding the most effective therapies for patients with MPM

Zebrafish-Based Screening Models for the Identification of Anti-Metastatic Drugs

Metastasis, a leading contributor to the morbidity of cancer patients, occurs through a multi-step process: invasion, intravasation, extravasation, colonization, and metastatic tumor formation. Each process is not only promoted by cancer cells themselves but is also affected by their microenvironment. Given this complexity, drug discovery for anti-metastatic drugs must consider the interaction between cancer cells and their microenvironments. The zebrafish is a suitable vertebrate animal model for in vivo high-throughput screening studies with physiological relevance to humans. This review covers the zebrafish model used to identify anti-metastatic drugs

Physiological and Morphological Changes during the Transition of Escherichia coli from Exponential Growth to Stationary Phase

Upon depletion of essential nutrients from the culture medium, the growth rate of bacteria slows down and eventually reaches zero. At this point the culture enters into the stationary phase, which has been operationally defined as the absence of increase in cell number. The transition from rapid growth, where cells grow with a generation time less than 20 min, into stationary phase is accompanied by adaptation of bacterial cells to the new state. In this period of the transition of Escherichia coli culture, a number of morphological and physiological changes take place, including a decrease of cell volume, an alteration of cell shape, a modulation of the nucleoid, an alteration in the components of cell wall, and an accumulation of several storage materials, and alteration of the transcription and the translation machineries. These changes are accompanied by changes in gene expression in such a way that the growth-coupled genes are mostly switched off while the stationary phase-specific genes are up-regulated. More than 100 stationary phase-specific genes have been identified, and these genes appear to be expressed sequentially in a definite order. These findings altogether mean that there is temporal alteration in the E.coli phenotype even after the cessation of cell growth. The morphological and physiological differentiation of E.coli during the growth transition was studied in terms of the global regulation of gene expression. Based on the transcriptome analysis by using a DNA microarray assay I identified more than 70 genes that were induced and other 70 repressed in the stationary-phase. Among the induced genes, those whose expression depends on the sigma S (RpoS) were identified by comparison of the transcriptome between the wild-type and a rpoS disruptant. To observe the changes in the promoter activities associated with these stationary-phase genes, a novel vector was constructed. It allows expression of two fluorescent proteins in different way: the green fluorescent protein under the control of a test promoter and the other, dsRed protein, under the control of reference promoter. Using this double-reporter vector, the levels and growth phase-dependent variations were determined by FACS for several representative promoters from the exponential phase- and stationary phase-specific genes. This analysis of the promoter activities indicated that the population heterogeneity of E.coli culture increases in the stationary phase. Attempts were then made to fractionate stationary-phase cultures into homogenous populations. Cultures of E.coli were separated into more than 15 cell populations, each forming a discrete band after centrifugation with Percoll gradient. The separation resulted from the difference in buoyant density but not the size difference. The cell density increased upon transition from exponential growth to stationary phase. Exponential-phase cultures formed at least 5 discrete bands with lower densities, whereas stationary-phase cultures formed more than 10 bands with higher densities. These findings altogether suggest that the growth phase-coupled transition of E.coli phenotype is discontinuous. Two molecular markers characterizing each cell population were identified: the functioning promoter species, as identified by measuring the expression of green fluorescent protein under the control of test promoters; and the expressed protein species, as monitored by quantitative-immunoblotting. The analysis of chemical composition revealed that significant increase was observed only for polysaccharides. In concert with this finding, glycogen granules were found to accumulate in the stationary-phase cells as revealed by thin section microscopy. This finding suggests that at least one component, which contributes the increase in cell density is polysacchatides. As an initial attempt to identify the gene or genes involved in each step of cell density increase, a random screening was performed, by analyzing the Percoll centrifugation pattern of a set of E.coli mutants, each with deletion of a large segment of the genome. Among of a total of mutant strains tested, the density increase stopped for mutants at specific steps, forming discrete intermediate bands along Percoll gradient. One or more genes within the deleted genome segments must be involved in the density shift during the growth transition of E. colt into stationary phase. In parallel, l also tested some of the known stationary-phase genes in the cell density shift. As an initial attempt, the role of RNA polymerase sigroa S. (RpoS) in the cell density shift was examined. The rpoS disruptant formed apparently a single low density band even in the stationary phase, and the growth phase-coupled density increase was small. Even after prolonged culture, no further increase in the cell density was observed for this rpoS disruptant. Thus I concluded that the growth phase-coupled increase in E.coli cell density ceased at an early stage for the rpoS disruptant. These findings of mutant studies indicate that a specific gene or a set of genes are involved in each step of the cell density increase during the transition period from exponential growth to stationary phase, and the RpoS sigma factor is one such factor that is needed at an early step of the cell density increase

Classification and Strength Measurement of Stationary-Phase Promoters by Use of a Newly Developed Promoter Cloning Vector

When an Escherichia coli culture changes from exponential growth to the stationary phase, expression of growth-related genes levels off, while a number of stationary-phase-specific genes are turned on. To gain insight into the growth phase-dependent global regulation of genome transcription, we analyzed the strength and specificity of promoters associated with the stationary-phase genes. For the in vivo assay of promoter activity, 300- to 500-bp DNA fragments upstream from the translation initiation codon were isolated and inserted into a newly constructed doubly fluorescent protein (DFP) vector. The activity of test promoters was determined by measuring the green fluorescent protein (GFP). To avoid the possible influence of plasmid copy number, the level of transcription of reference promoter lacUV5 on the same plasmid was determined by measuring the red fluorescent protein (RFP). Thus, the activities of test promoters could be easily and accurately determined by determining the GFP/RFP ratio. Analysis of the culture time-dependent variation of 100 test promoters indicated that (i) a major group of the stationary-phase promoters are up-regulated only in the presence of RpoS sigma; (ii) the phase-coupled increase in the activity of some promoters takes place even in the absence of RpoS; and (iii) the activity of some promoters increases in the absence of RpoS. This classification was confirmed by testing in vitro transcription by using reconstituted RpoD and RpoS holoenzymes

Plasma and Urinary Metabolomic Analysis of Gout and Asymptomatic Hyperuricemia and Profiling of Potential Biomarkers: A Pilot Study

Gout results from monosodium urate deposition caused by hyperuricemia, but most individuals with hyperuricemia remain asymptomatic. The pathogenesis of gout remains uncertain. To identify potential biomarkers distinguishing gout from asymptomatic hyperuricemia, we conducted a genetic analysis of urate transporters and metabolomic analysis as a proof-of-concept study, including 33 patients with gout and 9 individuals with asymptomatic hyperuricemia. The variant allele frequencies of rs72552713, rs2231142, and rs3733591, which are related to serum urate levels (SUA) and gout, did not differ between the gout and asymptomatic hyperuricemia groups. In metabolomic analysis, the levels of citrate cycle intermediates, especially 2-ketoglutarate, were higher in patients with gout than in those with asymptomatic hyperuricemia (fold difference = 1.415, p = 0.039). The impact on the TCA cycle was further emphasized in high-risk gout (SUA ≥ 9.0 mg/dL). Of note, urinary nicotinate was the most prominent biomarker differentiating high-risk gout from asymptomatic hyperuricemia (fold difference = 6.515, p = 0.020). Although urate transporters play critical roles in SUA elevation and promote hyperuricemia, this study suggests that the progression from asymptomatic hyperuricemia to gout might be closely related to other genetic and/or environmental factors affecting carbohydrate metabolism and urinary urate excretion

Growth Phase-Coupled Alterations in Cell Structure and Function of Escherichia coli

Escherichia coli cultures can be fractionated into more than 20 cell populations, each having a different bouyant density and apparently representing a specific stage of cell differentiation from exponential growth to stationary phase (H. Makinoshima, A. Nishimura, and A. Ishihama, Mol. Microbiol. 43:269-279, 2002). The density increase was found to be impaired at an early step for a mutant E. coli with the disrupted rpoS gene, which encodes the RNA polymerase RpoS (sigma-S) for stationary-phase gene transcription. This finding suggests that RpoS is need for the entire process of cell density increase. In the absence of RpoF sigma factor, the flagella are not formed as observed by electron microscopy, but the growth phase-coupled density increase takes place as in wild-type E. coli, confirming that the alteration in cell density is not directly correlated with the presence or absence of flagella. In the stationary-phase cells, accumulation of electron-dense areas was observed by electron microscopic observation of bacterial thin sections. By chemical determination, the increase in glycogen (or polysaccharides) was suggested to be one component, which contributes to the increase in weight-to-volume ratio of stationary-phase E. coli cells

PTPRZ1 regulates calmodulin phosphorylation and tumor progression in small-cell lung carcinoma

Abstract Background Small-cell lung carcinoma (SCLC) is a neuroendocrine tumor subtype and comprises approximately 15% of lung cancers. Because SCLC is still a disease with a poor prognosis and limited treatment options, there is an urgent need to develop targeted molecular agents for this disease. Methods We screened 20 cell lines from a variety of pathological phenotypes established from different organs by RT-PCR. Paraffin-embedded tissue from 252 primary tumors was examined for PTPRZ1 expression using immunohistochemistry. shRNA mediated PTPRZ1 down-regulation was used to study impact on tyrosine phosphorylation and in vivo tumor progression in SCLC cell lines. Results Here we show that PTPRZ1, a member of the protein tyrosine- phosphatase receptor (PTPR) family, is highly expressed in SCLC cell lines and specifically exists in human neuroendocrine tumor (NET) tissues. We also demonstrate that binding of the ligand of PTPRZ1, pleiotrophin (PTN), activates the PTN/PTPRZ1 signaling pathway to induce tyrosine phosphorylation of calmodulin (CaM) in SCLC cells, suggesting that PTPRZ1 is a regulator of tyrosine phosphorylation in SCLC cells. Furthermore, we found that PTPRZ1 actually has an important oncogenic role in tumor progression in the murine xenograft model. Conclusion PTPRZ1 was highly expressed in human NET tissues and PTPRZ1 is an oncogenic tyrosine phosphatase in SCLCs. These results imply that a new signaling pathway involving PTPRZ1 could be a feasible target for treatment of NETs.</p

A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis

10.7554/elife.70151eLife1

A chemical screen based on an interruption of zebrafish gastrulation identifies the HTR2C inhibitor Pizotifen as a suppressor of EMT-mediated metastasis

10.7554/elife.66973eLife10e6697