29aa42dc61c19382ab349d3901c476e2 pmc_10454412.pdf 24989fd25c00b2bcef705a74ac7be22280e5a708 pmc_10454412.pdf 65ee65f22193e71f516380aedcf54b3c40af7b09e873dbf7dccd12514d9b43f2 pmc_10454412.pdf Title: Purine Nucleotide Alterations in Tumoral Cell Lines Maintained with Physiological Levels of Folic Acid Subject: Most cancer cells have an increased synthesis of purine nucleotides to fulfil their enhanced division rate. The de novo synthesis of purines requires folic acid in the form of N10-formyltetrahydrofolate (10-formyl-THF). However, regular cell culture media contain very high, non-physiological concentrations of folic acid, which may have an impact on cell metabolism. Using cell culture media with physiological levels of folic acid (25 nM), we uncover purine alterations in several human cell lines. HEK293T, Jurkat, and A549 cells accumulate 5'-aminoimidazole-4-carboxamide ribonucleotide (ZMP), an intermediary of the de novo biosynthetic pathway, at physiological levels of folic acid, but not with the artificially high levels (2200 nM) present in regular media. Interestingly, HEK293T and Jurkat cells do not accumulate high levels of ZMP when AICAr, the precursor of ZMP, is added to medium containing 2200 nM folate; instead, ATP levels are increased, suggesting an enhanced de novo synthesis. On the other hand, HeLa and EHEB cells do not accumulate ZMP at physiological levels of folic acid, but they do accumulate in medium containing AICAr plus 2200 nM folate. Expression of SLC19A1, which encodes the reduced folate carrier (RFC), is increased in HEK293T and Jurkat cells compared with HeLa and EHEB, and it is correlated with the total purine nucleotide content at high levels of folic acid or with ZMP accumulation at physiological levels of folic acid. In conclusion, tumoral cell lines show a heterogenous response to folate changes in the media, some of them accumulating ZMP at physiological levels of folic acid. Further research is needed to clarify the ZMP downstream targets and their impact on cell function. Keywords: purines; nucleotides; ZMP; AICAR; folic acid; SLC19A1; cancer Author: Claudia Cano-Estrada, Lidia de Benito-Gómez, Paula Escudero-Ferruz, Neus Ontiveros, Daniel Iglesias-Serret and José M. López Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Aug 8 14:00:24 2023 CEST ModDate: Tue Aug 8 14:07:06 2023 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1952659 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- IODWWR+VnURWPalladioL Type 1 Custom yes yes no 10 0 LDRFDA+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 HCZSUT+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 BYEDRI+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 YFFKLH+CMSY10 Type 1 Builtin yes yes no 28 0 HJOMHV+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 50 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 53 0 ABMHBB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 82 0 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664 0 ABMHBC+PalatinoLinotype CID TrueType Identity-H yes yes yes 667 0 ABMHBD+PalatinoLinotype TrueType WinAnsi yes yes no 673 0 ABMHBF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 676 0 ABMHNB+PalatinoLinotype TrueType MacRoman yes yes no 679 0 CZKGAS+CMR10 Type 1 Builtin yes yes no 773 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-09-21 02:28:39 CEST RepresentationInformation: pmc_10454412.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-09-16 02:17:02 CEST Size: 1952659 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 1016 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Children: Item: Title: Physiological Levels of FA Induce ZMP Accumulation in Jurkat Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Physiological Levels of FA do Not Induce AMPK Activation in Jurkat Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Physiological Levels of FA Decrease Tetrazolium Dye Reduction by Jurkat Cells without Changes in Mitochondrial Membrane Potential Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: ZMP Levels in Different Human Cell Lines Maintained with Physiological Folate Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Differential Effect of AICAr in Purine Nucleotide Levels of Tumoral Cell Lines Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Expression of the Reduced Folate Carrier (RFC) SLC19A1 in Human Cell Lines Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Children: Item: Title: Jurkat Cells Maintained with Physiological Folate Accumulate ZMP, but AMPK Is Not Activated Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Differential ZMP Accumulation in Human Cell Lines Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Folate Transport and ZMP Accumulation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Could ZMP, or Any of Its Derivatives, Be Used as a Tumoral Marker? Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Children: Item: Title: Cell Culture Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Cell Growth Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Purine Measurement Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Protein Quantification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: MTT Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Mitoprobe DilC1(5) Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: Western Blot Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: qRT-PCR Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: Statistics Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1b26f7b2 Info: Title: Purine Nucleotide Alterations in Tumoral Cell Lines Maintained with Physiological Levels of Folic Acid Author: Claudia Cano-Estrada, Lidia de Benito-Gómez, Paula Escudero-Ferruz, Neus Ontiveros, Daniel Iglesias-Serret and José M. López Subject: Most cancer cells have an increased synthesis of purine nucleotides to fulfil their enhanced division rate. The de novo synthesis of purines requires folic acid in the form of N10-formyltetrahydrofolate (10-formyl-THF). However, regular cell culture media contain very high, non-physiological concentrations of folic acid, which may have an impact on cell metabolism. Using cell culture media with physiological levels of folic acid (25 nM), we uncover purine alterations in several human cell lines. HEK293T, Jurkat, and A549 cells accumulate 5'-aminoimidazole-4-carboxamide ribonucleotide (ZMP), an intermediary of the de novo biosynthetic pathway, at physiological levels of folic acid, but not with the artificially high levels (2200 nM) present in regular media. Interestingly, HEK293T and Jurkat cells do not accumulate high levels of ZMP when AICAr, the precursor of ZMP, is added to medium containing 2200 nM folate; instead, ATP levels are increased, suggesting an enhanced de novo synthesis. On the other hand, HeLa and EHEB cells do not accumulate ZMP at physiological levels of folic acid, but they do accumulate in medium containing AICAr plus 2200 nM folate. Expression of SLC19A1, which encodes the reduced folate carrier (RFC), is increased in HEK293T and Jurkat cells compared with HeLa and EHEB, and it is correlated with the total purine nucleotide content at high levels of folic acid or with ZMP accumulation at physiological levels of folic acid. In conclusion, tumoral cell lines show a heterogenous response to folate changes in the media, some of them accumulating ZMP at physiological levels of folic acid. Further research is needed to clarify the ZMP downstream targets and their impact on cell function. 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63, 309, 74 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 460, 722, 497, 733 Annotation: Subtype: Link Rect: 504, 722, 540, 733 Annotation: Subtype: Link Rect: 377, 699, 415, 710 Annotation: Subtype: Link Rect: 421, 699, 457, 710 Annotation: Subtype: Link Rect: 418, 676, 455, 687 Annotation: Subtype: Link Rect: 461, 676, 498, 687 Annotation: Subtype: Link Rect: 387, 652, 425, 664 Annotation: Subtype: Link Rect: 502, 629, 540, 640 Annotation: Subtype: Link Rect: 219, 607, 256, 617 Annotation: Subtype: Link Rect: 331, 583, 369, 594 Annotation: Subtype: Link Rect: 431, 560, 468, 571 Annotation: Subtype: Link Rect: 474, 560, 510, 571 Annotation: Subtype: Link Rect: 181, 527, 219, 537 Annotation: Subtype: Link Rect: 225, 527, 261, 537 Annotation: Subtype: Link Rect: 59, 492, 96, 502 Annotation: Subtype: Link Rect: 102, 492, 138, 502 Annotation: Subtype: Link Rect: 456, 468, 494, 479 Annotation: Subtype: Link Rect: 248, 434, 285, 445 Annotation: Subtype: Link Rect: 250, 399, 288, 410 Annotation: Subtype: Link Rect: 122, 366, 159, 376 Annotation: Subtype: Link Rect: 445, 342, 483, 353 Annotation: Subtype: Link Rect: 220, 320, 258, 330 Annotation: Subtype: Link Rect: 100, 285, 137, 295 Annotation: Subtype: Link Rect: 322, 261, 360, 272 Annotation: Subtype: Link Rect: 366, 261, 402, 272 Annotation: Subtype: Link Rect: 520, 238, 557, 249 Annotation: Subtype: Link Rect: 59, 228, 95, 238 Annotation: Subtype: Link Rect: 59, 205, 96, 215 Annotation: Subtype: Link Rect: 59, 182, 96, 192 Annotation: Subtype: Link Rect: 102, 182, 138, 192 Checksum: de350060 Type: CRC32 Checksum: 29aa42dc61c19382ab349d3901c476e2 Type: MD5 Checksum: 24989fd25c00b2bcef705a74ac7be22280e5a708 Type: SHA-1