Inositol Pyrophosphate Profiling of Two HCT116 Cell Lines Uncovers Variation in InsP₈ Levels

<div><p>The HCT116 cell line, which has a pseudo-diploid karotype, is a popular model in the fields of cancer cell biology, intestinal immunity, and inflammation. In the current study, we describe two batches of diverged HCT116 cells, which we designate as HCT116^NIH and HCT116^UCL. Using both gel electrophoresis and HPLC, we show that HCT116^UCL cells contain 6-fold higher levels of InsP₈ than HCT116^NIH cells. This observation is significant because InsP₈ is one of a group of molecules collectively known as ‘inositol pyrophosphates’ (PP-InsPs)—highly ‘energetic’ and conserved regulators of cellular and organismal metabolism. Variability in the cellular levels of InsP₈ within divergent HCT116 cell lines could have impacted the phenotypic data obtained in previous studies. This difference in InsP₈ levels is more remarkable for being specific; levels of other inositol phosphates, and notably InsP₆ and 5-InsP₇, are very similar in both HCT116^NIH and HCT116^UCL lines. We also developed a new HPLC procedure to record 1-InsP₇ levels directly (for the first time in any mammalian cell line); 1-InsP₇ comprised <2% of total InsP₇ in HCT116^NIH and HCT116^UCL lines. The elevated levels of InsP₈ in the HCT116^UCL lines were not due to an increase in expression of the PP-InsP kinases (IP6Ks and PPIP5Ks), nor to a decrease in the capacity to dephosphorylate InsP₈. We discuss how the divergent PP-InsP profiles of the newly-designated HCT116^NIH and HCT116^UCL lines should be considered an important research opportunity: future studies using these two lines may uncover new features that regulate InsP₈ turnover, and may also yield new directions for studying InsP₈ function.</p></div

Synthesis of InsP₇s and InsP₈ by IP6Ks and PPIP5Ks.

<p>The Fig describes the synthesis of 1-InsP₇, 5-InsP₇ and 1,5-InsP₈ in both yeasts and mammalian cells. IP6K1/2/3 = isoforms 1, 2 and 3 of inositol hexakisphosphate kinase (Kcs1 is the single yeast isoform); PPIP5K1/2 = isoforms 1 and 2 of diphosphoinositol pentakisphosphate kinase (Asp1 and Vip1 are the single isoforms in <i>Schizosaccharomyces pombe</i> and <i>Saccharomyces cerevisiae</i>, respectively).</p

Characterization of <i>D. discoideum</i> IP₅ species.

<p>Half of the acidic cell extract (from 5 ml culture) of WT <i>D. discoideum</i> was incubated on ice (-) while the second half was incubated at 90°C for 20 min (Acid). Both samples were thenneutralised and resolved on 35.5% PAGE together with the six possible IP₅ isomers. Inositol phosphates were visualised by Toluidine staining. Densitometry analysis of treated versus untreated sample was performed and IP₆ and IP₅s bands intensity compared. (A). Acidic treatment reveals the distinct presence of three IP₅ species, which are otherwise barely detectable, indicating that <i>D. discoideum</i> possesses a complex IP₅-derived inositol pyrophosphate metabolism. (B) Schematic representation of inositol pyrophosphate metabolism in <i>D. discoideum</i>. The gray arrow to (PP)₂-IP₃ indicates a likely potentiallye step. The dashed arrow from inositol to IP₃ indicates uncharacterized enzymatic steps. The figure shows the result of a representative experiment that was repeated three times.</p

No alteration of IP₇ and IP₈ metabolism after cAMP treatment.

<p>Vegetative growing <i>D. Discoideum</i> were incubated for the indicated time with 50 µM cAMP. The incubation was terminated with equal volume of 2M Percloric acid to extract the inositol phosphates. These were resolved on 35% PAGE and stained with Touilidine blue. Two independent experiments are shown with short (left) and long (right) cAMP incubation time. The figure shows the result of a representative experiment that was repeated three times.</p

PAGE analysis of <i>D. discoideum</i> cell extract reveal the presence of three major bands.

<p>Inositol phosphates were extracted from 10(WT) <i>D. discoideum</i> (AX2 strain) and the IP₆-Kinase (ip6k null) and phospholipase C mutant (plc null) grown at a density of 2–4×10⁶_. About 30–40 microliters of neutralised cell extract (equivalent to 1/20 of the total volume) was resolved on 35.5% PAGE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085533#pone.0085533-Losito1" target="_blank">[26]</a> and visualized with Toluidine blue (A) and DAPI staining (B). The figure shows the result of a representative experiment that was repeated three times.</p

Analysis of <i>Dictyostelium discoideum</i> Inositol Pyrophosphate Metabolism by Gel Electrophoresis

<div><p>The social amoeba <i>Dictyostelium discoideum</i> was instrumental in the discovery and early characterization of inositol pyrophosphates, a class of molecules possessing highly-energetic pyrophosphate bonds. Inositol pyrophosphates regulate diverse biological processes and are attracting attention due to their ability to control energy metabolism and insulin signalling. However, inositol pyrophosphate research has been hampered by the lack of simple experimental procedures to study them. The recent development of polyacrylamide gel electrophoresis (PAGE) and simple staining to resolve and detect inositol pyrophosphate species has opened new investigative possibilities. This technology is now commonly applied to study <i>in vitro</i> enzymatic reactions. Here we employ PAGE technology to characterize the <i>D. discoideum</i> inositol pyrophosphate metabolism. Surprisingly, only three major bands are detectable after resolving acidic extract on PAGE. We have demonstrated that these three bands correspond to inositol hexakisphosphate (IP₆ or Phytic acid) and its derivative inositol pyrophosphates, IP₇ and IP₈. Biochemical analyses and genetic evidence were used to establish the genuine inositol phosphate nature of these bands. We also identified IP₉ in <i>D. discoideum</i> cells, a molecule so far detected only from <i>in vitro</i> biochemical reactions. Furthermore, we discovered that this amoeba possesses three different inositol pentakisphosphates (IP₅) isomers, which are largely metabolised to inositol pyrophosphates. Comparison of PAGE with traditional Sax-HPLC revealed an underestimation of the cellular abundance of inositol pyrophosphates by traditional methods. In fact our study revealed much higher levels of inositol pyrophosphates in <i>D. discoideum</i> in the vegetative state than previously detected. A three-fold increase in IP₈ was observed during development of <i>D. discoideum</i> a value lower that previously reported. Analysis of inositol pyrophosphate metabolism using ip6k null amoeba revealed the absence of developmentally-induced synthesis of inositol pyrophosphates, suggesting that the alternative class of enzyme responsible for pyrophosphate synthesis, PP-IP₅K, doesn’t’ play a major role in the IP₈ developmental increase.</p></div

Separation of 1-InsP₇ and 5-InsP₇ by CarboPac HPLC.

<p>Standards of [³H]InsP₆, 1-[³H]InsP₇, 5-[³H]InsP₇, and [³H]InsP₈ (1 nmol of each) were chromatographed on a CarboPac HPLC column. Panels A and B show HPLC runs in which either 1-[³H]InsP₇ or 5-[³H]InsP₇ were added individually, while Panel C shows an HPLC run in which both [³H]InsP₇ isomers were added together. Panel D, the mass amount of 5-InsP₇ was increased to 20 nmol.</p

Treatment by <i>Phytase</i>, Ddp1 and acidic degradation define IP₆, IP₇, and IP₈, in <i>D. discoideum</i> cell extract.

<p>Wild type <i>D. discoideum</i> cell extract (-) was incubated with phytase (Phy) (A), recombinant diphosphoinositol polyphosphate phosphohydrolase (DDP1) (B) or treated with acid at high temperature (C). The inositol phosphate nature of the three major bands detectable by Toluidine stain is demonstrated by the Phytase treatment (A), an enzyme able to remove the phosphate group from any position of the inositol rings. The pyrophosphate nature of the two slower migrating bands is demonstrated by their disappearance after DDP1 treatment (B) and by the well known acidic sensitivity of the phosphoanhydride bond (C). The figure shows the result of a representative experiments repeated three to four times.</p

STR profiles of HCT116^NIH and HCT116^UCL cell-lines, compared with HCT116 cells curated at ATCC.

<p>The loci for eight core short tandem repeats plus Amelogenin were derived by ATCC for their curated HCT116 cell line (catalogue number CCL-247) and the HCT116^NIH and HCT116^UCL cells. The HCT116^NIH and HCT116^UCL cells had an 83% and 89% match with the parental HCT116 line, above the 80% minimum that designates common lineage.</p

PAGE analysis of inositol pyrophosphate during <i>D. Discoideum</i> development.

<p>Amoeba development program was induced as described in material and methods. The inositol phosphates extracted at the indicated time points were resolved on 35% PAGE and visualised with Toluidine. (A) The analysis of wild type (WT) <i>D. discoideum</i> developmental program reveal a 2.6fold increase in the IP₈/IP₆ ratio at the late stage of development, as quantified by densitometry quantified (Bottom), average +/− SD of four independent experiments. (B) To the contrary inositol pyrophosphates are not induced during IP₆-Kinase (ip6k null) developmental program. The figure shows the result of a representative experiment that was repeated four times for the WT and two times for ip6k1 null.</p