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

Abstract

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 HCT116NIH and HCT116UCL. Using both gel electrophoresis and HPLC, we show that HCT116UCL cells contain 6-fold higher levels of InsP8 than HCT116NIH 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 HCT116NIH and HCT116UCL 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 HCT116NIH and HCT116UCL lines. The elevated levels of InsP8 in the HCT116UCL 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 HCT116NIH and HCT116UCL 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.

The Significance of the Bifunctional Kinase/Phosphatase Activities of Diphosphoinositol Pentakisphosphate Kinases (PPIP5Ks) for Coupling Inositol Pyrophosphate Cell Signaling to Cellular Phosphate Homeostasis

“…3, A, B, C, and D). The depiction of InsP 7 as being the 5-isomer is based on previous work with HCT116 cells in which our HPLC procedures were shown to resolve 5-InsP 7 from 1-InsP 7 ; the latter comprises Ͻ2% of total InsP 7 (27). Indeed, it is generally believed that most of the cell's InsP 8 is synthesized from 5-InsP 7 rather than 1-InsP 7 (28,29).…”

“…75 h followed by 2 mM P i for 0.25 h; D, no P i for 3 h followed by 2 mM P i for 3 h. Next, cells were quenched, and the levels of the indicated inositol phosphates (InsP 5 , and H (InsP 8 ). The identification of InsP 7 as the 5-isomer is based on our previous work with HCT116 cells in which our HPLC procedures were shown to resolve 5-InsP 7 from 1-InsP 7 ; the latter is a minor isomer that comprises Ͻ2% of total InsP 7 (27). The graphics below the x axes correspond to those described above that depict the various extracellular [P i ] conditions.…”

“…In the assays of phosphatase activities toward InsP 8 described above, we incubated the PPIP5Ks with 0.05 M InsP 8 , which is the estimated concentration of this inositol pyrophosphate in HCT116 cells cultured in DMEM/F-12 (27). In this reaction condition, the InsP 8 phosphatase activities (0.8 -1.5 nmol/mg of protein/min; Fig.…”

“…For the phosphatase assays (Fig. 6D), we initially used a concentration of 0.05 M 1-InsP 7 (because cellular levels of the latter are 2-10% of total InsP 7 (27,28)). The maximum rates of the kinase activities toward InsP 6 (0.2-1 nmol/mg of protein/min; Fig.…”

“…In view of how acutely and specifically InsP 8 responds to high [P i ], we sought further evidence for the participation of PPIP5Ks in this event. We gener- ated an HCT116 cell line in which both PPIP5Ks have been knocked out using CRISPR (27). These cells do not synthesize InsP 8 irrespective of the concentration of extracellular [P i ] (Fig.…”