The ENTPD5/mt-PCPH oncoprotein is a catalytically inactive member of the ectonucleoside triphosphate diphosphohydrolase family

Abstract

Expression of the ENTPD5/mt-PCPH onco-protein and overexpression of the normal ENTPD5/PCPH protein contribute to the malignant transformation of diverse mammalian cell types, and PCPH is mutated and/or deregulated in various human tumor types. Expression of PCPH or mt-PCPH caused similar phenotypes, yet the effects promoted by mt-PCPH expression were consistently and substantially greater. ATP depletion and increased stress‑resistance are phenotypes commonly associated with PCPH and mt-PCPH expression. It was suggested that the intrinsic nucleoside triphosphate diphosphohydrolase (NTPDase) activity of PCPH and mt-PCPH may be responsible for these phenotypes, but direct supporting evidence remains to be established. Results from experiments designed to test such hypothesis demonstrate that, as expected, mt-PCPH expression in human colorectal carcinoma (CRC) cells decreased their ATP levels and conferred resistance to oxaliplatin, a colorectal cancer-relevant chemotherapeutic agent. Using a combination of site-directed mutagenesis, immunoprecipitation methods, in vitro enzyme activity assays and in situ enzyme activity determinations in live cells, this report also demonstrates that the mt-PCPH oncoprotein lacks detectable NTPDase activity, indicating that direct ATP cleavage by mt-PCPH did not cause the ATP depletion observed in mt-PCPH-expressing CRC cells. These results strongly suggest that the mt-PCPH oncoprotein may regulate the cellular energy levels and subsequent chemoresistance by an NTPDase-independent mechanism. Understanding possible alternative mechanisms will be essential to devise strategies for the successful treatment of predictably therapeutically resistant tumors expressing either increased PCPH levels or, particularly, the mt-PCPH oncoprotein.

Figure 1.

Effect of mt-PCPH on the ATP levels and the chemo-response of colon carcinoma cells. (A) Western blot analysis of lysates from HCT116 and HCT15 cells stably transfected with empty vector (pcDNA) or myc-tagged mt-PCPH ; GAPDH was used as loading control. (B) ATP levels of cells from panel A. ATP measurements were normalized using ATP standard curves and lysate total protein concentration. Measurements were performed in triplicate from three cell passages (n=3). (C) Cell viability of cells in panel A in response to oxaliplatin treatment. Experiments were repeated for two cell passages in triplicate (n=2). (D) ATP levels of empty vector (pcDNA), PCPH or mt-PCPH transfected HCT116 cells were determined (n=2) at the beginning (time zero) and the end (8 h) of the treatment with empty or ATP-encapsulated liposomes, and again 24 h after completion of the treatment, removal of liposomes and incubation in fresh liposome-free medium (32 h). (E) Cell viability of HCT116 cells stably transfected with empty vector (pcDNA) or mt-PCPH from (D), treated with oxaliplatin (n=2) after exposure to empty or ATP-containing liposomes.

Figure 2.

Design, expression and GDPase activity of apyrase mutants. (A) Schematic illustrating the mutagenesis of tryptophan 177 to alanine (W177A) and glycine 201 to alanine (G201A) to generate putative inactive NTPDase variants of PCPH or mt-PCPH; ACRs, apyrase conserved regions. (B) Western blot analysis of cell lysates from HCT116 cells 48 h after transient transfection with W177A, G201A or W177A+G201A (double) mutated PCPH or mt-PCPH constructs. (C) GDPase activity assay of cell lysates from HCT116 cells transiently transfected with native (nat.), W177A, G201A or W177A+G201A (double) PCPH mutants. Assays included 5 μ g of total protein and reaction mix containing 2 mM GDP, and were incubated at 37°C for 5 min. Reactions without protein (mix) and lysates from empty vector-transfected cells (pcDNA) were included as controls. (D) GDPase assays of 5 μ g of cell lysates from empty vector (pcDNA), native (nat.) or W1771A mutated PCPH or mt-PCPH-transfected HCT116 cells. Reactions executed as described for (C) were run in duplicate, and one control set was boiled prior to incubation with GDP.

Figure 3.

In vitro NTPDase activity of PCPH and mt-PCPH expressed in HCT116 cells. (A) NTPDase assay of lysates from cells transiently transfected with empty vector (pcDNA), PCPH or mt-PCPH; duplicate lysates were boiled or left untreated, incubated with GDP and released phosphate was detected by a malachite green colorometric assay. Each transfection was tested twice and experiments repeated three times (n=3). (B) Western blot analysis of input (20%) and eluates from myc-tag IPs of HCT116 cells carried out 48 h after transient transfection with empty vector (V), PCPH (P) or mt-PCPH (mt). Proteins eluted using LSB and the myc peptide. Immunoglobulin heavy (HC) and light chains (LC) are detected on blots of IPs eluted with LSB. (C) NTPDase assays were performed as previously described on IPs from C. One way ANOVA ^* p≤0.05, ^** p≤0.005, ^*** p≤0.0005.

Figure 4.

Effect of protein expression level and substrate nature and concentration on the catalytic activity of PCPH and mt-PCPH. (A) Western blot analysis of lysates prepared from HCT116 cells 48 h after being transiently transfected with increasing amounts of PCPH or mt-PCPH DNA probed for the myc-tag, using GAPDH as the loading control. Relative densitometric values are provided below the blot images. (B) NTPDase activity in lysates (5 μ g total protein) of cells described for panel A. Transfections were performed on cells at two different passages (n=2). (C) NTPDase activity of the lysates (5 μ g total protein) from HCT116 cells described in Fig. 3 on the indicated NDPs and NTPs. (D) NTPDase activity assays of the same lysates over a range of GDP concentrations (1 μ M to 2 mM). Cells were transfected at two different passages and the samples were run in duplicate (n=2).

Figure 5.

In situ NTPDase activity of PCPH and mt-PCPH expressed in HCT116 cells. (A) Phase contrast images of GDPase activity (brown) and fluorescence (IF) images (×20) of myc-tagged protein expression (green) in HCT116 cells 48 h after transfection with empty vector, PCPH or mt-PCPH . Transfections were executed on two cell passages (n=2) and transfected cells were tested in duplicate. The blue box shows a ×40 magnification of a field of PCPH transfected cells. (B) Densitometry of in situ NTPDase activity.