Correlation of intracellular diadenosine triphosphate (Ap3A) with apoptosis in Fhit-positive HEK293 cells

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Cancer Letters 259 (2008) 186–191 www.elsevier.com/locate/canlet

Correlation of intracellular diadenosine triphosphate (Ap3A) with apoptosis in Fhit-positive HEK293 cells David I. Fisher, Alexander G. McLennan

*

Cell Regulation and Signalling Group, School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, UK Received 7 September 2007; received in revised form 8 October 2007; accepted 8 October 2007

Abstract The pro-apoptotic Fhit tumor suppressor protein binds and hydrolyses diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) in vitro. We have measured the level of both these nucleotides in Fhit-positive HEK293 cells exposed to various apoptosis inducers. Cold shock, anti-Fas, cadmium ions and etoposide all increased the basal level of Ap4A of 0.500 pmol/106 cells by about 50%. However, the corresponding increases in Ap3A from a basal 0.079 pmol/ 106 cells correlated closely with the degree of apoptosis produced, up to a maximum of 0.510 pmol/106 cells with etoposide. These results support the view that Ap3A is the in vivo Fhit ligand and that an inhibition of Fhit activity is a key element in Fhit-mediated apoptosis.  2007 Elsevier Ireland Ltd. All rights reserved. Keywords: FHIT; Diadenosines; Tumor suppression; Apoptosis

1. Introduction The 1.7 Mb fragile histidine triad (FHIT) gene encodes a dimeric, 34 kDa tumor suppressor protein, Fhit, that in vitro hydrolyses the nucleotides diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) [1–3]. Deletion or loss of expression of the FHIT gene and consequent loss or inactivation of Fhit plays an important, early role in the development of many common human cancers and evidence now supports diverse roles for Fhit in cell cycle control, sensitivity to DNA-damaging agents and pro-apoptotic signalling [3,4]. * Corresponding author. Tel.: +44 151 795 4426; fax: +44 151 795 4406. E-mail address: [email protected] (A.G. McLennan).

Fhit-negative tumor cells are resistant to apoptosis but re-expression of FHIT in such cells leads to growth suppression and restoration of caspasedependent apoptosis, and reduces their tumorigenicity when transplanted into nude mice [5–7]. Curiously, the hydrolytic activity of Fhit is not required for tumor suppression as an active site mutant that binds but does not hydrolyse diadenosine nucleotides is as effective a suppressor as the wild type [8]. Instead, the pro-apoptotic ability of Fhit correlates with substrate binding. A series of Fhit mutants with progressively increasing Km values showed a corresponding functional loss when expressed in Fhit-negative tumor cells [9]. It has therefore been suggested that a Fhit-substrate complex is a key element of one or more pro-apoptotic signalling pathways in normal cells. In this scenario,

0304-3835/$ - see front matter  2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.10.007

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some upstream signal promotes formation or stabilization of this complex which then recruits the next downstream component via its negatively charged, nucleotide-bound surface [10,11]. Hydrolysis of the substrate would then terminate signal transduction. The molecular details of these pathways, and the identity of the proposed binding partner(s), have yet to be fully established; however, evidence is accumulating that they are involved in the DNA damage checkpoint response [12,13], modulation of cell cycle control via p21waf1 [14] and cyclophilin A [15], and in suppression of the PI3K-Akt/survivin anti-apoptotic pathway [16]. The identity and function of the nucleotide component of the Fhit-substrate complex is of particular interest but to date little attention has been focussed on it. Previously we have shown that the level of Ap3A, but not Ap4A, inversely correlated with Fhit status in a series of Fhit-positive and wholly or partially Fhit-negative normal and tumor cell lines, suggesting that Ap3A is the preferred Fhit ligand and substrate in vivo. [17]. In contrast, Ap4A is controlled by the unrelated NUDT2 Ap4A hydrolase [2]. However, evidence has also been presented implicating Ap4A as an inducer of apoptosis, with a high Ap4A/Ap3A ratio associated with cell death and a high Ap3A/Ap4A ratio associated with cell differentiation [18,19]. To further our understanding of the role of these nucleotides in apoptosis, we have examined the levels of Ap3A and Ap4A in Fhit-positive HEK293 cells exposed to various stresses that activate both intrinsic and extrinsic apoptotic pathways (etoposide, Cd2+, anti-Fas antibody, sorbitol and cold shock) and have found that in all cases bar one (hyperosmotic shock), the degree of apoptosis induced by these treatments shows a strong positive correlation with the level of Ap3A, but not Ap4A. This confirms and extends our earlier conclusion that Ap3A is the key ligand in the Fhit-substrate pro-apoptotic signalling complex. 2. Materials and methods 2.1. Cell growth and treatment HEK293 human adenovirus-transformed embryonic kidney epithelial cells were cultured at 37 C in 5% CO2 in MEM containing 10% fetal bovine serum and 1% non-essential amino acids. Cells to be subjected to apoptotic treatments were grown either for 16–19 h after seeding for 24 h treatments or for a further 24 h for shorter treatments. Cellular stress was applied by supplementation of the growth medium as follows: 25 lM cadmium

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acetate, 24 h: 0.44 mM etoposide, 24 h; 3 lg/ml anti-Fas antibody, 24 h; 0.2 M sorbitol, 45 min. Cells were coldshocked by replacement of the medium for 30 min with ice-cold cold shock buffer (10 mM Tris–HCl, pH 7.8, 0.25 M sucrose, 30 mM 2-mercaptoethanol, 10 mM EDTA, 4 mM MgCl2) [18]. Cells were harvested for analysis immediately after each treatment. 2.2. Live cell imaging For microscopy, HEK293 cells were cultured in 35 mm dishes and treated as above. Post-treatment, the medium was removed from the dishes and replaced with normal growth medium. Annexin V-conjugated fluorescein (1/500 dilution, Biosource) and propidium iodide (1 lg/ml) were added to the medium and cells visualised by confocal microscopy using a Zeiss LSM510 META confocal laser scanning microscope with a 20 · 0.5 NA objective. FITC fluorescence was excited with the 488 nm line from an argon ion laser and detected off a 545 nm dichroic mirror through a 500–550 band pass filter. PI fluorescence was excited with a 543 nm Helium Neon laser and detected through the 545 nm dichroic mirror and a 560 nm long pass filter. Five fields were chosen per dish with a mean number of 127 ± 21 (n = 35) cells per field and images taken at 12 min intervals for 12 h. Analysis of the fields was carried out using Kinetic Imaging Tracking software to identify new stain binding events. Data for annexin VFITC (green) and propidium iodide (red) binding were correlated to determine new apoptotic events occurring within the 12 h analysis. Apoptotic cells were defined as those that fluoresced green first and red later; cells that fluoresced red first, red and green simultaneously or not at all were not scored. 2.3. Diadenosine nucleotide assay Ap3A and Ap4A were extracted and assayed by a modification of our previously published procedure [17]. For each determination, six 90 mm dishes of cells at about 80% confluence were used. Cells were counted in two dishes and the remaining four separately extracted as follows. The cell layer was washed briefly with 4 ml warm PBS, the PBS rapidly removed, and 3 ml ice-cold 0.4 M TCA added. Cells were scraped into a cold tube, the dishes rinsed with two 1 ml portions of TCA and the combined 5 ml extract left at 4 C for 15 min. Ten picomoles each of Ap3A and Ap4A were added to two of the four extracts as internal standards. Five milliliters of 0.6 M tri-n-octylamine in 1,1,2-trichlorotrifluoroethane were added, the tube shaken for 5 min and then centrifuged at 1000g for 5 min. A sample (50 ll) of the upper aqueous layer was removed for ATP and ADP determination and the remainder (4.4 ml) mixed with 110 ll 2 M Tris–HCl, pH 8.5, 0.2 M Mg acetate and 10 U shrimp alkaline phosphatase (Roche) and incubated for 60 min at 37 C to

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hydrolyse mononucleotides. Next, 100 ll of a 50% (v/v) DEAE-Sephacel suspension in 20 mM Tris–HCl, pH 7.6 was added to adsorb the remaining nucleotides. After 10 min shaking, the suspension was centrifuged for 1 min at 10,000g. After discarding the supernatant, the pellet was washed with three 1.5 ml portions of water then shaken for 5 min with 0.5 ml 1.0 M triethylammonium bicarbonate, pH 7.5 to elute the dinucleotides. After centrifugation for 1 min, the supernatant was removed, the pellet re-extracted in the same manner, and the combined supernatants freeze–dried then dissolved in 100 ll 25 mM Hepes–NaOH, pH 7.8, 5 mM Mg acetate. Each sample was split into two 45 ll aliquots. 10 U shrimp alkaline phosphatase was added to both (tubes A and B) and 225 ng recombinant human Ap4A hydrolase [20] to one (tube A) to destroy the Ap4A and ATP product. The samples (now 50 ll) were incubated for 30 min at 37 C then for 15 min at 65 C to inactivate the enzymes. Ap4A was specifically measured in tube B as follows. 50 ll BacTiter-Glo luciferase reagent (Promega) was added and the background luminescence recorded in a BioOrbit 1250 luminometer. Then, 225 ng human Ap4A hydrolase was added and the increased luminescence due to Ap4A hydrolysis and subsequent production of ATP measured. Ap3A was specifically measured in tube A as follows. Twenty-five millimolar PEP (18 ll) was added followed by 5 lg pyruvate kinase (Roche) and 50 ll BacTiter-Glo reagent. After measurement of the background, 225 ng human Ap4A hydrolase was added to ensure no endogenous Ap4A remained, as this is a Fhit-substrate. Finally, 5.6 lg recombinant human Fhit protein [1] was added and the increased luminescence due to Ap3A hydrolysis and subsequent conversion of the ADP product to ATP by pyruvate kinase and PEP measured. Yields were determined from those extracts containing internal standards and results adjusted for yield and expressed as pmol nucleotide per 106 cells. Mean recoveries were 38.3% and 29.3% for Ap3A and Ap4A standards, respectively. Note that the dinucleotides assayed in this way are referred to as Ap3A and Ap4A to be consistent with most of the literature. However, this assay will also measure nucleotides of the form Ap3N and Ap4N where N is any nucleoside, e.g. Ap3G, Ap4C. The data quoted here do not take this into account as the changes observed are all relative. However, based on previous calculations, the ‘‘true’’ Ap3A and Ap4A concentrations will be roughly 0.75 times the values quoted [21]. 2.4. Mononucleotide assay The 50 ll samples of neutralized cell extracts were assayed for ATP and ADP content as follows. Samples were split into two 20 ll aliquots. Five microliters of buffer (200 mM Hepes–NaOH, pH 7.8, 40 mM Mg acetate) were added to both. Twenty-five millimolar PEP (14.5 ll) and 0.5 ll (5 lg) pyruvate kinase were added to

one (tube A) and 15 ll H2O to the other (tube B). Both tubes were incubated for 15 min at 37 C. In two separate tubes, 45 ll buffer (40 mM Hepes–NaOH, pH 7.8, 8 mM Mg acetate) was mixed with 50 ll CellTiter-Glo reagent (Promega) and the background luminescence determined at 25 C. To measure ATP, 5 ll from tube B was transferred to one of these tubes and the increase in luminescence measured. To measure ADP + ATP, 5 ll from tube A was transferred to the other tube. All measurements were performed on triplicate 5 ll samples. 3. Results In our previous study, the level of intracellular Ap3A in most Fhit-positive cells, including HEK293 cells, was too low to be measured, indicating that active Fhit normally maintains an extremely low level of this substrate. In contrast, Fhit-negative cells had high, readily measurable levels [17]. Using a modified and more sensitive version of this assay, we have now found that HEK293 cells not exposed to any form of cellular stress contain a low but measurable 0.079 ± 0.012 pmol/106 cells (n = 8) of Ap3A (approx. 90 nM, if evenly distributed), compared to 0.500 ± 0.037 pmol/106 cells (n = 8) of Ap4A (Table 1). To determine whether apoptotic stresses had any effect on these levels, cells were exposed to Cd2+ ions, etoposide, anti-Fas antibody, sorbitol and cold shock and both nucleotides measured. Each of these stresses was shown to induce a significant degree of apoptosis by imaging of cells using annexin V-FITC and propidium iodide. Apart from hyperosmotic shock (sorbitol), each of these stresses induced a virtually identical, 55% increase in Ap4A from 0.500 ± 0.037 pmol/106 cells to 0.778 ± 0.046 pmol/106 cells (Table 1). However, the increases in Ap3A were significantly greater (2- to 7-fold) and depended on the nature of the stress. Most strikingly, the increases in Ap3A and, consequently, the Ap3A/Ap4A ratio correlated positively with the degree of apoptosis induced by each stress (Fig. 1), suggesting a cause–effect relationship. The one exception was hyperosmotic shock which resulted in a 3-fold increase in Ap4A to 1.486 ± 0.305 pmol/106 cells and a 2.5-fold reduction in Ap3A to 0.030 ± 0.017 pmol/106 cells. Thus, compared to etoposide, which produced a 4-fold increase in the Ap3A/ Ap4A ratio, hyperosmotic shock resulted in an 8-fold decrease in this ratio (Table 1). Ap3A and Ap4A are thought to be made in vivo principally as constitutive by-products of protein synthesis via the adenylylation of ADP and ATP, respectively, by aminoacyl-tRNA synthetases, although there may be other sources [22,23]. However, the changes observed in the dinucleotides were not simply a reflection of changes in the ADP and ATP pools as the ATP/ADP ratio never varied more than 2-fold and did not correlate with the corresponding dinucleotide ratios (not shown).

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Table 1 Intracellular levels of Ap4A and Ap3A in HEK293 cells exposed to different pro-apoptotic stresses Treatment

Ap4Aa(pmol/106 cells)

Ap3Aa(pmol/106 cells)

Ap3A/Ap4A

Control Cold shock (4 C, 30 min) Anti-Fas (3 lg/ml, 24 h) Cd2+ (25 lM, 24 h) Etoposide (0.44 mM, 24 h) Sorbitol (0.2 M, 45 min)

0.500 ± 0.037 0.757 ± 0.025** 0.793 ± 0.127** 0.783 ± 0.196* 0.776 ± 0.134** 1.486 ± 0.305**

0.079 ± 0.012** 0.161 ± 0.032**,## 0.224 ± 0.023**,# 0.321 ± 0.024**,## 0.510 ± 0.044**,## 0.030 ± 0.017**

0.164 ± 0.038 0.210 ± 0.034b 0.290 ± 0.027** 0.462 ± 0.107** 0.678 ± 0.062** 0.018 ± 0.007**

a

Dinucleotides were measured as described in Section 2. Data are means ± SD. For controls, n = 8 and for treated cells, n = 6. < 0.001 and **P < 0.0001 compared to control; #P < 0.001 and ## P < 0.0001 compared to previous treatment (Student’s unpaired t-test). b The change in Ap3A/Ap4A ratio after cold shock is significant at P = 0.034. *P

0.8

Ap3A/Ap4A ratio

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0

1

2

3

4

5

6

Apoptotic events/100 cells

Fig. 1. Correlation between apoptosis and Ap3A/Ap4A ratio. Live cell imaging data for apoptotic cells were gathered as described in Section 2; dinucleotide data are from Table 1. The exceptional data for hyperosmotic shock are excluded. R2 = 0.98.

4. Discussion Our finding that apoptosis is generally associated with an increase in the Ap3A/Ap4A ratio contrasts with the results of Vartanian and co-workers. For example, they found that treatment of HL60 cells with etoposide for 20 h caused an increase in Ap4A from 0.7 to 2.7 pmol/106 cells and a reduction in Ap3A from 5.2 to 1.5 pmol/106 cells [19], leading to a 13-fold reduction in Ap3A/Ap4A, compared to the 4-fold increase that we observed in HEK293 cells. However, the high level of Ap3A in their study indicates that the subline of HL60 cells used must have been Fhit-negative. Previously, we found a very low level of Fhit expression (and consequently measurable Ap3A) in HL60 cells, although others have reported high expression, suggesting that sublines do vary [17,24,25]. Therefore, the increasingly quoted assertion that apoptosis is associated with a reduction in the Ap3A/Ap4A ratio is not a general phenomenon and must be revised. The factor(s) responsible for the changes observed in HL60 cells

remain unknown. It is also pertinent to point out that, in agreement with other studies [26,27], cold shock induced apoptosis in HEK293 cells. Previous work showing that Ap4A induced apoptosis in various cell lines used cells that had been permeabilised by cold shock [18], which may have sensitized them. With regard to pro-apoptotic Fhit signalling, it has been proposed that prolonging the lifetime of the Fhit-Ap3A complex is a key element in the transduction of an upstream signal via this complex to a downstream interacting partner. The results presented here are entirely consistent with this hypothesis. Normally, Fhit maintains a very low level of intracellular Ap3A; however, if some signal-mediated modification were to reduce the rate of hydrolysis, Ap3A would accumulate, as shown here, and so increase the likelihood of Fhit-Ap3A complexing with its partner. One such modification could be Src kinase-mediated phosphorylation of Tyr114 [28]. Phosphorylation of this residue on both subunits of the Fhit dimer yields an enzyme with biphasic kinetics for Ap3A hydrolysis, with one component having a 60-fold higher Km and 6-fold lower kcat than the unmodified protein [29]. These changes would strongly favor reduced Ap3A hydrolysis. However, it has been reported that replacement of Tyr114 with the phosphotyrosine mimic, aspartate, actually abolishes Fhit functionality [16], so possibly the interaction of phospho-Fhit with Src kinase itself (which would not be mimicked by aspartate replacement) is required. In support of this it has recently been shown that growth-factor stimulated complexing of phospho-Fhit with Src targets Fhit for proteasome-mediated degradation, leading to transmission of the mitogenic signal [30], so protein–protein interactions are clearly important for Fhit’s various functions. Interaction of Fhit with the SUMO-1 conjugating protein Ubc9 has also been reported to inhibit Ap3A hydrolysis [31].

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Whatever the reason, our results demonstrate for the first time that an increase in intracellular Ap3A, most probably mediated by a change in the hydrolytic activity of Fhit, correlates with the degree of apoptosis induced by different stresses. Whether this increased Ap3A has functions in addition to promoting Fhit interactions remains to be determined. Finally, it is not clear why hyperosmotic shock, which caused the same degree of apoptosis as etoposide, should be associated with a reduction in Ap3A and an increase in Ap4A. Hyperosmotic stress is know to activate both extrinsic and intrinsic apoptotic pathways and Src kinase [32,33] and so might be expected to increase Ap3A. Possibly the changes observed reflect a compensatory survival mechanism, such as regulatory volume increase, in those cells yet to succumb to apoptosis [32] or they may be part of an unrelated, stress-specific response. Acknowledgements The financial support of the North West Cancer Research Fund (Grant CR627) is gratefully acknowledged. Dave Spiller is thanked for help with the live cell imaging.

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