Enhanced suicidal erythrocyte death in mice carrying a loss-of-function mutation of the adenomatous polyposis coli gene

Abstract Loss-of-function mutations in human adenomatous polyposis coli (APC) lead to multiple colonic adenomatous polyps eventually resulting in colonic carcinoma. Similarly, heterozygous mice carrying defective APC (apcMin/+) suffer from intestinal tumours. The animals further suffer from anaemia, which in theory could result from accelerated eryptosis, a suicidal erythrocyte death triggered by enhanced cytosolic Ca2+ activity and characterized by cell membrane scrambling and cell shrinkage. To explore, whether APC-deficiency enhances eryptosis, we estimated cell membrane scrambling from annexin V binding, cell size from forward scatter and cytosolic ATP utilizing luciferin–luciferase in isolated erythrocytes from apcMin/+ mice and wild-type mice (apc+/+). Clearance of circulating erythrocytes was estimated by carboxyfluorescein-diacetate-succinimidyl-ester labelling. As a result, apcMin/+ mice were anaemic despite reticulocytosis. Cytosolic ATP was significantly lower and annexin V binding significantly higher in apcMin/+ erythrocytes than in apc+/+ erythrocytes. Glucose depletion enhanced annexin V binding, an effect significantly more pronounced in apcMin/+ erythrocytes than in apc+/+ erythrocytes. Extracellular Ca2+ removal or inhibition of Ca2+ entry with amiloride (1 mM) blunted the increase but did not abrogate the genotype differences of annexin V binding following glucose depletion. Stimulation of Ca2+-entry by treatment with Ca2+-ionophore ionomycin (10 μM) increased annexin V binding, an effect again significantly more pronounced in apcMin/+ erythrocytes than in apc+/+ erythrocytes. Following retrieval and injection into the circulation of the same mice, apcMin/+ erythrocytes were more rapidly cleared from circulating blood than apc+/+ erythrocytes. Most labelled erythrocytes were trapped in the spleen, which was significantly enlarged in apcMin/+ mice. The observations point to accelerated eryptosis and subsequent clearance of apcMin/+ erythrocytes, which contributes to or even accounts for the enhanced erythrocyte turnover, anaemia and splenomegaly in those mice.


Introduction
The APC protein binds the oncogenic protein ␤-catenin and favours its degradation [1][2][3][4]. Lack of APC is followed by accumulation of ␤-catenin, which enters the nucleus and stimulates the expression of several genes involved in the regulation of cell proliferation [5,6]. Loss-of-function mutations affecting APC lead to the development of multiple colonic adenomatous polyps, which eventually results in colonic carcinoma [7,8]. Mice carrying a mutation in the APC gene (apc Min/ϩ ), which leads to truncation of the APC protein at amino acid 850, develop multiple intestinal tumours [9]. Beyond that the mice suffer from enhanced gastric acid secretion [10], hyperaldosteronism and increased blood pressure [11]. Moreover, the animals were shown to suffer from anaemia, a disorder considered to be secondary to blood loss [12].
This study was performed to elucidate whether the anaemia and reticulocytosis in apc Min/ϩ mice is secondary to increased eryptosis. Thus, eryptosis was determined in erythrocytes from apc Min/ϩ mice and from wild-type mice (apc ϩ/ϩ ).

Mice
Mice with mutated APC (apc Min/ϩ ) and wild-type mice (apc ϩ/ϩ ) were generated by breeding of apc Min/ϩ mice initially obtained from the Jackson Laboratory. The mice (four to eight per experiment, sex-matched, age as indicated) were fed a control diet (C1314; Altromin, Heidenau, Germany) and had access to drinking water ad libitum. Unless otherwise stated, 9-to 26-week-old mice were used.

Blood count and reticulocyte estimation
Blood was withdrawn into heparinized capillaries by puncturing the retrobulbar plexus. To this end, the mice were anaesthetized with diethylether (Roth, Karlsruhe, Germany). Anaesthesia was verified by testing the hind limb reflex. Then, 50 l of blood was taken by puncturing the retrobulbar plexus. For all experiments except for the blood count heparin blood was obtained. For the blood count, EDTA blood was analysed using an electronic haematology counter (scil VET abc, Weinheim, Germany). Relative reticulocyte numbers were determined using the Retic-COUNT reagent (BD, Heidelberg, Germany) according to the manufacturer's instructions.

FACS analysis of annexin V-binding and forward scatter
For FACS analysis of annexin V-binding and forward scatter 50 l cell suspensions were washed in Ringer solution containing 5 mM CaCl2 and then stained with Annexin-V-FITC (1:250 dilution; Immunotools, Friesoythe, Germany) in this solution for 20 min. at 37ЊC under protection from light. In the stained erythrocyte cell suspensions, forward scatter of the cells was determined, and annexin V fluorescence intensity was measured in FL-1 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS calibur (BD, Heidelberg, Germany).

Measurement of the in vivo clearance of fluorescence-labelled erythrocytes
The in vivo clearance of fluorescence-labelled erythrocytes was determined as described previously [33]. Briefly, erythrocytes (obtained from 200 l blood) were fluorescence labelled by staining the cells with 5 M carboxyfluorescein-diacetate-succinimidyl-ester (CFSE; Molecular Probes, Leiden, The Netherlands) in PBS and incubated for 30 min. at 37ЊC. After washing twice in PBS containing 1% FCS, the pellet was resuspended in Ringer solution (37ЊC), and 100 l of the CFSE-labelled erythrocytes were injected into the tail vein of the recipient mouse. As indicated, blood was retrieved from the tail veins of the mice, and CFSE-dependent fluorescence intensity of the erythrocytes was measured in FL-1 as described earlier. The percentage of CFSE-positive erythrocytes was calculated in percentage of the total labelled fraction determined 10 min. after injection.

Confocal microscopy and immunofluorescence
For the detection of annexin V-binding and CFSE-dependent fluorescence of erythrocytes in splenic tissue, the mice were deeply anaesthetized with diethyleter. Then, they were killed by cervical dislocation. After laparotomy, the spleens of apc Min/ϩ and of apc ϩ/ϩ mice were removed, weighed and mechanically homogenized in 1 ml cold PBS. The suspension was then centrifuged at 500 ϫ g for 10 min. at 4ЊC. The cell pellet was resuspended in 200 l cold PBS. Five microlitres of Annexin V-APC (BD) were added, and incubation was carried out for 20 min. at 37ЊC protected from light. The suspension was then transferred onto a glass slide and mounted with Prolong ® Gold antifade reagent (Invitrogen). Images were taken on a Zeiss LSM 5 EXCITER Confocal Laser Scanning Microscope (Carl Zeiss MicroImaging GmbH, Germany) with a water immersion Plan-Neofluar 63/1.3 NA DIC.

Estimation of intracellular ATP concentration
For determination of erythrocyte ATP, 80 l of erythrocyte pellets were incubated for 12 hrs at 37ЊC in Ringer solution with or without glucose (final haematocrit 5%). All manipulations were then performed at 4ЊC to avoid ATP degradation. Cells were lysed in distilled water, and proteins were precipitated by addition of HClO4 (5%). After centrifugation, an aliquot of the supernatant (400 l) was adjusted to pH 7.7 by addition of saturated KHCO3 solution. After dilution of the supernatant, the ATP concentration of the aliquots was determined utilizing the luciferinluciferase assay kit (Roche Diagnostics, Mannheim, Germany) on a luminometer (Berthold Biolumat LB9500, Bad Wildbad, Germany) according to the manufacturer's protocol. ATP concentrations refer to the cystol of erythrocytes.

Statistics
Data are expressed as arithmetic means Ϯ S.E.M., and statistical analysis was made by non-parametric Mann-Whitney test as indicated in the figure legends using GraphPad InStat Version 3.06 (San Diego, CA, USA); n denotes the number of different erythrocyte specimens studied.

Blood count and percentage of reticulocytes
A blood count revealed moderate anaemia of the apc Min/ϩ mice. Erythrocyte count, haemoglobin concentration and haematocrit were significantly smaller in apc Min/ϩ than in apc ϩ/ϩ mice ( Table 1). The mean corpuscular volume was, however, significantly increased. According to FACS analysis, the reticulocyte number was significantly higher in apc Min/ϩ than in apc ϩ/ϩ mice at differ-ent ages (4, 6, 8 and 12 weeks), pointing to enhanced erythrocyte formation (Table 1).

In vivo clearance of CFSE-labelled erythrocytes
The enhanced reticulocyte number accompanied by moderate anaemia points to enhanced erythrocyte turnover. To determine the life span of circulating erythrocytes blood was drawn from apc Min/ϩ and apc ϩ/ϩ (control) mice, erythrocytes were labelled with CFSE and the labelled apc Min/ϩ erythrocytes and apc ϩ/ϩ erythrocytes were injected into the same mice. As illustrated in Figure 1, labelled apc Min/ϩ erythrocytes disappeared from circulating blood of apc Min/ϩ mice significantly more rapidly than labelled apc ϩ/ϩ erythrocytes. Thus, the life span of apc Min/ϩ erythrocytes in apc Min/ϩ mice was significantly shorter than the life span of apc ϩ/ϩ erythrocytes in apc ϩ/ϩ mice. The percentage of apc Min/ϩ erythrocytes remaining in circulating blood of apc ϩ/ϩ mice within 24 hrs tended to be higher (62.1 Ϯ 7.4, n ϭ 3) than the respective percentage of apc Min/ϩ erythrocytes in apc Min/ϩ mice (50.3 Ϯ 4.9, n ϭ 4), a difference, however, not reaching statistical significance. The percentage of apc ϩ/ϩ erythrocytes remaining within 24 hrs in circulating blood of apc Min/ϩ mice tended to be lower (84.2 Ϯ 3.2, n ϭ 3) as the respective percentage of apc ϩ/ϩ erythrocytes in apc ϩ/ϩ mice (89.4 Ϯ 1.2, n ϭ 4), a difference, however, again not statistically significant.

Analysis of the spleen and splenic erythrocytes
As evident from Figure 2A and B, the labelled erythrocytes were mainly trapped in the spleen, which was significantly larger in apc Min/ϩ mice than in apc ϩ/ϩ mice. A detailed non-quantitative analysis revealed that the number of fluorescent annexin V binding ) and wild-type mice (apc ϩ/ϩ ) as a function of age. The data are compared to the normal range in mice [67,68]. * indicates significant differences between genotypes (Mann-Whitney test; P ϭ 0.0286). and thus phosphatidylserine-exposing erythrocytes was higher in the spleens from apc Min/ϩ mice than from apc ϩ/ϩ mice (control; Fig. 2C). CFSE accumulates in the cytosol, whereas annexin V binds to phosphatidylserine in the cell membrane.

؉/؉ and apc Min/؉ erythrocytes
In view of the accelerated clearance of circulating erythrocytes in the spleen of apc Min/ϩ mice and their enhanced phosphatidylserine exposure at the cell surface, additional experiments were performed to determine annexin V binding of apc Min/ϩ erythrocytes and apc ϩ/ϩ erythrocytes in FACS analysis. The experiments were performed in the presence and absence of glucose, as energy depletion is known to foster eryptosis [34]. As shown in Figure 3A and B, annexin V binding reflecting phosphatidylserine exposure at the erythrocyte surface was significantly higher in apc Min/ϩ erythrocytes than in apc ϩ/ϩ erythrocytes following energy depletion.

Role of Ca
2؉ for cell membrane scrambling of apc ؉/؉ and apc Min/؉ erythrocytes As cytosolic Ca 2ϩ is important for triggering of eryptosis, the Ca 2ϩ sensitivity of annexin V binding was tested by exposing apc Min/ϩ and apc ϩ/ϩ erythrocytes to the Ca 2ϩ ionophore ionomycin (10 M). As illustrated in Figure 4A, the ionomycin effect on annexin V binding was significantly stronger in apc Min/ϩ erythrocytes than in apc ϩ/ϩ erythrocytes pointing to higher Ca 2ϩ sensitivity of apc Min/ϩ erythrocytes. To define the role of Ca 2ϩ entry for the triggering of energy depletion-induced eryptosis, apc Min/ϩ and apc ϩ/ϩ erythrocytes were incubated with glucosefree Ringer in the presence and absence of amiloride (1 mM), an inhibitor of the non-specific cation conductance in erythrocytes. As a result, amiloride significantly attenuated the increase in annexin V binding following energy depletion in both apc Min/ϩ erythrocytes and apc ϩ/ϩ erythrocytes (Fig. 4B). However, amiloride did not abolish the differences between apc Min/ϩ erythrocytes and apc ϩ/ϩ erythrocytes of the annexin V binding following energy depletion. Similar observations were made in the absence of extracellular Ca 2ϩ . As shown in Figure 4C, removal of extracellular Ca 2ϩ tended to attenuate the increase in annexin V binding following energy depletion in both apc Min/ϩ erythrocytes and apc ϩ/ϩ erythrocytes. However, similar to amiloride administration, Ca 2ϩ removal did not abolish the differences between apc Min/ϩ erythrocytes and apc ϩ/ϩ erythrocytes of the annexin V binding following energy depletion. Collectively, these observations point to enhanced susceptibility of apc Min/ϩ erythrocytes to the cell membrane scrambling effect of energy depletion and enhanced cytosolic Ca 2ϩ activity.

Cytosolic ATP concentration in apc
؉/؉

and apc Min/؉ erythrocytes
Glucose deprivation is likely to affect the intracellular ATP content. Hence, the intracellular ATP concentration of erythrocytes  , white bars) exposed for 10 hrs to Ringer solution with (left bars) or without (right bars) glucose in the absence and presence of 1 mM amiloride. **, *** indicate significant (P Ͻ 0.01, P Ͻ 0.001) difference between genotypes, # significant (P Ͻ 0.05) difference from absence of amiloride, § § § indicates significant (P Ͻ 0.001) difference from the presence of glucose (Mann-Whitney test). (C) Arithmetic mean Ϯ S.E.M. (n ϭ 5) of the percentage of annexin V-binding erythrocytes from APC-deficient mice (apc Min/ϩ , black bars) and wild-type mice (apc ϩ/ϩ , white bars) exposed for 10 hrs to Ringer solution with (left bars) or without (right bars) glucose in the absence (ϪCa 2ϩ ) and presence (ϩCa 2ϩ ) of 1 mM extracellular Ca 2ϩ . *, ** indicate significant (P Ͻ 0.05, P Ͻ 0.001) difference between genotypes; § § indicates significant (P Ͻ 0.01) difference from the presence of glucose (Mann-Whitney test). incubated in the presence or absence of glucose for 12 hrs was determined. As shown in Figure 5, glucose depletion indeed decreased the intracellular ATP concentration of erythrocytes from both genotypes. In the presence of glucose, cytosolic ATP content was significantly lower in apc Min/ϩ erythrocytes than in apc ϩ/ϩ erythrocytes. Following glucose depletion cytosolic ATP content tended to be lower in apc Min/ϩ erythrocytes than in apc ϩ/ϩ erythrocytes, a difference, however, not reaching statistical significance (Fig. 5).

Cell volume of apc ؉/؉ and apc
Min/؉ erythrocytes To depict cell shrinkage, another hallmark of eryptosis, forward scatter of apc Min/ϩ erythrocytes and apc ϩ/ϩ erythrocytes was determined in FACS analysis. As shown in Figure 6, the forward scatter was significantly reduced by energy depletion in erythrocytes from both genotypes, an effect not significantly different between apc Min/ϩ erythrocytes and apc ϩ/ϩ erythrocytes.

Discussion
According to the present observations, heterozygous mice carrying defective APC (apc Min/ϩ ) suffer from mild anaemia with decreased erythrocyte count, haemoglobin concentration and haematocrit. The anaemia occurs despite significantly higher reticulocyte count in apc Min/ϩ mice, pointing to enhanced formation of new erythrocytes. Accordingly, the anaemia is secondary to enhanced turnover of apc Min/ϩ erythrocytes, which is further apparent from accelerated in vivo clearance of CFSE-labelled erythrocytes. The erythrocytes are to a large part trapped in the spleen. The splenic accumulation of eryptotic erythrocytes presumably accounts for the splenomegaly of those mice. Conversely, splenomegaly may foster splenic trapping of erythrocytes.
The accelerated clearance of the apc Min/ϩ erythrocytes is presumably due to enhanced phosphatidylserine exposure at their surface. Phosphatidylserine-exposing cells are trapped by macrophages [35], engulfed and degraded [36]. Phosphatidylserine-exposing erythrocytes are thus rapidly cleared from circulating blood [32]. To the extent that the accelerated loss of circulating erythrocytes is not matched by a similarly enhanced formation of new erythrocytes, the accelerated eryptosis leads to anaemia.
The present observations do not allow safe conclusions as to the mechanism linking APC deficiency to eryptosis. Clearly, erythrocytes from APC-deficient mice are more susceptible to the eryptotic effects of increased cytosolic Ca 2ϩ activity, a property unmasked by the enhanced eryptosis of those erythrocytes following treatment with the Ca 2ϩ ionophore ionomycin. The Ca 2ϩ Fig. 5 Intracellular ATP content in erythrocytes from APC-deficient and wild-type mice. Arithmetic mean Ϯ S.E.M. (n ϭ 4) of the cytosolic ATP concentration in erythrocytes from APC-deficient mice (apc Min/ϩ , black bars) and wild-type mice (apc ϩ/ϩ , white bars) exposed for 12 hrs to Ringer solution without (left bars) or with (right bars) glucose. # indicates significant (P Ͻ 0.05) difference between genotypes; * indicates significant (P Ͻ 0.05) difference from presence of glucose (Mann-Whitney test).

Fig. 6
Forward scatter in erythrocytes from APC-deficient and wild-type mice. (A) Histogram of forward scatter as a measure of cell volume in a representative experiment of erythrocytes from APC-deficient mice (Min/ϩ) and wild-type mice (ϩ/ϩ) exposed for 8 hrs to glucose-depleted Ringer. (B) Arithmetic mean Ϯ S.E.M. (n ϭ 8) of forward scatter of erythrocytes from 8-week-old APC-deficient mice (apc Min/ϩ , black bars) and wild-type mice (apc ϩ/ϩ , white bars) exposed for 8 hrs to glucosecontaining (left bars) or glucose-depleted (right bars) Ringer. *** indicate significant (P Ͻ 0.001) difference from glucose-containing Ringer (Mann-Whitney test).
ionophore should increase cytosolic Ca 2ϩ levels to similarly high values in APC-deficient and wild-type erythrocytes. Thus, at least part of the defect must be downstream of cytosolic Ca 2ϩ . Along those lines, even in the absence of extracellular Ca 2ϩ , eryptosis was enhanced in APC-deficient erythrocytes.
Erythrocytes from APC-deficient mice have lower cytosolic ATP levels, which should render them indeed more vulnerable to eryptosis. Energy depletion is known to stimulate protein kinase C, which in turn has been shown to trigger cell shrinkage and cell membrane scrambling [34].
Eryptosis is typically paralleled by decrease of cell volume [24]. However, no significant differences were observed in forward scatter between apc Min/ϩ and apc ϩ/ϩ erythrocytes. Possibly, the energy depletion of apc Min/ϩ erythrocytes impairs the activity of the Na ϩ /K ϩ ATPase leading to cellular loss of K ϩ and cellular gain of Na ϩ . K ϩ depletion expectedly blunts the K ϩ exit following activation of K ϩ channels and thus compromises the cellular KCl loss and cell shrinkage following Ca 2ϩ entry. Eryptosis has been determined in Ringer, indicating that the enhanced eryptosis was a property of the erythrocytes rather than a result from direct effects of plasma components on erythrocyte survival. Moreover, the clearance of CSFE-labelled erythrocytes from apc Min/ϩ mice is enhanced even in wild-type mice. The present observation could be explained by a role of APC in the maintenance of cytosolic ATP levels and survival of erythrocytes. Alternatively, the erythrocytes have been rendered more vulnerable to eryptotic stimuli by some component in circulating blood prior to the experiments. It is noteworthy that reticulocytosis increases with age of the animals and may at least in part be related to tumour growth. Anaemia is a well-known complication of malignancy [37,38] including familial adenomatous polyposis [12,39]. In view of the present observations, tumour anaemia may at least in part be due to enhanced eryptosis followed by accelerated clearance of eryptotic cells from circulating blood. In patients with malignancy, the eryptosis may be further triggered by cytostatic treatment, as several cytotoxic drugs have been shown to stimulate eryptosis [13]. Eryptosis is triggered by a wide variety of further anaemia-causing xenobiotics and endogeneous substances [40][41][42][43][44][45][46][47], and accelerated eryptosis has been observed in anaemia of several clinical disorders [13], including iron deficiency [32], phosphate depletion [48], haemolytic uraemic syndrome [49], sepsis [50], malaria [51][52][53][54] or Wilson's disease [55]. It is considered likely that APC deficiency enhances the susceptibility to the eryptotic effect of those xenobiotics, endogeneous substances and clinical disorders. In view of the rapid clearance of erythrocytes, the splenomegaly and the profound anaemia despite reticulocytosis in apc Min/ϩ mice, confounding triggers of eryptosis may be present in the blood of those mice.
Phosphatidylserine-exposing erythrocytes have been shown to adhere to the vascular wall [56][57][58][59][60], and to stimulate blood clotting [56,61,62]. Accordingly, excessive eryptosis due to oxidative stress may lead to derangements of microcirculation and enhanced eryptosis has been suggested to participate in the vascular injury of metabolic syndrome [63], a chronic clinical condition consisting of the clustering of cardiovascular risk factors including hypertension, that in humans relates also to colo-rectal cancer, and other forms of malignancies [64]. Intriguingly, the apc Min/ϩ mice suffer from hyperaldosteronism and hypertension [11], an observation similarly made in APC patients [65]. Oxidative stress has further been shown to be relevant for ageing of stored red blood cells [66].
In conclusion, lack of APC leads to enhanced suicidal erythrocyte death or eryptosis. The effect contributes to the anaemia in APC-deficient mice and presumably in patients carrying a loss-offunction mutation of the gene encoding the APC protein. Future studies may explore whether eryptosis is similarly enhanced in human patients suffering from APC.