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 (apc(Min/+)) 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 Ca(2+) 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 apc(Min/+) mice and wild-type mice (apc(+/+)). Clearance of circulating erythrocytes was estimated by carboxyfluorescein-diacetate-succinimidyl-ester labelling. As a result, apc(Min/+) mice were anaemic despite reticulocytosis. Cytosolic ATP was significantly lower and annexin V binding significantly higher in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Glucose depletion enhanced annexin V binding, an effect significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Extracellular Ca(2+) removal or inhibition of Ca(2+) entry with amiloride (1 mM) blunted the increase but did not abrogate the genotype differences of annexin V binding following glucose depletion. Stimulation of Ca(2+) -entry by treatment with Ca(2+) -ionophore ionomycin (10 μM) increased annexin V binding, an effect again significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Following retrieval and injection into the circulation of the same mice, apc(Min/+) erythrocytes were more rapidly cleared from circulating blood than apc(+/+) erythrocytes. Most labelled erythrocytes were trapped in the spleen, which was significantly enlarged in apc(Min/+) mice. The observations point to accelerated eryptosis and subsequent clearance of apc(Min/+) erythrocytes, which contributes to or even accounts for the enhanced erythrocyte turnover, anaemia and splenomegaly in those mice.

Fig 1

Accelerated clearance of erythrocytes in APC-deficient mice. Time-dependent decay of CFSE-labelled circulating erythrocytes drawn from 20-week-old APC-deficient mice (black squares) and wild-type mice (black diamonds) and injected into the same mice. The percentage of CFSE-labelled circulating cells is plotted against time after injection. Values are normalized arithmetic mean ± S.E.M. (n =4) of the percentages of CFSE-labelled erythrocytes. * indicates significant difference between genotypes (Mann–Whitney test; P < 0.05).

Fig 2

Splenomegaly associated with increased number of annexin V binding erythrocytes in APC-deficient mice. (A) Photograph of spleens from 20-week-old APC-deficient mice (right) and wild-type mice (left). (B) Arithmetic mean ± S.E.M. (n = 4) of the spleen/body weight ratios of 20-week-old APC-deficient mice (apc^Min/+, black bar) and wild-type mice (apc^+/+, white bar), * indicates significant difference between genotypes (Mann-Whitney test; P < 0.05;). (C) Confocal microscopy of CFSE dependent (middle panels), annexin V-APC (right panels) dependent and merged fluorescence (left panels) of erythrocytes from the spleens of APC-deficient (apc^Min/+, lower panels) mice and wild-type mice (apc^+/+, upper).

Fig 3

Enhanced phosphatidylserine abundance at the surface of erythrocytes from APC-deficient mice. (A) Histogram of annexin V binding reflecting phosphatidylserine exposure in a representative experiment of erythrocytes from APC-deficient mice (apc^Min/+) and their wild-type littermates (apc^+/+) exposed for 8 hrs to glucose-depleted Ringer. (B) Arithmetic mean ± S.E.M. (n = 4) of the percentage of annexin V-binding erythrocytes from 8-week-old APC-deficient mice (apc^Min/+, black bars) and wild-type mice (apc^+/+, white bars) exposed for 8 hrs to glucose-containing (left bars) or glucose-depleted (right bars) Ringer. Annexin V bound to the cell membrane whereas CFSE accumulated in the cytosol. * indicates significant (P < 0.05) difference from glucose-containing Ringer; ^# indicates significant difference (P < 0.05) between genotypes (Mann–Whitney test).

Fig 4

Ca²⁺ sensitivity of erythrocytes from APC-deficient and wild-type mice. (A) Arithmetic mean ± S.E.M. (n = 5) of the percentage of annexin V-binding erythrocytes from APC-deficient mice (apc^Min/+, black bars) and wild-type mice (apc^+/+, white bars) exposed for 30 min. to Ringer solution without (left bars) or with (right bars) 10 μM ionomycin. *, ** indicate (P < 0.05, P < 0.01) significant difference between genotypes; ^## indicates (P < 0.01) significant difference from absence of ionomycin (Mann–Whitney test). (B) Arithmetic mean ± S.E.M. (n = 7 experiments with samples from four different mice) of the percentage of annexin V-binding erythrocytes from APC-deficient mice (apc^Min/+, black bars) and wild-type mice (apc^+/+, 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 < 0.01, P < 0.001) difference between genotypes, ^# significant (P < 0.05) difference from absence of amiloride, ^§§§ indicates significant (P < 0.001) difference from the presence of glucose (Mann–Whitney test). (C) Arithmetic mean ± S.E.M. (n = 5) of the percentage of annexin V-binding erythrocytes from APC-deficient mice (apc^Min/+, black bars) and wild-type mice (apc^+/+, white bars) exposed for 10 hrs to Ringer solution with (left bars) or without (right bars) glucose in the absence (−Ca²⁺) and presence (+Ca²⁺) of 1 mM extracellular Ca²⁺. *, ** indicate significant (P < 0.05, P < 0.001) difference between genotypes; ^§§ indicates significant (P < 0.01) difference from the presence of glucose (Mann–Whitney test).

Fig 5

Intracellular ATP content in erythrocytes from APC-deficient and wild-type mice. Arithmetic mean ± S.E.M. (n = 4) of the cytosolic ATP concentration in erythrocytes from APC-deficient mice (apc^Min/+, black bars) and wild-type mice (apc^+/+, white bars) exposed for 12 hrs to Ringer solution without (left bars) or with (right bars) glucose. ^# indicates significant (P < 0.05) difference between genotypes; * indicates significant (P < 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/+) and wild-type mice (+/+) exposed for 8 hrs to glucose-depleted Ringer. (B) Arithmetic mean ± S.E.M. (n = 8) of forward scatter of erythrocytes from 8-week-old APC-deficient mice (apc^Min/+, black bars) and wild-type mice (apc^+/+, white bars) exposed for 8 hrs to glucose-containing (left bars) or glucose-depleted (right bars) Ringer. *** indicate significant (P < 0.001) difference from glucose-containing Ringer (Mann–Whitney test).