Heat shock proteins and Bcl-2 expression and function in relation to the differential hyperthermic sensitivity between leukemic and normal hematopoietic cells.

A major problem in autologous stem cell transplantation is the occurrence of relapse by residual neoplastic cells from the graft. The selective toxicity of hyperthermia toward malignant hematopoietic progenitors compared with normal bone marrow cells has been utilized in purging protocols. The underlying mechanism for this selective toxicity has remained unclear. By using normal and leukemic cell line models, we searched for molecular mechanisms underlying this selective toxicity. We found that the differential heat sensitivity could not be explained by differences in the expression or inducibility of Hsp and also not by the overall chaperone capacity of the cells. Despite an apparent similarity in initial heat-induced damage, the leukemic cells underwent heat-induced apoptosis more readily than normal hematopoietic cells. The differences in apoptosis initiation were found at or upstream of cytochrome c release from the mitochondria. Sensitivity to staurosporine-induced apoptosis was similar in all cell lines tested, indicating that the apoptotic pathways were equally functional. The higher sensitivity to heat-induced apoptosis correlated with the level of Bcl-2 protein expression. Moreover, stable overexpression of Bcl-2 protected the most heat sensitive leukemic cells against heat-induced apoptosis. Our data indicate that leukemic cells have a specifically lower threshold for heat damage to initiate and execute apoptosis, which is due to an imbalance in the expression of the Bcl-2 family proteins in favor of the proapoptotic family members.

Differential role for very late antigen-5 in mobilization and homing of hematopoietic stem cells.

The role of very late antigen-5 (VLA-5) in homing and mobilization of hematopoietic stem cells from normal bone marrow (NBM) and bone marrow (MBM) and peripheral blood (MPB) from mobilized mice was investigated. We found a decreased number of VLA-5-expressing cells in the lineage-negative fraction of MPB. However, virtually all stem/progenitor cells were present in the VLA-5(+) fraction and hence mobilization of hematopoietic stem cell subsets does not coincide with a downregulation of VLA-5. Stem/progenitor cells from MPB and MBM demonstrated enhanced stromal-derived factor-alpha-induced migration. This enhanced migration correlates with an improved hematopoietic reconstitution potential, with the migrated MPB cells showing the fastest reconstitution. Interestingly, homing of MPB, MBM and NBM stem/progenitor cells in bone marrow and spleen did not differ and is therefore not responsible for the differences in hematopoietic reconstitution. The observed increase in VLA-5(+) cells in the recipients after transplantation can most probably be attributed to selective homing of VLA-5(+) cells instead of an upregulation of VLA-5. Treatment with an antibody to VLA-5 partially inhibited bone marrow homing of progenitor cells, whereas homing in the spleen was hardly affected. These data indicate a differential role for VLA-5 in the movement of stem cells from and toward bone marrow.

Purging of acute myeloid leukaemia cells from stem cell grafts by hyperthermia: enhancement of the therapeutic index by the tetrapeptide AcSDKP and the alkyl-lysophospholipid ET-18-OCH(3).

Hyperthermia has been shown to be a potential purging modality in autologous stem cell transplantation settings owing to its selective toxicity towards leukaemic cells. We describe two approaches to further increase the therapeutic index of the hyperthermic purging modality by using normal murine bone marrow cells and a murine model for acute myeloid leukaemia. First, the tetrapeptide AcSDKP was used to protect the normal haematopoietic progenitor cells against hyperthermic damage. Pretreatment for 8 h at 37 degrees C with 1 x 10(-9) mol/l AcSDKP resulted in a decrease in hyperthermic sensitivity of only normal haematopoietic progenitor cells. This combined treatment protocol revealed a therapeutic index (ratio of surviving fractions of normal vs. leukaemic cells) of > 500, which was considered to be sufficient for purging. This was confirmed in vivo by the survival of lethally irradiated recipients transplanted with purged simulated remission bone marrow (1 x 10(6) normal bone marrow cells and 5 x 10(4) leukaemic cells). A further increase of the therapeutic index cells was achieved by the alkyl-lysophospholipid ET-18-OCH(3). An incubation for 4 h at 37 degrees C with 25 microg/ml in the presence of 5% fetal calf serum preferentially enhanced the cytotoxic effect towards the leukaemic stem cell. The combination of AcSDKP and ET-18-OCH(3) with hyperthermia resulted in a therapeutic index of > 5000. This enabled a reduction of the hyperthermic treatment and will further minimize the toxicity to normal haematopoietic stem cell subsets, while a therapeutic index far above the required value is achieved. This tripartite purging treatment therefore offers a safe and fast purging protocol for the elimination of residual leukaemic cells in autografts.

In vitro effect of acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) analogs resistant to angiotensin I-converting enzyme on hematopoietic stem cell and progenitor cell proliferation.

The tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP), an inhibitor of hematopoietic stem cell proliferation, is known to reduce in vivo the damage resulting from treatment with chemotherapeutic agents or ionizing radiation on the stem cell compartment. Recently, AcSDKP has been shown to be a physiological substrate of the N-active site of angiotensin I-converting enzyme (ACE). Four analogs of the tetrapeptide expressing a high stability towards ACE degradation in vitro have been synthesized in order to provide new molecules likely to improve the myeloprotection displayed by AcSDKP. These analogs are three pseudopeptides with a modified peptidic bond, Ac-Serpsi(CH2-NH)Asp-Lys-Pro, Ac-Ser-Asppsi(CH2-NH)Lys-Pro, Ac-Ser-Asp-Lyspsi(CH2-N)Pro, and one C-terminus modified peptide (AcSDKP-NH2). We report here that these analogs reduce in vitro the proportion of murine colony-forming units-granulocyte/macrophage in S-phase and inhibit the entry into cycle of high proliferative potential colony-forming cells. The efficacy of AcSDKP analogs in preventing in vitro primitive hematopoietic stem cells from entering into cycle suggests that these molecules could be new candidates for the powerful inhibition of hematopoietic stem and progenitor cell proliferation in vivo.

Artemisinin-derived sesquiterpene lactones as potential antitumour compounds: cytotoxic action against bone marrow and tumour cells.

We determined the in vitro cytotoxic activity of the sesquiterpene lactone endoperoxide artemisinin (1) and some chemically prepared derivatives, which have been found to display cytotoxicity to cloned murine Ehrlich ascites tumour (EAT) cells and human HeLa cells and against murine bone marrow using a clonogenic assay for committed progenitor cells of the granulocyte-monocyte lineage (CFU-GM assay). Comparing artemisinin (1) to deoxyartemisinin (2), the endoperoxide group appeared to play a role in cytotoxicity to CFU-GM cells. Dimers of dihydroartemisinin and dihydrodeoxyartemisinin revealed that the stereochemistry of the ether linkage of the dimers was a more important determinant for this cytotoxic activity. The nonsymmetrical dimer of dihydroartemisinin (3) and the corresponding endoperoxide-lacking dimer of dihydrodeoxyartemisinin (5) were equally cytotoxic to CFU-GM cells. Despite the differences between both systems, it may be stated that most compounds displayed higher cytotoxicity to CFU-GM cells than to EAT cells. Dimers of dihydroartemisinin (3, 4) were selected as potential antitumour compounds and subjected to the National Cancer Institute drug-screening programme consisting of about sixty human cancer cell lines derived from nine different tissues. Both compounds displayed the same specific cytotoxicity pattern. Throughout the screen dimer 3 was more active than 4.

Enhanced selectivity of hyperthermic purging of human progenitor cells using Goralatide, an inhibitor of cell cycle progression.

Recurrence of leukemia is a major problem after autologous stem cell transplantation. One potential means of reducing this risk is to purge the autologous transplant in vitro by hyperthermia. We have demonstrated that after a hyperthermic treatment of 120 min at 43 degrees C, the leukemic progenitor cells (CFU-AML) are decreased by 5-log but the normal hematopoietic committed progenitor cells (CFU-GM, BFU-E and CFU-E) are reduced by only 1-log. Moreover, the hyperthermic sensitivity coincides with the stem cell hierarchy, ie CFU-GM are less heat sensitive than BFU-E, while CFU-E are the most sensitive. The impact of pretreatment with the tetrapeptide AcSDKP (Goralatide) on the proliferative activity and heat sensitivity of the normal and leukemic progenitor cells was determined. An incubation of 21 h at 37 degrees C with 10(-9) M Goralatide reduces the number of normal hematopoietic progenitor cells in S-phase and concomitantly decreases their hyperthermic sensitivity. This effect implies that the proliferative activity is the major determinant for the detected differences in hyperthermic sensitivity of the subsets in the normal hematopoietic stem cell compartment. In contrast, the cell cycle progression of leukemic progenitor cells is not affected and hence these cells are not protected from hyperthermia-induced cell killing after preincubation with Goralatide. Thus, the treatment with Goralatide increases the therapeutic window of hyperthermia and increases the potential value of this physical purging technique.

Reduction of heat-induced haemotoxicity in a hyperthermic purging protocol of murine acute myeloid leukaemic stem cells by AcSDKP.

The tetrapeptide AcSDKP (Goralatide) is a cytokine with known inhibitory effects on cell proliferation. Many purging agents used in autologous bone marrow transplantation protocols, including hyperthermia, preferentially kill cycling cells. A pretreatment with Goralatide offers a possibility to reduce the haemotoxicity in many purging settings. The impact of Goralatide on the hyperthermic purging protocol was investigated in normal and myeloid leukaemic (SA8) murine cells. The median survival time after transplantation (i.e. leukaemia incidences) was used as an in vivo parameter to determine the effects on leukaemic cells. The hyperthermic effect on normal and leukaemic cells was also investigated in vitro using the cobblestone area-forming cell (CAFC) assay. A heat treatment of 90 min at 43 degrees C resulted in a 4-log depletion of leukaemic stem cells. For normal progenitor cells (CFU-GM) a 2-log cell kill was shown. The reduction in proliferative activity of the CFU-GM after an 8 h incubation with 10(-9) M Goralatide resulted in a decrease in the heat sensitivity of the progenitor subset to approximately a 1-log cell kill. The leukaemic precursor cells seem insensitive to Goralatide inhibition, implicating an increase in the therapeutic window of the hyperthermic purging protocol. Finally, simulated remission bone marrow (5% leukaemic blasts) was incubated with Goralatide followed by a heat treatment of 90 min at 43 degrees C. Lethally irradiated (10 Gy) mice transplanted with heat-treated remission bone marrow (10(6) normal bone marrow cells versus 5 x 10(4) leukaemic cells) died of aplasia while Goralatide-pretreated remission bone marrow could rescue the irradiated mice without revealing leukaemic engraftment. These findings confirmed the enhanced protection against hyperthermia of the normal haemopoietic subsets by Goralatide and thus increased the success of the hyperthermic purging protocol.

Goralatide (AcSDKP) selectively protects murine hematopoietic progenitors and stem cells against hyperthermic damage.

Hyperthermic purging procedures may be improved by methods that selectively inhibit the proliferative activity of normal hematopoietic progenitors and stem cells, since active proliferation of these subsets is accompanied by increased heat sensitivity. For this reason, bone marrow cells from CBA/H mice were incubated with Goralatide (tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro), a well-known inhibitor of normal hematopoietic progenitor cells to enter the S phase of the cell cycle. Subsequently, the cell suspensions were heat-treated at 43 degrees C for up to 90 minutes. After an exposure of 8 hours to 10(-9) M Goralatide, the number of CFU-GM cells in S phase decreased from 30 to 10%, resulting in an almost 10-fold increase in survival after 90 minutes at 43 degrees C. No effect on the primitive subsets could be detected because of their quiescent cell cycle state in normal bone marrow. To investigate the potential vulnerability of these subsets for Goralatide, bone marrow cells from 5-fluorouracil (5-FU)-pretreated mice were used. 5-FU induced increase in proliferative activity of the CFU-S-12 (day-12 colony-forming units-spleen), and the stem cell with marrow repopulating ability could be abolished by an incubation period with 10(-9) M Goralatide for 16 and 24 hours, respectively. Hence, this decrease in proliferative activity confers a decrease in hyperthermic sensitivity for the primitive hematopoietic subsets. The cytotoxic effect of the incubation on the absolute number of the hematopoietic progenitors and stem cells was <10%. Goralatide treatment (10(-8), 10(-9), and 10(-10) M) up to 24 hours had no effect on the growth kinetics and cell cycle distribution and consequently on the hyperthermic sensitivity of L1210 cells. Based on these results, it can be concluded that Goralatide will have a positive effect on the survival of hematopoietic progenitors and stem cells after hyperthermia and may lead to a gain in the therapeutic window of this purging modality.

Studies on the hyperthermic sensitivity of the murine hematopoietic stem cell compartment. II. Heat effect on donor stem cells with long-term repopulating ability.

Variations in hyperthermic sensitivity among different hematopoietic progenitor and stem cell populations of the bone marrow have been previously described for clonogenic subsets responsible for short-term hematopoiesis. However, less is known of the heat sensitivity of more primitive stem cells capable of long-term repopulation in irradiated recipients. In the present study, control and heat-treated (60 minutes at 43 degrees C) donor bone marrow cells from congenic B6-Gpi-1a mice were transplanted at different cell doses (10(4), 10(5), 10(6), and 10(7) nucleated cells) in pre-irradiated (6 Gy) B6-Gpi-1b mice. The development and levels of donor marrow engraftment were determined from blood Gpi phenotyping, and the bone marrow dose required for equivalent long-term engraftment at 20 weeks provided an estimate of the surviving fraction corresponding to primitive stem cells of long-term repopulating ability (LTRA). Comparison with previous bone marrow cell survival values demonstrates that LTRA cells are less sensitive to hyperthermic treatment than other hematopoietic subsets, confirming a relationship between the heat sensitivity and the hierarchical structure of the hematopoietic stem cell compartment.

Intracellular free calcium concentrations in cell suspensions during hyperthermia.

The aim of this study was to explore the possibility that heat-induced alterations in calcium homeostasis are the cause of hyperthermic cell killing. Therefore, the intracellular free calcium concentration ([Ca2+]i) was determined spectrofluorometrically, using the fluorescent calcium probe fura-2/acetoxy methylester (AM), at both physiological and hyperthermic temperatures in cell suspensions from six different tumor cell lines. For all cell lines fura-2 leakage appears to contribute to a change in the fluorescence signal and hence leads to a false indication of an increase in [Ca2+]i, especially at the hyperthermic temperature. Two methods were introduced that circumvent this problem and results in true values of [Ca2+]i. Also, measurements of [Ca2+]i in single cells using a fluorescent microscopical technique (not affected by dye leakage) were used for comparison. All three approaches show that a hyperthermic treatment that kills > 90% of the cells does not lead to changes in the [Ca2+]i in most cell lines. Therefore, heat-induced alterations of calcium homeostasis cannot be considered the general cause for hyperthermic cell killing.

Hyperthermia, intracellular free calcium and calcium ionophores.

It is shown that heat-induced increase of intracellular calcium does not correlate with hyperthermic cell killing. Six different cell lines were investigated; in four (EAT, HeLa S3, L5178Y-R and L5178Y-S) heat treatments killing 90% of the cells did not affect the levels of intracellular free calcium ([Ca2+]i). In one cell line (3T3) a heat-induced increase in [Ca2+]i was observed. LM cells showed a heat-induced increase of the ratio of the fluorescent signals, but this may be explained by Fura-2 leakage out of the cells. Calcium ionophores are used to address the question whether rises in [Ca2+]i might cause cell killing. To investigate the existence of sensitization to Ca2+ toxicity by heat, ionophore treatments are combined with hyperthermia. Both ionophores used, A23187 and ionomycin, cause cell killing corresponding with increases in [Ca2+]i at 37 degrees C in EAT cells. In HeLa S3 cells, substantial increases in [Ca2+]i due to the action of ionomycin were observed without corresponding cell killing. This indicates the presence of a threshold concentration of [Ca2+]i in HeLa S3 cells before the treatment becomes toxic. Both ionophores show synergism with hyperthermia for cell killing as well as at the level of increased [Ca2+]i. The synergistic action may be explained as thermal enhancement of calcium toxicity.

Synergistic action of calcium-ionophores and hyperthermia is best interpreted as thermal enhancement of calcium toxicity.

It has been shown that no relation exists between [Ca2+]i and hyperthermic cell killing, although heat-induced increase of [Ca2+]i can be observed in some cell lines. When ionophores are used, dose-dependent rises in [Ca2+]i may be found. Beyond a certain threshold of ionophore-induced increases in [Ca2+]i, cells may be killed. Different threshold levels of [Ca2+]i exist in different cell lines. Hyperthermia can act synergistically with calcium ionophores to potentiate cell killing. Since there is no causal relation between [Ca2+]i and heat toxicity, this synergism can be explained as heat enhanced Ca2+ toxicity. In the current report, it is shown that both ionophore-induced Ca2+ toxicity (37 degrees C) and its potentiation by heat are dependent on extracellular calcium and related to sustained increases in [Ca2+]i. With ionomycin concentrations up to 15 microM, no increase in [Ca2+]i was seen in cells maintained in medium without Ca2+. Ionomycin effects on intracellular compartments were absent, and the drug seemed to act solely on the level of the plasmamembrane. Also, the synergism of heat and ionomycin appeared to act at the plasmamembrane, because depletion of extracellular calcium completely abolished this synergistic effect. The data presented are also discussed in the light of controversies existing in the literature for the role of calcium in hyperthermic cell killing.

Studies on the hyperthermic sensitivity of the murine hematopoietic stem cell compartment. I. Heat effects on clonogenic stem cells and progenitors.

The heat sensitivity at 42 degrees, 43 degrees and 44 degrees C of various hematopoietic subsets in murine bone marrow (MRA, CFU-S-12, CFU-S-8, CFU-GM, BFU-E and CFU-E) was investigated in order to determine whether there is a relationship between heat sensitivity and the position of cells within the stem cell hierarchy. The results show that the primitive stem cell with marrow repopulating ability (MRA) is extremely heat-resistant compared with the most differentiated hematopoietic progenitor (colony-forming unit-erythroid [CFU-E]). The proliferative activity of the hematopoietic subsets was determined from the number of cells killed by hydroxyurea (HU). It is demonstrated that there is a progressive increase in the proportion of hematopoietic subset cells in S-phase with maturation. The various heat sensitivities among the different hematopoietic subsets appear to be related to their proliferative activity. This relationship may have relevance to the clinical application of hyperthermia as a purging modality.

Thermal sensitivity of the murine CFU-S-12: role of environmental cells.

The hyperthermic sensitivity of the CFU-S-12 in bone marrow from normal and anaemic mice was determined. The terminal slope of the survival curves, demonstrated by the T0 values, does not significantly differ in the resting and active cycling stem cells. In the active cycling stem cells the initial shoulder region was less dominant compared with the resting stem cells. The difference in heat sensitivity between resting and active proliferating CFU-S-12 might be explained by a difference in the accumulation of damage before lethality becomes manifest. The difference in heat sensitivity appears to be independent of the environmental accessory cells, demonstrated by a similar hyperthermic effect of the purified stem cells from bone marrow and spleen and the stem cells in the total cell suspensions. Therefore the heat sensitivity of the haemopoietic stem cell is not mediated by a release of injurious substances from environmental heat-damaged cells. The heat treatment does not result in a selection of macroscopic detectable colonies 12 days after inoculation, as is demonstrated by the same morphology of the spleen colonies from the stem cells before and after the hyperthermic treatment.

Effect of a hyperthermic treatment on the pluripotent haemopoietic stem cell in normal and anaemic mice.

Up to now, the hyperthermic sensitivity of pluripotent haemopoietic stem cells is unknown, and the few existing data from reports in the literature are conflicting. There are two main drawbacks in the set-up of those studies: (1) only CFU-S day 9 results were presented, whereas it is questionable if this assay gives a true reflection of the pluripotent stem cell, and (2) no attention has been paid to heat effects on the seeding efficiency, i.e. the amount of stem cells which will lodge in the spleen. The present study focused on the procedural differences and compared the results of a hyperthermic treatment (60 min, 42 degrees C) on the stem cells, assayed with the CFU-S day 9 and the CFU-S day 12 method, using the following three stem cell suspensions, all differing in their proliferative activity: bone marrow from normal mice and bone marrow and spleen cells from anaemic mice. Furthermore, we investigated the seeding efficiency before and after heat treatment. Resting stem cells, assayed with the CFU-S day 12 method, turned out to be resistant to hyperthermia as compared with the active cycling stem cells, while with the CFU-S day 9 assay the stem showed the same thermosensitivity in the two bone marrow suspensions. The active cycling stem cells do not significantly differ in thermosensitivity, in CFU-S day 9 and day 12 assays, although there is a difference between bone marrow and spleen. Hyperthermia appears to influence the seeding efficiency for spleen CFU-S; an increase of 1.73 was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Biogenesis of the red cell membrane and cytoskeletal proteins during erythropoiesis in vitro.

Purified erythroid progenitor cells (CFU-E) were used to study in vitro the production of the proteins present in the plasma membrane and the membrane skeleton. At different stages of erythropoiesis incorporation of [35S]methionine was measured and membranes were isolated. Whereas incorporation in the total protein mass of the cells increased during erythropoiesis, the labeling of the membrane protein fraction decreased. The major erythrocyte membrane proteins were synthesized already in the CFU-E and continued to be made till the orthochromatic erythroblast stage. Band 3 protein, however, was made at a much lower rate. The incorporation in the late stages was only 5% of that in the CFU-E. The major changes in the protein composition of the membrane and its adherent skeleton occurred at the enucleation step.

Expression of the 5'-flanking region of the beta major-globin gene in cultured murine erythroid precursor cells.

The amounts of mouse beta major-globin mRNA and globin gene upstream RNA polymerase III transcripts were compared during the differentiation of purified erythroid colony-forming progenitor cells (CFU-E) in vitro. The accumulation of each RNA was determined relative to the 100% levels in fetal liver erythroid cells. The mRNA level was low in freshly isolated CFU-E and began to accumulate only after a lag period of approximately 2-4 h. It continued to accumulate for approximately 40 h thereafter, at which point it was comparable to that in the fetal liver. In contrast, in three of four CFU-E preparations, the relative level of upstream RNAs was high in freshly isolated CFU-E and reached the maximal level (defined as 100% of the fetal liver level) more rapidly than did the mRNA. The early induction and accelerated accumulation of upstream RNAs in immature erythroid cells suggest some role for these RNAs at an early stage of globin gene activation.

Induction of globin mRNA transcription by erythropoietin in differentiating erythroid precursor cells.

The effects of erythropoietin (epo) on the proliferation of late erythroid progenitor cells (CFU-E) and on the formation of hemoglobin and of globin mRNA in these cells are described. CFU-E were isolated from thiamphenicol-pretreated anemic mice by elutriation and Percoll density gradient methods. These CFU-E are restricted in their capacity to proliferate in vitro without added epo. The epo dependence in vitro was not absolute. With no epo in the culture medium the first cell division was unimpaired, whereas the third division was only 1%-2% of the control. In the absence of epo the synthesis of hemoglobin is very low in CFU-E, but is increased significantly after about 5 h of incubation with epo present. In epo deprived cells there was considerable hemoglobin formed at about 14 h, but not earlier. The presence as detected by the Northern blot technique of globin mRNA, isolated from CFU-E, was variable, probably depending on the presence of some more mature erythroid cells. By an extrapolation method we show evidence that pure CFU-E would have virtually no detectable globin mRNA. The production of globin mRNA is rapidly (2 h) induced in cells incubated with epo. We conclude that epo, besides having a mitogenic effect on CFU-E, induces the rapid expression of the globin genes.

The transbilayer distribution of phosphatidylethanolamine in erythroid plasma membranes during erythropoiesis.

Fluorescamine was used to assess the transbilayer distribution of phosphatidylethanolamine in the plasma membrane of murine erythroid progenitor cells, CFU-E (colony-forming unit erythroid), at different stages of their differentiation pathway. Intact cells were exposed to increasing concentrations of fluorescamine and the amount of labeled phosphatidylethanolamine was determined by measuring the fluorescence intensity of its fluorescamine derivative. A semilogarithmic plot of the dose-response curve revealed three different pools of phosphatidylethanolamine, representing its fractions in, respectively, the inner- and outer monolayers of the plasma membrane and subcellular membrane systems. These results show that 9-11% of the total cellular phosphatidylethanolamine is present in the outer leaflet and 9-10% of it is located in the inner leaflet of the plasma membrane in early as well as late erythroblasts. This symmetric distribution of phosphatidylethanolamine over the two halves of the bilayer in the plasma membrane of CFU-E is very similar to that observed earlier in the plasma membrane of friend erythroleukaemic cells (Rawyler, Van der Schaft, Roelofsen and Op den Kamp (1985) Biochemistry 24, 1777-1783). These observations imply that the characteristic asymmetric distribution of phosphatidylethanolamine, as is found in mature erythrocytes, is accomplished at a very late stage of erythropoiesis and possibly during enucleation of the cells or shortly thereafter.