[Late and slow diagnosis of acute promyelocytic leukemias--the main cause of early death]. / Pozdní a pomalá diagnostika akutních promyelocytárních leukémií--hlavní prícina casných úmrtí.

Acute hypergranular promyelocytic leukemia (AML M3) belongs to malignant diseases leading very rapidly to death. Immediate treatment based on early diagnosis may cure one third of patients. The typical finding in peripheral blood of patients is pancytopenia with or without atypical promyelocytes. In published studies only 15-25% patients exhibit leukocyte counts above 10 x 10(9)/l. Five of our ten patients studied had leukocyte count above 10 x 10(9)/l. The difference might be in connection with late and slow diagnosis of AML M3. AML is not taken into consideration during medical examination even if the disease occurs in medical family. Thus we describe clinical signs of AML M3 that could be divided into three main groups: bleeding, infections and anemia. In patients with bleeding or anemia or with infections repeating within a short period or with an infection and concurrent signs of bleeding or anemia the complete blood cell count should be examined immediately. If blood cell count abnormalities are found the patient should be sent immediately to hematology unit for further examination and treatment. Early diagnosis enables to start "differentiation therapy" with all-trans retinoic acid that could be administered as monotherapy only in patients with leukocytes below 5 x 10(9)/l. Early diagnosis of AML M3 might ameliorate the fate of patients, since four of our five patients referred to us with elevated leukocyte counts expired in the first five days.

Red cell ferritin and iron stores in chronic granulocytic leukemia.

Basic red cell ferritin was investigated in 28 patients with different phases of chronic granulocytic leukemia (CGL). Red cell ferritin was significantly decreased in remission after busulphan treatment and significantly elevated in the blast crisis as compared to healthy controls. Bone marrow stainable iron was decreased or absent in 86% of patients in the initial phase at the time of diagnosis and in 92% of those in remission. Red cell ferritin correlated with serum ferritin, however, serum ferritin level remained above normal range during all phases of the disease. A negative correlation between red cell ferritin and hemoglobin (Hb) (r = -0.605, p < 0.001) suggested that red cell ferritin level reflected the rate of iron utilization for heme synthesis. Decreased red cell iron stores observed in the remission may be explained by regression of dyserythropoiesis and by restoration of normal Hb synthesis after busulphan treatment. A progressive dyserythropoiesis in the blast crisis may lead to an increased red cell ferritin level.

Transferrin and iron distribution in subcellular fractions of K562 cells in the early stages of transferrin endocytosis.

Iron distribution in subcellular fractions was investigated at different times after a single cohort of 59Fe-125 I-labeled transferrin (Tf) endocytosis in K562 cells. Cell homogenates prepared by hypotonic lysis and deoxyribonuclease (DNAase) treatment were fractionated on Percoll density gradients. Iron-containing components in the postmitochondrial supernatant were further fractionated according to their molecular weight using gel chromatography and membrane filtration. In the initial phases of endocytosis, both iron and Tf were found in the light vesicular fraction. After 3 min the labels diverged, with iron appearing in the postmitochondrial supernatant and Tf in the heavy fraction containing mitochondria, lysosomes and nuclei. Iron released from Tf-containing vesicles appeared both in low- and high-molecular-weight fractions in the postmitochondrial supernatant. After 5 min of endocytosis 59Fe activity in the low-molecular-weight fraction remained constant and 59Fe accumulated in a high-molecular-weight fraction susceptible to desferrioxamine chelation. After 10 min, 59Fe radioactivity in this fraction decreased and a majority of cytosolic 59Fe was found in ferritin. These results do not support the concept of the cytosolic low-molecular-weight iron pool as a kinetic intermediate between transferrin and ferritin iron in K562 cells.

Inhibition of heme synthesis decreases transferrin receptor expression in mouse erythroleukemia cells.

The coordination of transferrin receptor (TfR) expression and heme synthesis was investigated in mouse erythroleukemia (MEL) cells of line 707 treated with heme synthesis inhibitors or in a variant line Fw genetically deficient in heme synthesis. Cells of line 707 were induced for differentiation by 5 mM hexamethylene bisacetamide (HMBA). TfR expression increased in the course of induction, as judged by increased TfR mRNA synthesis, increased cytoplasmic TfR mRNA level, and by the increased number of cellular 125I-Tf binding sites. Addition of 0.1 mM succinylacetone (SA) decreased cellular TfR to the level comparable with the uninduced cells. The decrease was reverted by the iron chelator desferrioxamine (DFO) but not by exogenous hemin. In short-term (1-2 hours) incubation, SA inhibited 59Fe incorporation from transferrin into heme, whereas total cellular 59Fe uptake was increased. A decrease in TfR mRNA synthesis was apparent after 2 hours of SA treatment. Conversely, glutathione peroxidase mRNA synthesis, previously shown to be inducible by iron, was increased by SA treatment. Cells of heme deficient line Fw did not increase the number of Tf binding sites after the induction of differentiation by 5 mM sodium butyrate. SA had no effect on TfR expression in Fw cells. The results suggest that the depletion of cellular non-heme iron due to the increase in heme synthesis maintains a high level of transferrin receptor expression in differentiating erythroid cells even after the cessation of cell division.

The regulation of transferrin receptor and glutathione peroxidase mRNAs synthesis by changes in intracellular iron levels.

The effect of changes of iron availability in the culture medium on the expression of TfR (transferrin receptor) and glutathione peroxidase (GSHPx) genes was investigated in uninduced or induced murine erythroleukemia (Friend) cells of lines 707 and Fw labeled with (3H)uridine for 3 h. The level of the labeled cytoplasmic TfR mRNA exhibited about 2-3-fold increase and the level of the labeled GSHPx mRNA about 2-fold increase in induced Friend 707 cells in comparison with uninduced cells. Raising the levels of intracellular iron by treatment of Friend 707 cells with either hemin, Fe-pyridoxal isonicotinoyl hydrazone (PIH) or diferric transferrin (Tf) resulted in decreased levels of the labeled TfR mRNA. On the other hand, hemin and Fe-PIH caused an increase in the labeled cytoplasmic GSHPx mRNA. Conversely, treatment with PIH or desferrioxamine stimulated synthesis of TfR mRNA and decreased the levels of the labeled GSHPx mRNA. In Fw cells we did not find any difference between the levels of the labeled cytoplasmic TfR mRNA in induced and uninduced cells and the levels of labeled cytoplasmic GSHPx mRNA were only slightly increased by induction. Changes of the intracellular iron pool caused the same effect as in Friend 707 cells.

The relationship between heme synthesis and iron uptake in erythroid cells.

Hemin inhibited 59Fe uptake from transferrin (Tf) by mouse erythro-leukemia cells (MELC) induced for differentiation by hexamethylene bisacetamide (HMBA), but the rate and the extent of 125I-Tf endocytosis was unaffected. Hemin inhibited 59Fe incorporation into heme by a greater proportion than the overall uptake of 59Fe from Tf. Desferrioxamine (DFO) reverted the inhibition of 59Fe transport into MELC caused by hemin. Exogenous 5-aminolevulinic acid (ALA) stimulated 59Fe utilization for heme synthesis in MELC but did not revert inhibition induced by hemin. No evidence for a direct effect of heme on the Tf cycle or iron release was found.

Examination of serum ferritin and erythrocyte ferritin--its role in the blood transfusion service.

Blood donors were examined for serum ferritin values and concentration of ferritin in the erythrocytes. The group of male and female donors without previous donations showed average values of 102.27 ng and 51.75 ng of ferritin per one ml of serum, respectively. Males with over 20 donations had 68.04 ng ferritin per one ml, females 37.14 ng of ferritin per one ml. The reduced serum ferritin values in multiple male and female donors is statistically significant. Serum ferritin values in women of the two groups are lower than those of males, the difference also being statistically significant. In male and female blood donors, irrespective of the number of donations, average values of 13.74 ag and 12.07 ag of ferritin per erythrocyte, respectively, were established. The difference in ferritin concentration in the erythrocytes between males and females is statistically insignificant. The correlation coefficient failed to demonstrate any dependence between erythrocyte ferritin concentration and concentration of ferritin in the serum. The object of serum ferritin determination in blood donors is to detect the earliest stage of storage iron deficiency in the organism. For the latter purpose, the determination of erythrocyte ferritin is ineffective.

Inhibition of cellular iron uptake by haem in mouse erythroleukaemia cells.

Haemin inhibited iron uptake from transferrin (Tf) by mouse erythroleukaemia cells (MELC) induced for differentiation by hexamethylene bisacetamide (HMBA). The rate of 59Fe internalization was decreased, but the rate and the extent of 125I-Tf endocytosis was unaffected by the addition of haemin. Haemin inhibited 59Fe incorporation into haem by a greater proportion than the overall uptake of 59Fe from Tf. The reduction of total cellular 59Fe uptake was more pronounced at 59Fe-Tf concentrations closer to saturation. Exogenous 5-aminolaevulinic acid stimulated 59Fe utilization for haem synthesis in MELC but did not revert the inhibition induced by haemin. Haem synthesis measured by 14C-glycine incorporation into haem was maintained for at least 1 h without an external transferrin iron source and was inhibited by the addition of haemin equally over the whole range of Tf concentrations studied. Desferrioxamine (DFO) stimulated cellular uptake of 59Fe by the uninduced cells and reverted the inhibition of 59Fe transport into HMBA treated cells caused by haemin. Addition of DFO within a short-term incubation had no effect on haem synthesis measured by 14C-glycine incorporation into haem. No evidence for a direct effect of haem on the transferrin cycle or iron release was found. It was concluded that the reduction of iron uptake by haemin treated MELC is secondary to the decrease in iron utilization for haem synthesis.

Inhibitor of normal granulopoiesis produced by cells of MDS patients.

Low-density blood cells from patients with refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T) release a high molecular weight inhibitory substance that reduces the entry of normal progenitor cells of granulocytes and macrophages (CFU-GM) into the S-phase. Out of 20 patients with refractory anemia (RA and RAS) only 3 were positive. One patient with CMML was negative. Serial examination of 3 patients (two RA and one CMML) revealed that the production of the inhibitory activity preceded the development of the disease into RAEB, RAEB-T, or AML. With one exception, the inhibitory activity in positive cases was neutralized by antiserum against human placental ferritin.

Non-transferrin donors of iron for heme synthesis in immature erythroid cells.

The mechanism of iron uptake from several iron-containing compounds by transferrin-depleted rabbit reticulocytes and mouse spleen erythroid cells was investigated. Iron complexes of DL-penicillamine, citrate and six different aroyl hydrazones may be utilized by immature erythroid cells for hemoglobin synthesis, although less efficiently than iron from transferrin. HTF-14, a monoclonal antibody against human transferrin, reacts with rabbit transferrin and inhibits iron uptake and heme synthesis by rabbit reticulocytes. HTF-14 had no significant effect on iron uptake and heme synthesis when non-transferrin donors of iron were examined. Ammonium chloride (NH4Cl) increases intracellular pH and blocks the release or utilization of iron from the internalized transferrin. NH4Cl only slightly affected iron incorporation and heme synthesis from non-transferrin donors of iron. Hemin inhibited transferrin iron uptake and heme synthesis, but had a much lesser effect on iron incorporation and heme synthesis from non-transferrin donors of iron. These results allow us to conclude that transferrin-depleted reticulocytes take up iron from all of the examined non-transferrin iron donors without the involvement of the transferrin/transferrin receptor pathway.

Iron uptake from transferrin and transferrin endocytic cycle in Friend erythroleukemia cells.

Several aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of hemoglobin synthesis by dimethyl sulfoxide. The maximal rate of iron uptake from 59Fe-labeled transferrin, 1.5 X 10(6) atoms of Fe/cell per 30 min in uninduced cells, increased to 3 X 10(6) atoms/cell after 5 days of induction. The increase in iron uptake was not accompanied by a proportional increase in the number of transferrin receptors detected by 125I-labeled transferrin binding, suggesting a more efficient iron uptake by transferrin receptors in induced cells, with the rate of about 26 iron atoms per receptor per hour, compared to 15 atoms in uninduced cells. In agreement with this conclusion are results of the study of cellular 125I or 59Fe labeled transferrin kinetics. In the induced cells transferrin endocytosis and release proceeded with identical rates and all the endocytosed iron was retained inside the cell. On the other hand, transferrin release by uninduced cells was significantly slower and a substantial part of internalized 59Fe was released. On the basis of these results, different efficiency of iron release from internalized transferrin, accompanied by changes in cellular transferrin kinetics, is proposed as one of the factors determining the rate of iron uptake by developing erythroid cells.

Leukocyte-derived inhibitory activity in patients with myelodysplastic syndrome.

Leukocyte-derived inhibitory activity inhibiting the entry of normal progenitor cells of granulocytes and macrophages (CFU-GM) into the S-phase of a cell cycle was investigated in 16 patients with different forms of myelodysplastic syndrome (MDS). The presence of this inhibitory activity was analysed in medium conditioned with low-density cells obtained from peripheral blood of MDS patients. The inhibition rate was measured by 3H-thymidine suicide technique with subsequent cultivation of pretreated cells in semisolid agar medium. Low-density cells from MDS patients of various types were studied: from the twelve patients with refractory anaemia (RA or RAS) only three were positive, one patient with chronic myelomonocytic leukaemia (CMML) was negative while one patient with refractory anaemia with excess of blasts (RAEB) and two patients with RAEB in transformation (RAEB-T) were positive with respect of the described test. In two patients with RA, who underwent a long-term investigation, the production of leukocyte-derived inhibitory activity preceded the development of disease into RAEB or RAEB-T. In five positive cases, supernatants were incubated with antiserum against human placental ferritin; with one exception, the inhibitory activity was neutralized.

Cytogenetic study of circulating blasts in leukemias.

A special cultivation technique of separated blasts of peripheral blood in suspension culture has been used for cytogenetic diagnosis of patients suffering from acute nonlymphocytic leukemia, chronic myeloid leukemia, and refractory anemia with excess of blasts. Twenty-three patients were examined; in ten cases isolation of blasts was performed on Ficoll-Verografin and in the remaining 13 patients further separation of T-lymphocyte precursors by means of sheep erythrocytes was performed. Remarkably, a 100% success rate was attained in all cultivations. The optimum harvesting time was 72-96 hours; the rate of cell division per cultivation was determined by means of bromodeoxyuridine incorporation; second mitoses were revealed only after 96-hour cultivation. In all patients, except one, abnormal karyotypic changes were ascertained in separated blasts of peripheral blood cultivations. In most cases the morphology of chromosomes obtained from separated blasts of peripheral blood cultivations was of excellent quality.

Regulation of iron uptake in erythroid and non-erythroid cells.

Iron uptake and the transferrin endocytotic cycle were studied in Friend erythroleukemia 707 cells cultured over a period of 5 days after induction with dimethylsulphoxide, or without it. The increase in iron uptake observed 5 days following induction with dimethylsulphoxide is not associated with a corresponding increase in the number of transferrin receptors. While the uptake rate of induced cells is 26 atoms of iron per one transferrin receptor per hour, in non-induced cells, the rate is only 15 atoms of iron. Induced cells retain all iron, with endocytosis and transferrin release occurring at the same rate. By contrast, the rate of iron release from non-induced cells is slower and part of the iron bound to transferrin returns from the cell. Haem inhibits iron uptake in induced cells and has no effect on non-induced cells. Its regulatory role is apparently confined to the erythroid cell with massive haemoglobin synthesis. The differences in the efficacy of iron release from induced and non-induced erythroleukemia cells are, possibly, apart from the changes in the number of transferrin receptors, an additional factor involved in the control of cellular iron uptake.

Iron uptake and transferrin endocytosis in undifferentiated and differentiated erythroid cells.

Iron uptake from transferrin (Tf) by Friend erythroleukemia cells was studied before and after induction of the cells by dimethyl sulfoxide (DMSO) in culture. The increase in iron uptake after 5 days of induction was not accompanied by a proportional increase in the number of transferrin receptors detected by 125I-labeled Tf binding, resulting in DMSO induced cells taking up iron with the rate of about 26 iron atoms per receptor per hour, compared to 15 atoms in uninduced cells. In agreement with this finding are results of the study of cellular 125I or 59Fe-labeled Tf kinetics. In the induced cells Tf endocytosis and release proceeded with identical rates and all the endocytosed iron was retained inside the cell. Tf release by uninduced cells was slower and a part of internalized 59Fe was released. Hemin inhibits Tf-bound iron uptake by DMSO induced cells but not by the uninduced cells. Correspondingly, addition of hemin had little effect on Tf kinetics in the uninduced cells, while the release of Tf was slower in hemin-treated DMSO induced cells. Different efficiency of iron release from internalized Tf, accompanied by changes in cellular Tf kinetics is therefore proposed as a factor determining the rate of iron uptake by developing erythroid cells.