Kazuyasu Endo

Two chronic myelogenous leultaemia cell lines which represent different stages of erythroid differentiation

Summary We established two cell lines, YN‐1 and Y‐1K. from the peripheral blood of two chronic myelogenous leukaemia patients in blastic crisis. Characterization of the YN‐1 and Y‐1K cells revealed that these cells expressed erythroid lineage markers. However, there was a marked difference in the level of γ‐globin mRNA and haenioglobin in YN‐1 and Y‐1K cells. YN‐1 contained approximately 1–5% benzidine‐positive staining cells, whereas no benzidine‐positive cells were observed in Y‐1K cells. Haemoglobin production in YN‐1 cells was markedly increased with various chemical inducers of erythroid differentiation, but was not in Y‐1K cells. In contrast, Y‐1K cells expressed CD34 stem cell antigen and CD41 megakaryocyte‐specific antigen. These observations suggested that, although both cell lines were committed to the erythroid lineage. each cell line represented a distinct differentiation stage in the erythroid differentiation programme. Y‐1K seemed to correspond to an early stage of cells in erythroid lineage, whereas YN‐1 represented a more advanced stage in human erythropoiesis.

Overexpression of AML1 renders a T hybridoma resistant to T cell receptor-mediated apoptosis

The AML1 gene, which encodes the DNA binding subunit of the heterodimeric transcription factor, PEBP2/CBF, is involved in several types of chromosomal translocations associated with human acute myeloid leukemia, and has been shown by gene targeting to be essential for the development of definitive hematopoiesis in the murine fetal liver. In addition, the gene is expressed abundantly in T lymphocytes and has been implicated in T cell specific gene expression. In the present study we examined the function of AML1 in T cell receptor (TCR)-mediated, Fas/Fas-ligand dependent apoptosis of a T hybridoma line, DO11.10. Several independent cell clones overexpressing the AML1 protein were isolated by transfecting AML1 cDNA into these cells. These clones possessed an increased level of PEBP2/CBF DNA binding activity and were found to be resistant to apoptosis induced by anti-CD3 antibody treatment. Northern blot analysis revealed that induction of the Fas-ligand transcript was markedly suppressed in the anti-CD3 treated clones. Instead, expression of IL-2 receptor α subunit (IL-2Rα), which is a manifestation of proliferative TCR signaling, was induced. This was in contrast to the parental, anti-CD3 treated DO11.10 cells where induction of Fas-ligand but not of IL-2Rα was observed. Resistance of the AML1 overexpressing cell clones to TCR-mediated apoptosis is most likely attributable to the lack of Fas-ligand induction, since simultaneous treatment with anti-CD3 and anti-Fas antibodies caused apoptosis of the clones. The overall results suggest that the AML1 protein may play a pivotal role in switching TCR signaling between apoptosis and cell proliferation in T lymphocytes.

T cell-mediated inhibition of erythropoiesis in aplastic anaemia: the possible role of IFN-γ and TNF-α

Summary. The inhibitory activity of T cells on autologous erythroid colony‐forming units (CFU‐E) (T cell inhibitory activity) in patients with aplastic anaemia (AA) was investigated. In 11 (32·4%) out of 34 AA cases, T cell inhibition on autologous CFU‐E growth was greater than that in normal individuals. In order to evaluate the mechanism of this inhibitory activity, T cell surface markers, interferon (IFN) production in peripheral blood mononuclear cell (PBMNC) liquid culture, and cytokine levels such as IFN and tumour necrosis factor‐α (TNF‐α) in CFU‐E clot cocultured with T cells, were measured in a portion of the patients. In five patients investigated for IFN production in PBMNC liquid culture, all produced statistically more IFN activity than normal individuals under phytohaemagglutinin (PHA‐P) stimulation (P<0·01) with no relation to T cell inhibitory activity. In only one patient whose T cells displayed increased CD8 and HLA‐DR antigen (CD8+HLA‐DR+) and inhibitory activity, a significant amount of IFN‐γ was observed in CFU‐E clot cocultured with T cells, and the addition of anti‐IFN‐γ antibody to the coculture resulted in recovered CFU‐E colony growth. These results suggest that IFN‐γ production by T cells may explain, at least in part, the pathogenesis of haematopoietic defects in AA. In other patients however, T cell inhibitory activity neither correlated to the T cell subpopulations (CD4+/CD8+, CD8+HLA‐DR+), IFN production in PBMNC liquid culture, nor to IFN and TNF‐α levels in CFU‐E clot culture. The roles played by cytokines other than IFN and TNF‐α on haematopoietic precursor cells require further evaluation in a larger sample of patients with AA.

A novel translocation involving chromosomes 2, 9, 14, and 22 in chronic myeloid leukemia

A 46-year-old man with chronic myelogenous leukemia was found to have a new complex translocation. In chronic phase, all of the bone marrow cells had a rearrangement of a t(2;9;14;22) (p21;q34;q32;q11). Southern blot analysis of leukocyte DNA revealed rearrangement of the breakpoint cluster region (bcr) within the 5.8-Kb bcr. The patient eventually died in blast crisis 28 months later. The cytogenetic findings of bone marrow cells showed a 46,XY,t(2;9;14;22)(p21;q34;q32;q11),add(1p),del(3q) karyotype in blast crisis.

Erythrocytosis in pseudohypoparathyroidism type I

Pseudohypoparathyroidism (PHP) is characterized by target-organ resistance to parathyroid hormone and is classified as type I or type II depending on the urinary cyclic AMP response to exogenous parathyroid hormone. Mild erythrocytosis has been observed in patients with PHP type I. Measurements of serum levels of erythropoietin (Epo) are useful for the differential diagnosis of subgroups of erythrocytosis [1]. We measured serum Epo to investigate the mechanism of erythrocytosis in PHP type I. PHP and idiopathic hypoparathyroidism were diagnosed as previously described [3], and the patients were treated with l~-hydroxylated metabolites of vitamin D 3 (Table 1).