41 research outputs found

    In vitro growth characteristics of human lymphoid malignancies in primary cell culture

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    The experimental work presented in this thesis deals with the analysis of the in vitro growth and differentiation characteristics of acute lymphoblastic leukemia ( T end non-T), T cell non Hodgkin's lymphoma and B cell chronic lymphocytic leukemia in primary cell culture. For these studies, reproducible cell culture assays for the growth of these lymphoid tumours first needed to be developed. Considerable attention has been paid to the response of these neoplasms to the polypeptide hormone interleukin 2. The experiments described in chapters 2 and 3 deal with the in vitro colony growth of non-T acute lymphoblastic leukemia (ALL) and with the effect of interleukin 2 on these cells in combination with a factor elaborated by feeder leukocytes. The requirements of the ALL cells for activation with a lectin (PHA) or a phorbol ester (TPA) for colony growth have also been investigated. To assess whether non-T ALL cells differentiate toward more mature cell types during in vitro growth, the morphological and immunological phenotypes of colony cells were determined. To compare the differentiation capacities of ALL with those of acute leukemia of the myeloid differentiation lineage (AML) the abilities of AML to produce more mature progeny under comparable in vitro conditions were studied (chapter 4). Chapter 5 deals with an analysis of growth requirements of 8 cell type chronic lymphocytic leukemia (CLL) in colony culture and specifically with the role of IL2 in the proliferation of B CLL cells. This analysis is extended in chapter 6, in which the results of binding experiments with radiolabeled IL2 are presented. These experiments were carried out to determine numbers and affinity of IL2 receptors expressed by B CLL cells- In addition, the hypothesis that certain CLL cells might be capable of self-stimulation via the autocrine production of IL2 is approached in this chapter. In chapter 7, culture characteristics of acute lymphoblastic leukemia and non Hodgkin's lymphoma (NHL) of the T cell differentiation lineage are presented. The studies described in chapter 8 are our first attempts toward the characterization of membrane phenotypes and growth requirements of normal T -lymphocytic precursor cells in the human bone marrow. For this purpose, we applied the human long-term bone marrow culture system. Knowledge of the growth end differentiation features of normal lymphoid progenitors is essential for our understanding of whether or how the leukemic counter parts of these cells reflect a modified response to growth and differentiation stimuli. In chapter 9 a brief overview of our current understanding of the role of IL2 in the proliferation of neoplastic T and B cells is presented. Moreover, the results of this thesis are discussed in this chapter with reference to their implications for our insight into the control of proliferation and differentiation of the different types of lymphoid leukemia/lymphom

    RUNX1 Mutations in the Leukemic Progression of Severe Congenital Neutropenia

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    Somatic RUNX1 mutations are found in approximately 10% of patients with de novo acute myeloid leukemia (AML), but are more common in secondary forms of myelodysplastic syndrome (MDS) or AML. Particularly, this applies to MDS/AML developing from certain types of leukemia-prone inherited bone marrow failure syndromes. How these RUNX1 mutations contribute to the pathobiology of secondary MDS/AML is still unknown. This mini-review focusses on the role of RUNX1 mutations as the most common secondary leukemogenic hit in MDS/AML evolving from severe congenital neutropenia (SCN)

    CD20 and CD40 mediated mitogenic responses in B-lineage acute lymphoblastic leukaemia

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    Activation of CD20, a cross-membrane ion channel, induces cell cycle progression from G0 to G1 in B lymphocytes. Subsequent activation of CD40, a membrane receptor of the nerve growth factor receptor superfamily, transits the B cells to the S phase. CD40 may also act synergistically in combination with IL-4 (B lymphocytes) or IL-3/IL-7 (B-cell precursors). We investigated the proliferative responses of B-lineage acute lymphoblastic leukaemia (ALL) cells to CD20/CD40 activation. In 18/56 ALL cases, CD20 activation resulted in significant increases in DNA synthesis. Similar, although more moderate, effects were seen of activation of CD40 in 10/44 cases. Responses to CD20 or CD40 activation were independent of co-stimulation with IL-3, IL-4 or IL-7, and various cocktails of the different growth stimuli did not act synergistically

    Mutations in the gene for the granulocyte colony-stimulating-factor receptor in patients with acute myeloid leukemia preceded by severe congenital neutropenia

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    BACKGROUND. In severe congenital neutropenia the maturation of myeloid progenitor cells is arrested. The myelodysplastic syndrome and acute myeloid leukemia develop in some patients with severe congenital neutropenia. Abnormalities in the signal-transduction pathways for granulocyte colony-stimulating factor (G-CSF) may play a part in the progression to acute myeloid leukemia. METHODS. We isolated genomic DNA and RNA from hematopoietic cells obtained from two patients with acute myeloid leukemia and histories of severe congenital neutropenia. The nucleotide sequences encoding the cytoplasmic domain of the G-CSF receptor were amplified by means of the polymerase chain reaction and sequenced. Murine myeloid 32D.C10 cells were transfected with complementary DNA encoding the wild-type or mutant G-CSF receptors and tested for their responses to G-CSF. RESULTS. Point mutations in the gene for the G-CSF receptor were identified in both patients. The mutations, a substitution of thymine for cytosine at the codon for glutamine at position 718 (Gln718) in one patient and at the codon for glutamine at position 731(Gln731) in the other, caused a truncation of the C-terminal cytoplasmic region of the receptor. Both mutant and wild-type genes for the G-CSF receptor were present in leukemic cells from the two patients. In one patient, the mutation was also found in the neutropenic stage, before the progression to acute myeloid leukemia. The 32D.C10 cells expressing mutant receptors had abnormally high proliferative responses but failed to mature when cultured in G-CSF. The mutant G-CSF receptors also interfered with terminal maturation mediated by the wild-type G-CSF receptor in the 32D.C10 cells that coexpressed the wild-type and mutant receptors. CONCLUSIONS. Mutations in the gene for the G-CSF receptor that interrupt signals required for the maturation of myeloid cells are involved in the pathogenesis of severe congenital neutropenia and associated with the progression to acute myeloid leukemia

    Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation

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    The granulocyte colony-stimulating factor receptor (G-CSF-R) transduces signals important for the proliferation and maturation of myeloid progenitor cells. To identify functionally important regions in the cytoplasmic domain of the G-CSF-R, we compared the actions of the wild-type receptor, two mutants, and a natural splice variant in transfectants of the mouse pro-B cell line BAF3 and two myeloid cell lines, 32D and L-GM. A region of 55 amino acids adjacent to the transmembrane domain was found to be sufficient for generating a growth signal. The immediate downstream sequence of 30 amino acids substantially enhanced the growth signaling in the three cell lines. In contrast, the carboxy-terminal part of 98 amino acids strongly inhibited growth signaling in the two myeloid cell lines but not in BAF3 cells. Truncation of this region lead to an inability of the G-CSF-R to transduce maturation signals in L-GM cells. An alternative carboxy tail present in a splice variant of the G-CSF-R also inhibited growth signaling, notably in both the myeloid cells and BAF3 cells, but appeared not to be involved in maturation

    G-CSF receptor truncations found in SCN/AML relieve SOCS3-controlled inhibition of STAT5 but leave suppression of STAT3 intact

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    Truncated granulocyte colony-stimulating factor receptors (G-CSF-Rs) are implicated in severe congenital neutropenia (SCN) and the consecutive development of acute myeloid leukemia (AML). Mice expressing G-CSF-R truncation mutants (gcsfr-d715) show defective receptor internalization, an increased signal transducer and activator of transcription 5 (STAT5)/STAT3 activation ratio, and hyperproliferative responses to G-CSF treatment. We determined whether a lack of negative feedback by suppressor of cytokine signaling (SOCS) proteins contributes to the signaling abnormalities of G-CSF-R-d715. Expression of SOCS3 transcripts in bone marrow cells from G-CSF-treated gcsfr-d715 mice was approximately 60% lower than in wild-type (WT) littermates. SOCS3 efficiently suppressed STAT3 and STAT5 activation by WT G-CSF-R in luciferase reporter assays. In contrast, while SOCS3 still inhibited STAT3 activation by G-CSF-R-d715, STAT5 activation was no longer affected. This was due mainly to loss of the SOCS3 recruitment site Tyr729, with an additional contribution of the internalization defects of G-CSF-R-d715. Because Tyr729 is also a docking site for the Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2), which binds to and inactivates STAT5, we suggest a model in which reduced SOCS3 expression, combined with the loss of recruitment of both SOCS3 and SHP-2 to the activated receptor complex, determine the increased STAT5/STAT3 activation ratio and the resulting signaling abnormalities projected by truncated G-CSF-R mutants
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