12 research outputs found
Molecular characterization of translocation (6;9) in acute nonlymphocytic leukemia
Specific chromosomal translocations are one of the defects associated
with leukemia. Isolation and characterization of genes affected by these
translocations may give insight into the processes of both leukemogenesis
and normal hematopoiesis. When the experiments described in this thesis
were started, several genes involved in translocations in lymphoid
leukemia were isolated. These genes were all translocated into T-cell
receptor and Immunoglobulin loci, which deregulated their expression. In
myeloid leukemia only translocation (9;22) was characterized molecularly
and the resulting bcr-abl gene was the only fusion gene known. Chapter 1
gives an overview of what is known to date about genes involved in
leukemogenesis. To extend the research on the molecular characterization
of translocations in myeloid leukemia, we decided to clone and
characterize the translocation breakpoints of t(6;9) that characterizes a
subtype of acute myeloid leukemia. Chapter 2 gives an introduction to
t(6;9) AML and reports the results of our investigations. Chapter 3
discusses these results in relation to our current understanding of
leukemogenesi
Stem cell factor induces phosphatidylinositol 3'-kinase-dependent Lyn/Tec/Dok-1 complex formation in hematopoietic cells
Stem cell factor (SCF) has an important role in the proliferation,
differentiation, survival, and migration of hematopoietic cells. SCF
exerts its effects by binding to cKit, a receptor with intrinsic tyrosine
kinase activity. Activation of phosphatidylinositol 3'-kinase (PI3-K) by
cKit was previously shown to contribute to many SCF-induced cellular
responses. Therefore, PI3-K-dependent signaling pathways activated by SCF
were investigated. The PI3-K-dependent activation and phosphorylation of
the tyrosine kinase Tec and the adapter molecule p62Dok-1 are reported.
The study shows that Tec and Dok-1 form a stable complex with Lyn and 2
unidentified phosphoproteins of 56 and 140 kd. Both the Tec homology and
the SH2 domain of Tec were identified as being required for the
interaction with Dok-1, whereas 2 domains in Dok-1 appeared to mediate the
association with Tec. In addition, Tec and Lyn were shown to phosphorylate
Dok-1, whereas phosphorylated Dok-1 was demonstrated to bind to the SH2
domains of several signaling molecules activated by SCF, including Abl,
CrkL, SHIP, and PLCgamma-1, but not those of Vav and Shc. These findings
suggest that p62Dok-1 may function as an important scaffold molecule in
cKit-mediated signaling
Tyrosine kinase receptor RON functions downstream of the erythropoietin
Erythropoietin (EPO) is required for cell survival during differentiation
and for progenitor expansion during stress erythropoiesis. Although
signaling pathways may couple directly to docking sites on the EPO
receptor (EpoR), additional docking molecules expand the signaling
platform of the receptor. We studied the roles of the docking molecules
Grb2-associated binder-1 (Gab1) and Gab2 in EPO-induced signal
transduction and erythropoiesis. Inhibitors of phosphatidylinositide
3-kinase and Src kinases suppressed EPO-dependent phosphorylation of Gab2.
In contrast, Gab1 activation depends on recruitment and phosphorylation by
the tyrosine kinase receptor RON, with which it is constitutively
associated. RON activation induces the phosphorylation of Gab1,
mitogen-activated protein kinase (MAPK), and protein kinase B (PKB) but
not of signal transducer and activator of transcription 5 (Stat5). RON
activation was sufficient to replace EPO in progenitor expansion but not
in differentiation. In conclusion, we elucidated a novel mechanism
specifically involved in the expansion of erythroblasts involving RON as a
downstream target of the Epo
Hydroxyurea responsiveness in β-thalassemic patients is determined by the stress response adaptation of erythroid progenitors and their differentiation propensity
β-thalassemia is caused by mutations in the β-globin locus resulting in loss of, or reduced, hemoglobin A (adult hemoglobin, HbA, α2β2) production. Hydroxyurea treatment increases fetal γ-globin (fetal hemoglobin, HbF, α2γ2) expression in postnatal life substituting for the missing adult β-globin and is, therefore, an attractive therapeutic approach. Patients treated with hydroxyurea fall into three categories: i) 'responders' who increase hemoglobin to therapeutic levels; (ii) 'moderate-responders' who increase hemoglobin levels but still need transfusions at longer intervals; and (iii) 'non-responders' who do not reach adequate hemoglobin levels and remain transfusion-dependent. The mechanisms underlying these differential responses remain largely unclear. We generated RNA expression profiles from erythroblast progenitors of 8 responder and 8 non-responder β-thalassemia patients. These profiles revealed that hydroxyurea treatment induced differential expression of many genes in cells from non-responders while it h
Grsf1-induced translation of the SNARE protein use1 is required for expansion of the erythroid compartment
Induction of cell proliferation requires a concomitant increase in the synthesis of glycosylated lipids and membrane proteins, which is dependent on ER-Golgi protein transport by CopII-coated vesicles. In this process, retrograde transport of ER resident proteins from the Golgi is crucial to maintain ER integrity, and allows for anterograde transport to continue. We previously showed that expression of the CopI specific SNARE protein Use1 (Unusual SNARE in the ER 1) is tightly regulated by eIF4E-dependent translation initiation of Use1 mRNA. Here we investigate the mechanism that controls Use1 mRNA translation. The 5'UTR of mouse Use1 contains a 156 nt alternatively spliced intron. The non-spliced form is the predominantly translated mRNA. The alternatively spliced sequence contains G-repeats that bind the RNA-binding protein G-rich sequence binding factor 1 (Grsf1) in RNA band shift assays. The presence of these G-repeats rendered translation of reporter constructs dependent on the Grsf1 concentration. Down regulation of either Grsf1 or Use1 abrogated expansion of erythroblasts. The 5'UTR of human Use1 lacks the splice donor site, but contains an additional upstream open reading frame in close proximity of the translation start site. Similar to mouse Use1, also the human 5'UTR contains G-repeats in front of the start codon. In conclusion, Grsf1 controls translation of the SNARE protein Use1, possibly by positioning the 40S ribosomal subunit and associated translation factors in front of the translation start site
Stem cell factor receptor (c-KIT) codon 816 mutations predict development of bilateral testicular germ-cell tumors
Testicular germ-cell tumors (TGCTs) of adolescents and adults originate
from intratubular germ cell neoplasia (ITGCN), which is composed of the
malignant counterparts of embryonal germ cells. ITGCN cells are
characterized, among others, by the presence of stem cell factor receptor
c-KIT. Once established, ITGCN will always progress to invasiveness.
Approximately 2.5-5% of patients with a TGCT will develop bilateral
disease and require complete castration, resulting in infertility, a need
for lifelong androgen replacement, and psychological stress. To date, the
only way to predict a contralateral tumor is surgical biopsy of the
contralateral testis to demonstrate ITGCN. We did a retrospective study of
224 unilateral and 61 proven bilateral TGCTs (from 46 patients, in three
independently collected series in Europe) for the presence of activating
c-KIT codon 816 mutations. A c-KIT codon 816 mutation was found in three
unilateral TGCT (1.3%), and in 57 bilateral TGCTs (93%; P < 0.0001). In
the two wild-type bilateral tumors for which ITGCN was available, the
preinvasive cells contained the mutation. The mutations were somatic in
origin and identical in both tumors. We conclude that somatic activating
codon 816 c-KIT mutations are associated with development of bilateral
TGCT. Detection of c-KIT codon 816 mutations in unilateral TGCT identifies
patients at risk for bilateral disease. These patients may undergo
tailored treatment to prevent the development of bilateral disease, with
retention of testicular hormonal function
The glucocorticoid receptor cooperates with the erythropoietin receptor and c-Kit to enhance and sustain proliferation of erythroid progenitors in vitro
Although erythropoietin (Epo) is essential for the production of mature
red blood cells, the cooperation with other factors is required for a
proper balance between progenitor proliferation and differentiation. In
avian erythroid progenitors, steroid hormones cooperate with tyrosine
kinase receptors to induce renewal of erythroid progenitors. We examined
the role of corticosteroids in the in vitro expansion of primary human
erythroid cells in liquid cultures and colony assays. Dexamethasone (Dex),
a synthetic glucocorticoid hormone, cooperated with Epo and stem cell
factor to induce erythroid progenitors to undergo 15 to 22 cell divisions,
corresponding to a 10(5)- to 10(6)-fold amplification of erythroid cells.
Dex acted directly on erythroid progenitors and maintained the
colony-forming capacity of the progenitor cells expanded in liquid
cultures. The hormone delayed terminal differentiation into erythrocytes,
which was assayed by morphology, hemoglobin accumulation, and the
expression of genes characteristic for immature cells. Sustained
proliferation of erythroid progenitors could be induced equally well from
purified erythroid burst-forming units (BFU-E), from CD34(+) blast cells,
and from bone marrow depleted from CD34(+) cells
Protein kinase C alpha controls erythropoietin receptor signaling.
Protein kinase C (PKC) is implied in the activation of multiple targets of
erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR)
signal transduction and in the regulation of erythroid proliferation and
differentiation remained elusive. We analyzed the effect of PKC inhibitors
with distinct modes of action on EpoR signaling in primary human
erythroblasts and in a recently established murine erythroid cell line.
Active PKC appeared essential for Epo-induced phosphorylation of the Epo
receptor itself, STAT5, Gab1, Erk1/2, AKT, and other downstream targets.
Under the same conditions, stem cell factor-induced signal transduction
was not impaired. LY294002, a specific inhibitor of phosphoinositol
3-kinase, also suppressed Epo-induced signal transduction, which could be
partially relieved by activators of PKC. PKC inhibitors or LY294002 did
not affect membrane expression of the EpoR, the association of JAK2 with
the EpoR, or the in vitro kinase activity of JAK2. The data suggest that
PKC controls EpoR signaling instead of being a downstream effector. PKC
and phosphoinositol 3-kinase may act in concert to regulate association of
the EpoR complex such that it is responsive to ligand stimulation. Reduced
PKC-activity inhibited Epo-dependent differentiation, although it did not
effect Epo-dependent "renewal divisions" induced in the presence of Epo,
stem cell factor, and dexamethasone