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Upregulation of alpha globin promotes apoptotic cell death in the hematopoietic cell line FL5.12

Brecht, Karin. Upregulation of alpha globin promotes apoptotic cell death in the hematopoietic cell line FL5.12. 2004, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_6971

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Abstract

The function of alpha globin in the context of oxygen transport in erythroid cells is well
described. There is growing evidence, however, that alpha globin is involved in processes
other than tissue oxygen supply. Recently the expression of alpha globin has been shown to
be induced upon specific apoptotic stimuli like cytokine deprivation or cisplatin treatment in
the IL-3 dependent hematopoietic pro B cell line, FL5.12. In the current work we confirm for
the first time that the upregulation of alpha globin is not restricted to the transcript, but is also
observed at protein level. Different from erythroid cells, which bear globin proteins at 14 kD,
alpha globin displayed two distinct bands of 14 and 14.5 kD molecular weight under normal
growth conditions. In healthy cells both forms of alpha globin were localized to the
cytoplasm, whereas in apoptotic cells the 14 kD alpha globin was partially localized to the
cytoskeleton. In contrast to alpha globin, beta globin was expressed at a very low level, while
other globins or globin-like genes were not expressed at all. Further, we found that alpha
globin was not associated with its prosthetic group heme. Apoptotic cells neither produced
hemoglobin nor displayed a phenotype of cells differentiating down the erythroid lineage.
Very interestingly, also other cell lines of variable differentiation status including NIH3T3,
HeLa, and K562 upregulated alpha globin during treatment with apoptosis-inducing agents
indicating that alpha globin upregulation in apoptosis is not unique to FL5.12 cells.
Enrichment of FL5.12 cells ectopically expressing GFP-alpha globin turned out to be difficult
even in the presence of IL-3. Under IL-3-deprived apoptotic conditions, GFP-alpha globin
accelerated the progression of cell death in a comparable fashion to GFP-Bax. In particular,
caspase-8, -9 and -3 as well as the proapoptotic factors Bid, Bax, and cytochrome c were
activated. In contrast, the expression of an antisense alpha globin construct exhibited a minor
effect on the progression of apoptotic cell death. Taken together these data indicate that alpha
globin is a new and crucial factor in apoptosis especially supporting the mitochondrial
pathway, and its upregulation is a widespread phenomenon in apoptosis.
To study how the transcription of alpha globin is placed in the broader context of apoptosis,
we searched for transcription factors, which were concomitantly upregulated with alpha
globin. In cytokine-deprived FL5.12 cells transcription factor GATA-2, containing binding
sites for regulatory sequences of globin and virtually all erythroid genes, was the most
prominently upregulated candidate as assessed by gene chip arrays and RT QPCR. GATA-1
was expressed at low levels and weakly induced, while GATA-3 was completely absent. In
FL5.12 cells treated with cisplatin or doxorubicin, GATA-2 levels remained unchanged. By
investigating other cell lines, which induced alpha globin in apoptosis, we found that GATA-
2 was also upregulated in NIH3T3 but not K562 and HeLa cells. To evaluate the influence of
GATA-2 on alpha globin expression and cell viability we overexpressed GATA-2 in FL5.12
and NIH3T3 cells. Interestingly, high expression of GATA-2 resulted in immediate cell death
in FL5.12 cells and caused a severe but transient stress phenotype in NIH3T3 cells. We
further found that alpha globin levels were indeed elevated in GATA-overexpressing FL5.12
but not NIH3T3 cells. Transduction of antisense GATA-2 in FL5.12 cells reduced both the
increase of GATA-2 and alpha globin under apoptotic conditions and delayed cell death. In
summary, our results suggest that the mechanisms to induce alpha globin under apoptotic conditions differ depending on the death stimulus and the cell line. The fact that
overexpressed GATA-2 promoted, while antisense GATA-2 delayed cell death in FL5.12
cells, suggests that the function of GATA-2 is not restricted to maintenance and proliferation
of immature hematopoietic progenitors but is also critical in apoptosis.
Advisors:Heim, Jutta
Committee Members:Erb, Peter and Bickle, Thomas A.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:6971
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:134
Language:English
Identification Number:
Last Modified:23 Feb 2018 11:40
Deposited On:13 Feb 2009 15:00

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