Apart from CAD, the transient liberation of AIF during day 6 of TF-1 differentiation could pose another threat to the genomic DNA in cells. We have demonstrated the absence of AIF in the nucleus of TF-1 cells despite its release from the mitochondria by using confocal studies. Moreover, the expression of heat shock protein 70 kDa (Hsp70), a well-known antagonist of AIF, was found to be temporarily increased at day 6. Taken together, our results implied a plausible retention of AIF in the cytoplasm by Hsp70. Although Hsp70 is commonly utilized by many cancer cells to counteract AIF and avoid DNA fragmentation, we are the first to demonstrate its role in suppressing AIF during normal erythroid maturation. / As a whole, we have illustrated that the activated caspase-3, mediated most likely by the mitochondrial pathway, is an essential component in the differentiation of TF-1 cells. Its activation was nevertheless not coupled with DNA fragmentation due to some protective mechanisms such as CAD downregulation, Hsp70 upregulation and overexpression of Bcl-XL. Our study therefore provides some insights in the understanding of the relationship between human erythropoiesis and apoptosis and a better understanding in this regard will undoubtedly facilitate the development of new drugs in the treatment of different hematopoietic diseases. / Caspases play a central role in apoptosis. Their activations during the process are accounted for different biochemical and morphological changes in apoptotic cells. Yet in recent years, increasing studies had shown that caspases were also involved in some non-apoptotic cellular events, including T and B-lymphocytes activation, as well as the terminal differentiation of lens cells, megakaryocytes and erythrocytes. / In order to find out other unknown cellular mechanisms in erythropoiesis, mRNA differential display was employed to compare the gene expression pattern of TF-1 cells at different stages of differentiation. Several differentially expressed genes were identified and subsequently confirmed by RT PCR. These genes include formin binding protein 3, destrin and T-complex protein-1 (TCP-1). Their involvement in erythroid differentiation was still not clear at the moment but would be investigated in the near future. Furthermore, aiming at identifying the interacting proteins or inhibitors of caspase-3 in the system, a pull down assay was developed by means of the bacterial expression of a recombinant human caspase-3 mutant protein. With the mutation in the active site, the binding of our recombinant caspase-3 mutant with two known partners ICAD and BIRII (Baculovirus Inhibitor of apoptosis protein Repeat II) domain has been demonstrated. We hope in the near future that it can be employed to fish out some novel caspase-3 substrates from the differentiating TF-1 cell lysate. / In the present study, the participation of caspase in in vitro erythropoiesis was investigated using a human erythroleukemia cell line TF-1. Erythropoietin (EPO) induced erythroid maturation of TF-1 as indicated by the expression of erythroid-lineage markers like glycophorin A (GPA), transferrin receptors (CD71) and synthesis of hemoglobin (Hb). Activation of caspase-3 was observed from day 6 to day 12 during TF-1 differentiation after EPO treatment. With the administration of caspase-3 specific inhibitor, expressions of GPA and CD71 were partially blocked, suggesting that caspase-3 activation is essential in erythropoiesis in our TF-1 model. / Possible involvement of the intrinsic and extrinsic apoptotic pathways was studied by investigating respectively the activation of pro-caspase-9 and -8. It was found that caspase-9, but not -8, was activated at the corresponding time point when caspase-3 was activated. Besides, a transient mitochondrial depolarization coupled with the release of cytochrome c and apoptosis inducing factor (AIF) were detected on day 6, strongly implying a role of mitochondria in triggering the activation of executioner caspase-3. On the other hand, GPA and CD71 expressions were blocked by the application of mitochondrial depolarization inhibitor cyclosporin A (CyA). Also, the recovery of mitochondrial membrane potential was found to be correlated with an overexpression of Bcl-XL at a late stage of TF-1 differentiation, and the role of Bcl-XL was subsequently manifested further by a significant retardation of erythroid differentiation in the siRNA Bcl-XL knocked down TF-1 cells. / The exact role of caspase-3 in erythroid differentiation is far from clear at this moment. Yet, its regulation in the process is equally intriguing. On the course of TF-1 maturation, activated caspase-3 was able to cleave and de-localize the Inhibitor of Caspase-activated DNase (ICAD) from the nucleus, but at the same time DNA fragmentation was not detected by TUNEL assay nor agarose electrophoresis. Furthermore, protection against DNA fragmentation was observed in the EPO-treated TF-1 cells when challenged with a potent apoptotic inducer staurosporine (STS). These observations are in contrast to our understanding that DNA is fragmented by CAD (Caspase-activated DNase) when ICAD in the ICAD-CAD complex is cleaved by caspase-3. For these apparently contradictory observations, we demonstrated that downregulation of CAD occurred at the mRNA and protein levels during the erythroid differentiation in TF-1. This provides a cell rescuing mechanism in non-apoptotic cells with activated caspases. / Lui Chun Kin Julian. / "September 2006." / Adviser: Siu Kai Kong. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1620. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 239-253). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_343908 |
| Date | January 2006 |
| Contributors | Lui, Chun Kin Julian., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, theses |
| Format | electronic resource, microform, microfiche, 1 online resource (xxvii, 253 p. : ill.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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