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Characterization of proteins involved in differentiation and apoptosis of human leukemia and epithelial cancer cells
Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Today, cancer is understood as an epigenetic as well as a genetic disease. The main epigenetic hallmarks of the cancer cell are DNA methylation and histone modifications. The latter changes may be an optimal target for novel anticancer agents. The main goal of using histone deacetylase inhibitors (HDACIs) would be restoration of gene expression of those tumor-suppressor genes that have been transcriptionally silenced by promoter-associated histone deacetylation. However, HDACIs have pleiotropic effects that we are only just starting to understand. These may also be responsible for the induction of differentiation, cell-cycle arrest and pro-apoptotic effects.

There are now so many HDACIs available, with such different chemical structures and biological and biochemical properties, that it is hopeful that at least some of them will succeed, probably in combination with other agents or therapies.

In our studies we focussed ourselves on studies some new HDACIs, that can be useful for treating cancers, including leukemia and epithelial cancer. To do that, we used novel HDACIs, like BML-210, and their combination with the differentiation inducer all-trans retinoic acid (ATRA). Cell differentiation and proliferation in general, and specific gene expression require de novo protein synthesis and/or post-translational protein modifications. So, we tried to identify proteins in general and specifically the proteins that could be important for the cell differentiation process, and when and where in the cell the proteins appear.

We delineated that HDACIs inhibited leukemia (NB4 and HL-60) cell growth in a time- and dose-dependent way. Moreover, BML-210 blocked HeLa cell growth and promoted apoptosis in a time-dependent way. Combining of BML-210 with ATRA induced a differentiation process in leukemia cell lines that lead to apoptosis. This correlated with cell cycle arrest in G0/G1 stage and changes in expression of cell cycle proteins (p21, p53), transcription factors (NF-κB, Sp1) and their binding activity to consensus or specific promoter sequences. We also assessed histone modifications, i.e. H3 phosphorylation and H4 hyperacetylation due to HDACI, leading to chromatin remodeling and changes in gene transcriptions.

We have also studied changes in protein maps caused by HDACIs and differentiation agents, identifying differences for a few proteins due to growth inhibition and induction of differentiation in NB4 cells using BML-210 alone or in combination with ATRA. These proteins are involved in cell proliferation and signal transduction, like Rab, actin and calpain. One of them was alpha-dystrobrevin (α-DB). To further study possible roles of the latter, we determined changes of α-DB protein isoform expression that correlated with induction of differentiation. We thus identified a novel ensemble of α-DB interacting proteins in promyelocytic leukemia cells, including tropomyosin 3, actin, tubulin, RIBA, STAT and others, being important in cytoskeleton reorganization and signal transduction. Using confocal microscopy, we determined that α-DB co-localizes with HSP90 and F-actin in NB4 and HeLa cells. We also revealed that it changes sub-cellular compartment after treatment with ATRA and/or BML-210. α-DB silencing affected F-actin expression in HeLa cells, further supporting the idea that α-DB is involved in cytoskeleton reorganization in cells. Altogether, our results suggest that α−DB may work as a structural protein during proliferation and differentiation processes of human cancer cells.

Based on our findings, we suggest that HDACIs, like BML-210, can be promising anticancer agents, especially in leukemia treatment, by inducing apoptosis and regulating proliferation and differentiation through the modulation of histone acetylations and gene expression.

Place, publisher, year, edition, pages
Linköping University Electronic Press, 2008.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1060
Keyword [en]
leukemia, histone deacetylase inhibitors, retinoic acid, differentiation, apoptosis
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-11741ISBN: 978-91-7393-916-4 (print)OAI: oai:DiVA.org:liu-11741DiVA: diva2:18161
Public defence
2008-05-14, Linden, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2008-05-30 Created: 2008-05-30 Last updated: 2015-11-19
List of papers
1. The novel histone deacetylase inhibitor BML-210 exerts growth inhibitory, proapoptotic and differentiation stimulating effects on the human leukemia cell lines
Open this publication in new window or tab >>The novel histone deacetylase inhibitor BML-210 exerts growth inhibitory, proapoptotic and differentiation stimulating effects on the human leukemia cell lines
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2006 (English)In: European Journal of Pharmacology, ISSN 0014-2999, Vol. 549, no 1-3, 9-18 p.Article in journal (Refereed) Published
Abstract [en]

Histone deacetylase inhibitors have a potent role in the strategy for the treatment of leukemias. BML-210 (N-(2-Aminophenyl)-N′ phenyloctanol diamine) is the novel histone deacetylase inhibitor, and its mechanism of action has not been characterized. In this study, we examined the in vitro effects of BML-210 on the human leukemia cell lines (NB4, HL-60, THP-1, and K562). We found that BML-210 inhibits the growth of all cell lines and promotes apoptosis in a dose- and time-dependent manner. BML-210 alone induces HL-60 and K562 cell differentiation (up to 30%) to granulocytes and erythrocytes, respectively, and in combination with differentiation agents — all-trans retinoic acid and hemin, markedly potentates it. Those treatments cause G1 arrest and histone H4 acetylation, affects transcription factor NF-κB and Sp1 binding activity to their consensus sequences, the p21 or the FasL promoters, and influences expression of Sp1, NF-κB, p21 and FasL. These findings suggest that BML-210 could be a promising antileukemic agent to induce apoptosis and to modulate differentiation through the modulation of histone acetylation and gene expression.

Keyword
Apoptosis; Differentiation; Histone deacetylase inhibitor; Leukemia; Transcription factors
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13262 (URN)10.1016/j.ejphar.2006.08.010 (DOI)
Available from: 2008-05-30 Created: 2008-05-30
2. Apoptotic effects of the novel histone deacetylase inhibitor BML-210 on HeLa cells
Open this publication in new window or tab >>Apoptotic effects of the novel histone deacetylase inhibitor BML-210 on HeLa cells
Manuscript (Other academic)
Identifiers
urn:nbn:se:liu:diva-13263 (URN)
Available from: 2008-05-30 Created: 2008-05-30 Last updated: 2010-01-13
3. Effects of retinoic acid and histone deacetylase inhibitor Bml-210 on protein expression in NB4 cells
Open this publication in new window or tab >>Effects of retinoic acid and histone deacetylase inhibitor Bml-210 on protein expression in NB4 cells
2005 (English)In: Biologija, ISSN 1392-0146, Vol. 4, 88-93 p.Article in journal (Refereed) Published
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13264 (URN)
Available from: 2008-05-30 Created: 2008-05-30
4. Multiple roles of alpha-dystrobrevin in human cancer cells during proliferation and differentiation processes
Open this publication in new window or tab >>Multiple roles of alpha-dystrobrevin in human cancer cells during proliferation and differentiation processes
Manuscript (Other academic)
Identifiers
urn:nbn:se:liu:diva-13265 (URN)
Available from: 2008-05-30 Created: 2008-05-30 Last updated: 2010-01-13
5. Retinoic acid and histone deacelytase inhibitor BML-210 inhibit proliferation of human cervical cancer HeLa cells
Open this publication in new window or tab >>Retinoic acid and histone deacelytase inhibitor BML-210 inhibit proliferation of human cervical cancer HeLa cells
2006 (English)In: Annals of the New York Academy of Sciences, ISSN 0077-8923, Vol. 1091, 346-355 p.Article in journal (Refereed) Published
Abstract [en]

Human papillomavirus (HPV) infection is believed to be the central cause of cervical cancer. The viral proteins E6 and E7 from high-risk HPV types prevent cells from differentiating apoptosis and inducing hyperproliferative lesions. Human cervical carcinoma HeLa cells contain integrated human papillomavirus type 18 (HPV-18). Retinoic acid (RA) is a key regulator of epithelial cell differentiation and a growth inhibitor in vitro of HeLa cervical carcinoma cells. Cellular responses to RA are mediated by nuclear retinoic acid receptors (RARs) and retinoid X receptors. On the other hand, histone deacetylase inhibitors have been shown to be chemopreventive agents for the treatment of cancer cells. In this article, we have examined the antiproliferative effect of RA and histone deacetylase inhibitor BML-210 on HeLa cells, and particularly the effects on protein expression that may be involved in the cell cycle control and apoptosis. Our data suggest that a combination of RA and BML-210 leads to cell growth inhibition with subsequent apoptosis in a treatment time-dependent manner. We confirm that BML-210 alone or in combination with RA causes a marked increase in the level of p21. The changes in the p53 level are under the influence of p38 phosphorylation. We also discovered that the histone deacetylase inhibitor BML-210 causes increased levels of anti-apoptotic protein Bcl-2 and phosphorylated p38 MAP Kinase; the latter link in cell cycle arrest with response to extracellular stimuli. Our results suggest that RA and BML-210 are involved in different signaling pathways that regulate cell cycle arrest and lead to apoptosis of HeLa cells.

Keyword
histone deacetylase inhibitor, proliferation, p53, p21
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13266 (URN)10.1196/annals.1378.079 (DOI)
Available from: 2008-05-30 Created: 2008-05-30 Last updated: 2009-05-07
6. The histone deacetylase inhibitor FK228 distinctly sensitizes the human leukemia cells to retinoic acid-induced differentiation
Open this publication in new window or tab >>The histone deacetylase inhibitor FK228 distinctly sensitizes the human leukemia cells to retinoic acid-induced differentiation
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2006 (English)In: Annals of the New York Academy of Sciences, ISSN 0077-8923, Vol. 1091, 368-384 p.Article in journal (Refereed) Published
Abstract [en]

FK228 (depsipeptide) is a novel histone deacetylase inhibitor (HDACI) that has shown therapeutical efficacy in clinical trials for malignant lymphoma. In this article, we examined in vitro effects of FK228 on human leukemia cell lines, NB4 and HL-60. FK228 alone (0.2–1 ng/mL) inhibited leukemia cell growth in a dose-dependent manner and induced death by apoptosis. FK228 had selective differentiating effects on two cell lines when used for 6 h before induction of granulocytic differentiation by retinoic acid (RA) or in combination with RA. These effects were accompanied by a time- and dose-dependent histone H4 hyper-acetylation or histone H3 dephosphorylation and alterations in DNA binding of NF-κB in association with cell death and differentiation. Pifithrin-α (PFT), an inhibitor of p53 transcriptional activity, protected only NB4 cells with functional p53 from FK228-induced apoptosis and did not interfere with antiproliferative activity in p53-negative HL-60 cells. In NB4 cells, PFT inhibited p53 binding to the p21 (Waf1/Cip1) promotor and induced DNA binding of NF-κB leading to enhanced cell survival. Thus, beneficial effects of FK228 on human promyelocytic leukemia may be exerted through the induction of differentiation or apoptosis via histone modification and selective involvement of transcription factors, such as NF-κB and p53.

Keyword
differentiation, histone deacetylase inhibitor, leukemia, NF-κB, p53
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13267 (URN)10.1196/annals.1378.081 (DOI)
Available from: 2008-05-30 Created: 2008-05-30 Last updated: 2011-01-11

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Borutinskaite, Veronika Viktorija

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