During cells response to external stimulus, signals are transmitted to the nucleus from the surface of the the cell during the processes that regulate the signalling events, during which the cascade of protekinases and proteinphosphatases are initiated and other regulatory proteins are activated (Hunter 1995; Karin et al. 1997). Long-term effects may alter the activity of receptors in the proliferating cells and effect on the differentiation or apoptosis pathways. Leukemic cell lines are applied as an in vitro model in leukemia, enabling the molecular mechanisms of cellular proliferation or differentiation processes to be investigated. Differentiation inducers (DMSO, ATRA, dbcAMP, etc.) can induce NB4 and HL-60 to granulocytic differentiation. The forbolmyristat acetate (PMA), vitamin D3 and sodium butyrate can induce differentiation to monocytes/marophages lineage of NB4, HL-60, and THP-1 cells (Breitman et al. 1980; Collins et al. 1977; Collins 1987). During differentiation, cell growth is stopped and specific proteins that are involved in phenotype formation are activated.

The cell differentiation process into granulocytes can be devided into several stages: the first—initiation of differentiation, the second—differentiation into granulocytes or monocytes depending on the used inducer, and the third- maturation, when the cell that has lost its ability to multiply, begins to produce the specific proteins. The inducer of granulocytic differentiation all-trans retinoic acid (ATRA) activates genes by binding to the ATRA receptors in the nuclei of cells through the all-trans retinoic acid response elements (RARE). Some of these genes’ products may be involved, directly or indirectly, in the process of differentiation induction (Sporn et al. 1994; Pawson 1995), others can be expressed in the cytoplasm or the nuclei of differentiated. Matured granulocytes induced to differentiation by ATRA, similar to neutrophils, underwent the programmed cell death (apoptosis). The signalling pathways and molecular mechanisms that are involved in the granulocytic differentiation of leukemic cells that leads to apoptosis remain unclear. The mechanisms of cellular proliferation, differentiation, and apoptosis involve various protein-modifying enzymes, including protein phosphatases and protein kinases that are responsible for phosphorylation or dephosphorylation of proteins (on threonine, serine, or tyrosine residues). The phosphorylation of tyrosine residues is particularly specific at the stage of transmission of the external signal to the DNA, so it is important not only to identify newly synthesized proteins, but also to determine where and when they are modified (e.g., thyrosine phosphorylated).

Global functional proteomic analysis helps to understand the molecular mechanisms of diseases, providing new possibilities for detecting specific regulatory proteins and new targets for the rational treatment of leukemias and cancer.

Proteomic changes in leukemia cells, promoted to granulocytic differentiation by only ATRA or together with epigenetic modifiers were investigated our study (Navakauskiene et al. 2002, 2003a,b, 2004a,b, 2012, 2014; Kulyte et al. 2001, 2002; Borutinskaite et al. 2011, 2005; Borutinskaite and Navakauskiene 2015; Treigyte et al. 2000b,a, 2004; Valiuliene et al. 2015).

Methods for computerized analysis of proteome maps have been developed (Matuzevicius et al. 2008). Such proteomic analysis serves to elucidate the network of protein interactions and proteins responsible for myeloid cell development and leukemia, as well as to discover new targets for rational cancer therapy.

1 Proteomic Analysis of Cytoplasmic and Nuclear Proteins in Human Hematopoietic CD34+, AML Cell Line KG1 and Mature Neutrophils

Cytoplasmic and nuclear proteins we isolated from cells of different state of hematopoietic differentiation: primary hematopoietic CD34+, cancerous cells arrested at the stage of incomplete differentiation KG1 (AML cell line) and healthy mature neutrophils. The isolated cytoplasmic and nuclear proteins were fractionated in the 2DE system (Figs. 4.1 and 4.2).

Fig. 4.1
figure 1

Proteomic analysis of cytoplasmic proteins isolated from human hematopoietic CD34+, incompletely differentiated KG1 cells, and mature human neutrophils (NF). The cytoplasmic proteins (Cyt P) are isolated and fractionated in the 2DE system. After visualization of the proteins by staining with Coomassie blue, protein comparative analysis was performed to select proteins with different expression in different cell differentiation states. These proteins were prepared for mass spectrometry analysis, identified and quantified by computational analysis (Table 4.1)

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Fig. 4.2
figure 2

Proteome analysis of nuclear proteins isolated from human hematopoietic CD34+, incompletely differentiated AML cell line KG1, and mature human neutrophils (NF). Nuclear proteins (Tr F) were isolated and fractionated in the 2DE system. After visualization of the proteins by staining with Coomassie blue, protein comparative analysis was performed to select proteins with different expression in different hematopoietic cell differentiation states. These proteins were analyzed by mass spectrometry, identified and quantified by computational analysis (Table 4.2)

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After protein visualization a comparative analysis of the protein maps was performed and proteins that could serve as potential markers of leukemia were selected for mass spectrometry analysis. Proteins were selected based on changes in their expression in different states of cell differentiation. The summarized analysis data are presented in Table 4.1. The computational methods for protein expression analysis in different hematopoietic cells were applied and fold change between protein expression calculated. The network of identified cytoplasmic proteins distinctive for human hematopoietic CD34+, incompletely differentiated KG1 cells, and mature human neutrophils is presented in Fig. 4.3.

Fig. 4.3
figure 3

Proteomic analysis of cytoplasmic proteins isolated from human hematopoietic CD34+, incompletely differentiated KG1 cells, and mature human neutrophils. The network of identified cytoplasmic proteins distinctive for human hematopoietic CD34+, incompletely differentiated KG1 cells, and mature human neutrophils

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Table 4.1 The summarized search results (by PepIdent, EMBL, MS-Tag software) of comparative expression analysis of cytoplasmic proteins from KG1, Neutrophils (NF) and CD34+
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It is noticeable that in the cytoplasm (Figs. 4.1 and 4.3) cancerous, non-differentiated KG1 cells contain increased levels of the following proteins: Rab GTPase-activating protein 1, WD repeat-containing protein 81, Syncoilin, Aspartyl-tRNA synthetase cytoplasmic (SYDC), proteasome activator complex subunit 1, fatty acid-binding protein epidermal (FABP5), and others. Proteins with markedly reduced expression in cancer cells have also been identified: CAMP (Cathelicidin antimicrobial peptide), annexin A13, 14-3-3 protein, tubulin alfa-4A chain and several others. These proteins are related to the cellular signaling system (Rab-GTPase-activating protein, 14-3-3, etc.), to the maintenance of cell structure (sinkoiline, annexin, tubulin, etc.), are also involved in cell signaling and other cellular functions. Nuclear proteins isolated from different stages of hematopoietic differentiation were fractionated in the 2DE system (Fig. 4.2), followed by protein 2DE map comparison and mass spectrometry analysis of selected proteins. Proteins for analysis were selected on changes in their expression in different states of cell differentiation. The identified nuclear proteins are presented in Table 4.2 and the network of identified nuclear proteins typical for human hematopoietic CD34+, incompletely differentiated AML cell line KG1, and mature human neutrophils was established (Fig. 4.4).

Fig. 4.4
figure 4

Proteome analysis of nuclear proteins isolated from human hematopoietic CD34+, incompletely differentiated AML cell line KG1, and mature human neutrophils. The network of identified nuclear proteins typical for human hematopoietic CD34+, incompletely differentiated AML cell line KG1, and mature human neutrophils

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Table 4.2 The summarized search results (by PepIdent, EMBL, MS-Tag software) of comparative expression analysis of identified nuclear proteins isolated from CD34+, KG1 cells, and human neutrophils (NF)
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Several specific proteins have been identified that are specific only to healthy mature neutrophils. Multiple protein expression is enhanced in KG1 cancerous non-differentiated cells: putative GTP-binding protein, Protein mab-21-like 1, etc. For example, the expression of the periodic AMP-dependent transcription factor ATF-3 is reduced in KG1 leukemic cells. Changes in the expression of these proteins could be related to blood cell differentiation.

2 Proteomic Analysis of APL Cells Induced to Differentiation

Our initial study (Navakauskiene et al. 2003a,b; Treigyte et al. 2000b,a, 2004; Borutinskaite et al. 2005) was concentrated on identification of proteins that can be involved in cell differentiation and proliferation in acute promyelocytic HL-60 and NB4 cells. The differentiation of leukemic cells was stimulated by using ATRA. The protein profile increase was observed afterwards.

The proteomics in NB4 cells was studied when induction of granulocytic differentiation with following growth inhibition was induced with all-trans retinoic acid and HDAC inhibitor BML-210. The alterations in protein expression at various times of treatment (2, 4, 8, 24, 48, 72, 96 h) were noticed (Fig. 4.5) (Borutinskaite et al. 2005).

Fig. 4.5
figure 5

Expression of proteins in NB4 cells treated with all-trans retinoic acid (ATRA) and histone deacetylase inhibitor BML-210. Total soluble (A) and insoluble (B) protein fractions were dissociated from control and treated with 1μM ATRA and 10μM BML-210 separately or in combination (1μM ATRA and 5μM BML-210) NB4 cells during the indicated period of time. Isolated proteins were fractionated by SDS-PAGE on an 8–16% acrylamide gradient gel and then stained with brilliant Blue G-Colloidal. Migration of the molecular size marker proteins is shown to the left (kDa values). The position of bands that were cut to MALDI-MS analysis is designated by arrows and numbers in the images. According Borutinskaite et al. (2005)

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Our studies demonstrated potential of combination of HDACi BML-210 with all-trans retinoic acid for differentiation induction in leukemic NB4 cells: the largest amount of differentiated cells after ATRA treatment was observed at 72–96 h (70–80%), and the combined treatment considerably improves granulocytic differentiation of NB4 cells (up to 90%).

Total soluble and less soluble proteins were isolated, fractionated, and identified by mass spectrometry. The less soluble (or insoluble) protein fraction of NB4 cells showed biggest differences in expression of proteins during treatments with ATRA and HDAC inhibitor BML-210. Using mass spectrometry, there were inspected proteins of cells with cancer and membrane or membrane-associated proteins. We identified (Figs. 4.6 and 4.7) such proteins like caspase-7, nesprin-2, RAB, USP6NL protein, Vav-3, ADAMTS-19, lipoprotein receptor-related protein, ADAM-17, actin-binding LIM protein 1, caldesmon (CDM) splice isoform 3, caldesmon, calpain 10, dystrobrevin-β, vimentin, ADAMTS-17, GEF-H1, calpain 9, calpain 1, actin, F-actin capping protein alfa-subunit (Borutinskaite et al. 2005). All functions of identified proteins are listed in Table 4.3.

Fig. 4.6
figure 6

Expression of proteins in NB4 cells treated with all-trans retinoic acid (ATRA) and histone deacetylase inhibitor BML-210. Represents the identified protein network. According Borutinskaite et al. (2005)

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Fig. 4.7
figure 7

Expression of proteins in NB4 cells treated with all-trans retinoic acid (ATRA) and histone deacetylase inhibitor BML-210. Represents the involvement of identified proteins into large scale of proteins network inclusive different cellular processes like apoptosis and cytoskeleton reorganization. According Borutinskaite et al. (2005)

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Table 4.3 The characteristics and functions of identified proteins in NB4 cells treated with ATRA and inhibitor BML-210. Proteins were estimated according to PeptIdent program and EXPASY database, experimental (gel position) and calculated Mw and pI, MOWSE SCORE and sequence coverage (PeptIdent) for each protein were represented: 1st column–-number of band in gels (Fig. 4.5B); 2nd column–-the protein Mw and pI, calculated from the amino acid sequence in the ExPASy database; 3rd column–-the name of identified proteins; 4th column–-general function(s) of a protein in Uniprot database. According Borutinskaite et al. (2005)
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  1. a https://clinicalepigeneticsjournal.biomedcentral.com/articles/10.1186/s13148-015-0158-1

The high expression of small GTPase, Rab2B was detected in insoluble fraction of untreated and treated cells for 8 h, and their decreased level was observed after 24 h of treatment. This indicate that Rab2B expression may be related with granulocytic differentiation or apoptosis of NB4 cells. It was shown that the main function of Rab2B protein is vesicle transport and membrane fusion regulation. Actin (ACTB) and actin-associated proteins (nesprin-2, actin-binding LIM protein, vimentin and caldesmon) were found in insoluble fraction of NB4 cells. These proteins are important for cytoskeletal reorganization during cell growth and differentiation/apoptosis. The higher expression of aforementioned proteins was in soluble fraction compared with insoluble fraction of NB4 cells.

Also we found two proteins (ADAM17 and ARHG2) that were upregulated both in control cells and induced to differentiation with following apoptosis by ATRA and HDAC inhibitor BML-210. It was showed that ADAMs have adhesive and proteolytic characteristics which result in regulation of such processes as cellular fate, proliferation, and growth. ADAM-17 is known as the one of the best investigated ADAM enzymes and is extensively expressed in different tissues including the brain, kidney, heart, and skeletal muscle. ADAM-17 is an important player in inflammation through TNF-alpha pathway, in the development of the nervous system, as it activates the neural cell adhesion, in carcinogenesis due to the enzyme which sheds growth factors required for tumor progression and growth. Another protein in the same band after fractionation in SDS/PAGE is ARHG2 (GEF-H1) that is involved in cellular processes, e.g., cell motility, cell-cycle regulation, polarization, epithelial barrier permeability, and cancer (Kashyap et al. 2019).

The other exciting protein that we identified—DTNB (dystrobrevin-β) protein in insoluble fraction. It was shown that dystrobrevin-βinteracts with dystrophin short form DP71 and syntrophins SNTG1 and SNTG2. It was demonstrated that DTNB-knockout mice were healthy and had no abnormality, however the level of DTNB binding proteins were reduced. This leads to the conclussion that DTNB can be an anchor or scaffold for dystrophin and syntrophins/other associating proteins at the basal membranes of kidney and liver.

Also we indentified the two proteins (calpain 1 and calpain 9) that belong to the calcium ion-dependent papain-like protease (Calpain) family. It is known that these proteins are critical mediators of the action of calcium and are tightly regulated by an endogenous inhibitor, calpastatin. It was showed that calpain can regulate the dystrophin, heat shock protein (HSP90), actin, caspases, phospholipase A/B/C, and other protein functions (Patterson et al. 2011).

In conclusion, in this study we have found differently expressed proteins in the soluble and less soluble (insoluble) fraction of the NB4 cells. These changes in protein expression can be associated with early changes in chromatin structure during the process of differentiation or apoptosis after the treatment with HDAC inhibitor BML-210 and ATRA. Identification of new proteins involved in the differentiation process can be helpful in finding new therapies for APL treatment (Borutinskaite et al. 2005).

3 Proteome Profile in APL Cells Induced with Histone Deacetylase Inhibitor BML-210

The study was dedicated to identify proteins whose expression changed after NB4 cells treatment with HDAC inhibitor BML-210 (Borutinskaite and Navakauskiene 2015). BML-210 at concentration up to 20μM was the reason of anti-proliferative and slight cytotoxic effects on NB4 cells in a dose- and time-dependent manner with accumulation of cells in G0/G1 cycle phase. We found out that after 20μM BML-210 treatment apoptotic population in cell culture was about 90% after 48 h of treatment. Proliferating (control) NB4 cells and cells treated with 20μM BML-210 for 24 h were lysed and soluble cell proteins were fractionated in 2DE gels and analysed by mass spectrometry (Fig. 4.8). Description of proteins identified by mass spectrometry in NB4 cells treated with HDACi BML-210 and fractionated by 2DE is presented in Appendix D (Borutinskaite and Navakauskiene 2015).

Fig. 4.8
figure 8

Proteomic analysis of proteins after NB4 cell treatment with HDAC inhibitor BML-210. Proteins from untreated NB4 cells and cells treated with 20μM BML-210 for 24 h fractionated in 2-DE system and visualized by Coomasie staining. Identified proteins are indicated in the Table 4.4. Migration of the molecular size marker proteins is shown at the center (kDa)

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We observed variations in protein expression profile while treating with BML-210. Computational analysis of protein expression changes are presented as fold change in comparisson with control cells (Table 4.4). The expression of proteins whose ratio G2/G1>0 is increased in treated (G2) cells more than in untreated (G1).

Table 4.4 Characterization of proteins identified in control and treated with HDAC inhibitor BML-210 NB4 cells. Computational analysis of protein expression changes are presented as fold change in comparisson with control cells (Ratio G2/G1). The expression of proteins whose Ratio G2/G1>0 is increased in treated (G2) cells than in untreated (G1)
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In total, 35 proteins were identified: 16 were down-regulated such as endoplasmin, ENPL; heat shock 84 kDa, HSP90B; 14-3-3 protein, 1433F; proliferating cell nuclear antigen, PCNA; calreticulin, CALR; and 19 proteins up-regulated (such as chloride intracellular channel protein 1, CLIC1; thioredoxin domain-containing protein 12, TXD12; lactoylglutathione lyase, LGUL) after treatment with BML-210. It is known that α/β-tubulin, β-actin, cofilin-1, myosin regulatory light chain 12A, tropomyosin, and gelsolin are involved in cell growth and/or homeostasis regulation. Other identified proteins participate in metabolism processes (disulphide isomerase, α-enolase), in protein folding (the heat shock proteins like endoplasmin, HSP90B, GRP75, GRP78 and CH60). Proteins of one more group, such as PCNA, nucleophosmin, 14-3-3 protein, prohibitin, guanine nucleotide-binding protein subunit α-11, chloride intracellular channel protein 1, and nucleoside diphosphate kinase A are in charge of signal transduction, apoptosis, cell differentiation and communication processes (Fig. 4.9). One of the proteins with detected expression changes is HSP90. It was observed that HDAC inhibitors can provoke hyperacetylation of HSP90 and its inactivation, resulting to the degradation of proteins that need the chaperone function of HSP90 (including some oncoproteins) (Bali et al. 2005). Furthermore it was demonstrated that HSP90 inhibition correlated with growth arrest followed by differentiation and apoptosis (Nawarak et al. 2009; Heller et al. 2009). Calreticulin function was investigated by the other group (Sheng et al. 2014). They have found that high expression of calreticulin was positively related with tumor and metastasis and that calreticulin regulated cell proliferation, migration and invasion of pancreatic cancer cells in a MEK/ERK pathway dependent manner. 14-3-3 proteins have a significant role in a broad spectrum of vital regulatory processes, especially regarding signal transduction, apoptosis, cell cycle progression, and DNA replication. It was shown that treatment of leukemic cells with HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) provoked cofilin phosphorylation, rised of the vimentin and paxillin expression, also lowered the expression of stathmin (Grebenova et al. 2012).

Fig. 4.9
figure 9

Involvement of identified proteins in control and treated with HDAC inhibitor BML-210 NB4 cells into the cellular processes network

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4 HDAC Inhibitor Belinostat Modulates Protein Profile in NB4 Cells

One of the most promising agents acting as epigenetic drug such as HDAC inhibitors—belinostat. It is an innovative and potent hydroxamate-type HDAC inhibitor that demonstrates potency to block enzymatic activity of 1-st and 2-nd class of HDACs (Khan et al. 2008). Belinostat exerts its anti-deacetylase action via its hydroxamic acid moiety binding to zinc ion in enzymes catalytic domain and blocking substrate access (Witter et al. 2007). In the others (Gravina et al. 2012) studies and we observed (Savickiene et al. 2014; Valiuliene et al. 2015; Valiulienė et al. 2016; Valiuliene et al. 2017; Vitkeviciene et al. 2019) its activity resulting in cell cycle arrest, apoptosis initiation, and cell proliferation inhibition. In addition, our group indicated that belinostat promotes APL granulocytic differentiation AML (Savickiene et al. 2014). Regarding belinostat’s activity on APL cells, we performed proteomic analysis after chromatin immunoprecipitation with hyperacetylated histone H4 (H4hyperAc) and established proteins being in the active complex with H4hyperAc (Valiuliene et al. 2015). Totaly we identified 68 proteins. We found that in untreated NB4 cells hyperacetylated histone H4 (H4hyperAc) associated with 45 different proteins (Table 4.5).

Table 4.5 Summary of identified NB4 cells proteins identified in complexes with hyperacetylated histone H4 in control (C) and belinostat (Bel) treated cells. According Valiuliene et al. (2015)
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The network of proteins related with hyperacetylated histone H4 in control NB4 cells is presented in Fig. 4.10 and proteins associated with hyperacetylated histone H4 in NB4 cells treated with HDAC inhibitor belinostat is presented in Fig. 4.11. Only in control cells, H4hyperAc was found related with proteins that participate in DNA replication (POLA2), transcription (GCOM1, POLR2M, NELFE, NCL), translation (RPL7) and RNA splising (SCNM1). Also it was estimated that H4hyperAc is associated with proto-oncogene SPECC1, regulator of apoptosis ADAMTSL4, together with proteins involved in various signaling cascades: NFκB, JAK2/STAT4, Ras and Hedgehog signal transduction pathways. Noteworthy, it was observed that H4hyperAc in control NB4 cells is associated with nucleophosmin (NPM), protein who is responsible for regulation of tumor suppressors TP53/p53 and ARF and is shown to be overexpressed in actively proliferating cells, like different cancer and stem cells (Lim and Wang 2006). Somewhat lesser extent of NPM was observed in complexes with H4hyperAc after treatment with 2μM belinostat.

Fig. 4.10
figure 10

Proteins identified in association with hyperacetylated histone H4 in control NB4 cells. Untreated NB4 cells were subjected to ChIP—MS analysis. Association network of identified proteins was studied and represented using STRING database (http://string.embl.de). According Valiuliene et al. (2015)

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Fig. 4.11
figure 11

Proteins identified in association with hyperacetylated histone H4 in belinostat treated NB4 cells. 2μM belinostat treated NB4 cells were subjected to ChIP—MS analysis. Association network of identified proteins was studied and represented using STRING database (http://string.embl.de). According Valiuliene et al. (2015)

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After 6 h treatment with 2μM belinostat (Table 4.5, Fig. 4.11) it was estimated that H4hyperAc is associated with proteins that are pro-apoptotic and required for apoptotic response (S100A9, S100A8, LGALS7, GOLGA3, PPT1). Tumor suppressor APC was indicated in immunoprecipitated complexes, too. It should be noted that H4hyperAc is as well related with proteins that participate in the defense against oxidative stress (TXNRD2) and access of all-trans retinoic acid to the nuclear retinoic acid receptors regulation (CRABP1). We also found that after 6 h treatment of NB4 cells with 2μM belinostat hyperacetylated histone H4 was no longer associated with proteins involved in gene transcription and/or translation. Nevereless it was found to associate with proteins, that are usually detected in cytosolic fraction as components of neutrophil extracellular traps (NETs). It is known that NETs are in association with DNA specific proteins, such as histones and antimicrobial proteins, that form an extracellular mesh able to trap and kill pathogens and they are released during a cell death that depends on ROS produced by the NADPH-oxidase complex (Valiuliene et al. 2015). After NB4 cells treatment with belinostat we identified calprotectin (S100A8 and S100A9) associated with hyperacetylted histone H4. Calprotectin is essential for the neutrophilic NADPH oxidase activation. Calprotectin is a protein complex composed of two calcium-binding proteins (S100A8 and S100A9) that are abundantly found in neutrophils cytosolic fraction and have shown to have apoptosis inducing activity. We also found a probable serine protease TMPRSS11A associated with hyperacetylated histone H4, which is in agreement with data, showing that NETs contain serine proteases, as they may execute antimicrobial functions in those structures. Taking all together, we assume that belinostat has cell death inducing activity in some manner may relate to NETs formation. Although, it is already known that belinostat triggers apoptosis in myeloid cells (Savickiene et al. 2014), not NETosis (cell death when NETs are released), the possibility that belinostat intervenes in NETs formation may not be rejected completely.

Regarding effect of belinostat on cell growth, differentiation, gene and protein expression, and on epigenetic modifications, which was identified by us, belinostat could have a potential value in APL therapy.

5 Proteomic Maps of Leukemia Cells Induced to Granulocytic Differentiation and Apoptosis

In our study published in 2004 year (Navakauskiene et al. 2004b) we tried to evaluate protein level changes in proliferating HL-60 cells compared to cells induced for apoptosis using etoposide/Z-VAD(OH)-FMK. Programmed cell death has a significant role in the development and maintenance of homeostasis within all cells. It is widely acknowledged that the physiological form of cell death in neutrophils is apoptosis. For quite a long time it was considered that aged neutrophils die within a short period of time by spontaneous apoptosis under healthy conditions, for the sake of maintaining of homeostatic cell numbers (Geering and Simon 2011). The aim of our study was to evaluate apoptosis—associated protein patterns in HL-60 cells that were provoked to apoptosis with etoposide and also with or without the presence of the broad caspase and apoptosis inhibitor Z-VAD(OH)-FMK. Cellular cytosolic and nuclear proteins were fractionated by 2DE and changes in protein expression were detected (Fig. 4.12). In our studies that are presented in following chapters (Treigyte et al. 2000a,b; Navakauskiene et al. 2012, 2004a), we have shown that the synthesis of new proteins and protein modification takes place when HL-60 cells are induced into granulocytic differentiation. We found some quantitative and qualitative differences in the cytosolic and nuclear protein patterns of control cells, cells treated with etoposide alone or together with Z-VAD(OH)-FMK for 6 h (Fig. 4.12). Analyses of cytosolic protein reference maps of control and drug-induced HL-60 cells revealed some new protein spots appeared, whereas the relative amount of others proteins markedly diminished or vanished after induction of apoptosis. The number of both cytosolic and nuclear polypeptides was different in HL-60 cells treated for 6 h with etoposide (60% apoptotic cells), by comparison with the control or 6 h etoposide/Z-VAD(OH)-FMK-treated cells (5–7% apoptotic cells) (Fig. 4.12). Proteins newly synthesized in HL-60 cells treated for apoptosis and absent in pan-caspase inhibitor-treated cells should have an apoptotic origin. Such proteins (marked by arrows in Fig. 4.12, 68μM etoposide, CytP) had mostly an acidic pI 4.5–5.1, with molecular masses between 25 kDa and 150 kDa.

Fig. 4.12
figure 12

Two-Dimensional electrophoretic maps of cytosolic and nuclear proteins of proliferating and induced to apoptosis HL-60 cells. Cytosolic (CytP) and nuclear (NuP) proteins of HL-60 cells treated for 6 h with 68μM of etoposide alone or in the presence of 25μM pan-caspase inhibitor Z-VAD(OH)-FMK were fractionated by 2D electrophoresis. The gels were stained by silver. Arrows show the position of proteins of apoptotic cell origin, appearing in cells treated with etoposide, but absent in broad caspase inhibitor-treated cells. According Navakauskiene et al. (2004b), License No 4796010620810

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Some proteins with acidic and neutral pI 4.5–7.0, which were detected in the cytoplasm both etoposide and etoposide/Z-VAD(OH)-FMK treated cells were translocated exclusively into the nucleus of etoposide treated cells (Fig. 4.12, NuP, marked by arrows). These proteins could be involved in regulation of genes required in apoptosis process. We implied that variations of protein synthesis and protein modifications which were observed in differentiating HL-60 cells reflect the formation of the differentiated granulocyte phenotype and the apoptotic process. Our current research demonstrates that some proteins are highly upregulated in the cytoplasm and afterwards accumulated in the nuclei after treatment of HL-60 cells with etoposide. These proteins could participate in regulation of genes needed for apoptosis. Proteins estimated both after treatment with etoposide alone and together with pan-caspase inhibitor Z-VAD(OH)-FMK show that they do not represent a downstream event of caspase activation. We suggest that these proteins may be required for the regulation of apoptosis.

Taken together, our present study shows that some proteins are strongly upregulated in the cytoplasm and subsequently accumulated in the nuclei after treatment of HL-60 cells with etoposide. These proteins could be involved in regulation of genes required for apoptosis. Proteins identified both after treatment with etoposide alone and together with pan-caspase inhibitor Z-VAD(OH)-FMK indicate that they do not represent a downstream event of caspase activation. Taken as a whole, obtained during our research results demonstrate that important alterations in both cytosolic and nuclear proteins take place in a highly regulated manner that leads to programmed cell death. Selective activation of some caspases is required to provoke apoptosis in HL-60 cells.