Cell Cycle-dependent Expression Research Articles

HMYH cell cycle-dependent expression, subcellular localization and association with replication foci: evidence suggesting replication-coupled repair of adenine:8-oxoguanine mispairs.

The human MutY homolog, hMYH, is an adenine-specific DNA glycosylase that removes adenines or 2-hydroxyadenines mispaired with guanines or 8-oxoguanines. In order to prevent mutations, this activity must be directed to the newly synthesized strand and not the template strand during DNA synthesis. The subcellular localization and expression of hMYH has been studied in serum-stimulated, proliferating MRC5 cells. Using specific antibodies, we demonstrate that endogenous hMYH protein localized both to nuclei and mitochondria. hMYH in the nuclei is distinctly distributed and co-localized with BrdU at replication foci and with proliferating cell nuclear antigen (PCNA). The levels of hMYH in the nucleus increased 3- to 4-fold during progression of the cell cycle and reached maximum levels in S phase compared to early G(1). Similar results were obtained for PCNA, while there were no notable changes in expression of 8-oxoguanine glycosylase or the human MutT homolog, MTH1, throughout the cell cycle. The cell cycle-dependent expression and localization of hMYH at sites of DNA replication suggest a role for this glycosylase in immediate post-replication DNA base excision repair.

Variations of components of the plasminogen activation system with the cell cycle in benign prostate tissue and prostate cancer.

Components of the fibrinolytic system are involved in tumor cell invasion and metastasis. Previous investigations suggested a cell cycle-dependent expression of urokinase-type plasminogen activator (u-PA) in epithelial cells. In order to determine a correlation of cell cycle phases with the fibrinolytic system, we investigated the expression of u-PA, tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor type 1 (PAI-1) in normal and tumor-containing prostate extracts and analyzed a possible relationship with flow cytometry-determined proliferative activity of the samples. Cell cycle phases were correlated with fibrinolytic parameters in prostate tissue. Samples were obtained from patients undergoing radical prostatectomy for prostate cancer and separated into two portions for DNA analysis and the detection of u-PA, t-PA, and PAI-1. Flow cytometric analysis was performed according to the Vindelov technique. The concentrations of u-PA, t-PA, and PAI-1 were determined from tissue extracts after homogenization by an enzyme-linked immunosorbent assay (ELISA) technique. Correlations of u-PA and t-PA expression with the frequency of G0/G1, S, G2M, S-phase fraction (SPF), and proliferation index (PI) for normal prostate and prostate cancer revealed no significant correlation. The only significant finding was observed in normal tissue revealing a positive correlation between PAI-1 expression and G0/G1 and a negative correlation with S-phase, SPF, and PI. No dependence of PAI-1 expression on different cell phases was found in prostate cancer. Furthermore, no significant correlation of u-PA, t-PA, and PAI-1 with cell cycles in organ-confined (<pT3a) and disseminated (> or = pT3a) tumors was found. No significant correlation in prostate cancer of components of the fibrinolytic system differentiated according to tumor grade or perineural tumor infiltration and cell cycle analysis was found. Only in highly differentiated G1 (Gleason 2-4) cancer, u-PA had a significant positive correlation with G2M-phase. Absence of a correlation between levels of components of the fibrinolytic system and cell cycle phases suggests that the reported association between increases of some of these components and aggressive biological behavior of prostate cancer is secondary to non-cell cycle-related mechanisms.

The human cytomegalovirus immediate early 2 protein dissociates cellular DNA synthesis from cyclin-dependent kinase activation.

Passage through the restriction point late in G1 normally commits cells to replicate their DNA. Here we show that the previously reported cell cycle block mediated by the human cytomegalovirus (HCMV) immediate early 2 (IE2) protein uncouples this association. First, IE2 expression leads to elevated levels of cyclin E-associated kinase activity via transcriptional activation of the cyclin E gene. This contributes to post-restriction point characteristics of IE2-expressing cells. Then these cells fail to undergo substantial DNA replication although they have entered S phase, and the induction of DNA replication observed after overexpression of cyclin E or D can be antagonized by IE2 without impinging on cyclin-associated kinase activities. These data suggest that IE2 secures restriction-point transition of cells before it stops them from replicating their genome. Our results fit well with HCMV physiology and support the view that IE2 is part of a viral activity which, on the one hand, promotes cell cycle-dependent expression of cellular replication factors but, on the other hand, disallows competitive cellular DNA synthesis.

YY1 as a Regulator of Replication-dependent Hamster Histone H3.2 Promoter and an Interactive Partner of AP-2

In analyzing cis-regulatory elements important for cell cycle control of the replication-dependent hamster histone H3.2 gene, we discovered a binding site for the transcription factor YY1 embedded within GC-rich sequences between the two tandem CCAAT repeats proximal to the TATA element. Base mutations that specifically eliminated YY1 binding resulted in suppression of the S phase induction of the H3.2 promoter. In addition, we discovered that YY1 is an interactive partner of AP-2, which also binds the H3.2 promoter and regulates its cell cycle-dependent expression. The critical domains for YY1 and AP-2A interaction are mapped, revealing that the N-terminal portion of YY1 (amino acids 1-300) and the DNA-binding/dimerization region of AP-2A are required. Our results suggest that YY1, acting as a transcription factor binding to its site on the promoter, or through protein-protein interaction with AP-2, may be part of a regulatory network including key cell cycle regulators such as c-Myc and Rb in controlling growth- and differentiation-regulated gene expression.

Investigation on cell proliferation with a new antibody against thymidine kinase 1.

The cytosolic thymidine kinase 1 (TK1) is one of the enzymes involved in DNA replication. Based on biochemical studies, TK1 is activated at late G1 of cell cycle, and its activity correlates with the cell proliferation. We have developed a polyclonal anti‐TK1 antibody against a synthetic peptide from the C‐terminus of human TK1. Using this antibody, here we demonstrate the exclusive location of TK1 in the cytoplasm of cells. Cell cycle dependent TK1 expression was studied by simultaneous fluorescence staining for TK1 and bromodeoxyuridine, by using elutriated cells, and by quantitation of the amount TK1 in relation to the cellular DNA content. TK1, which was strongly expressed in the cells in S+G2 period, raised at late G1 and decreased during mitosis. The amount of TK1 increased three folds from late G1 to G2. TK1 positive cells were demonstrated in areas of proliferation activity of various normal and malignant tissues. The new anti‐TK1 antibody works in archival specimens and is a specific marker of cell proliferation.

Overexpression of E2F-1 leads to cytokine-independent proliferation and survival in the hematopoietic cell line BaF-B03

AbstractCytokine receptors activate signals that regulate the transcription factor E2F-1, which then coordinates the expression of genes essential for DNA synthesis and cell cycle progression. Overexpression of E2F-1 most often induces S-phase entry followed by apoptosis, but in some cell types it leads to continuous proliferation and transformation. Here, it is shown that constitutive expression of E2F-1 promotes cytokine-independent proliferation in the murine pro-B cell line BaF-B03. There was no enhancement of apoptosis following cytokine withdrawal in these cells, despite the presence of intact p53-dependent apoptotic pathways. Notwithstanding the continuous presence of E2F-1, the cell cycle–dependent expression of cyclin A, cyclin B1, cyclin D1, cyclin E, and proliferating-cell nuclear antigen was restored with a pattern equivalent to that associated with cytokine stimulation. These findings provide evidence that, in the absence of cytokine, constitutive expression of E2F-1 can promote cell cycle progression and prevent apoptosis.

Cell cycle-dependent expression of CXC chemokine receptor 3 by endothelial cells mediates angiostatic activity.

Endothelial cell receptors for the angiostatic chemokines IFN-gamma-inducible protein of 10 kDa (IP-10) and monokine induced by IFN-gamma (Mig) have not yet been identified, and the mechanisms responsible for the effects of these chemokines on angiogenesis are still unclear. IP-10 and Mig share a common functional receptor on activated T lymphocytes, named CXC chemokine receptor 3 (CXCR3). Using in situ hybridization and immunohistochemistry, we show that CXCR3 is expressed by a small percentage of microvascular endothelial cells in several human normal and pathological tissues. Primary cultures of human microvascular endothelial cells (HMVECs) likewise express CXCR3, although this expression is limited to the S/G2-M phase of their cell cycle. Both IP-10 and Mig, as well as the IFN-gamma-inducible T-cell alpha chemoattractant (I-TAC), which all share high-affinity binding for CXCR3, block HMVEC proliferation in vitro, an effect that can be inhibited by an anti-CXCR3 antibody. These data provide definitive evidence of CXCR3 expression by HMVEC and open new avenues for therapeutic interventions in all conditions in which an angiostatic effect may be beneficial.

Chaperones in cell cycle regulation and mitogenic signal transduction: a review.

Chaperones/heat shock proteins (HSPs) of the HSP90 and HSP70 families show elevated levels in proliferating mammalian cells and a cell cycle-dependent expression. They transiently associate with key molecules of the cell cycle control system such as Cdk4, Wee-1, pRb, p53, p27/Kip1 and are involved in the nuclear localization of regulatory proteins. They also associate with viral oncoproteins such as SV40 super T, large T and small t antigen, polyoma large and middle S antigen and EpsteinBarr virus nuclear antigen. This association is based on a J-domain in the viral proteins and may assist their targeting to the pRb/E2F complex. Small HSPs and their state of phosphorylation and oligomerization also seem to be involved in proliferation and differentiation. Chaperones/HSPs thus play important roles within cell cycle processes. Their exact functioning, however, is still a matter of discussion. HSP90 in particular, but also HSP70 and other chaperones associate with proteins of the mitogen-activated signal cascade, particularly with the Src kinase, with tyrosine receptor kinases, with Raf and the MAP-kinase activating kinase (MEK). This apparently serves the folding and translocation of these proteins, but possibly also the formation of large immobilized complexes of signal transducing molecules (scaffolding function).

The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner.

Survivin, a human inhibitor of apoptosis protein (IAP), plays an important role in both cell cycle regulation and inhibition of apoptosis. Survivin is expressed in cells during the G(2)/M phase of the cell cycle, followed by rapid decline of both mRNA and protein levels at the G(1) phase. It has been suggested that cell cycle-dependent expression of survivin is regulated at the transcriptional level. In this study we demonstrate involvement of the ubiquitin-proteasome pathway in post-translational regulation of survivin. Survivin is a short-lived protein with a half-life of about 30 minutes and proteasome inhibitors greatly stabilise survivin in vivo. Expression of the survivin gene under the control of the CMV promoter cannot block cell cycle-dependent degradation of the protein. Proteasome inhibitors can block survivin degradation during the G(1) phase and polyubiquitinated derivatives can be detected in vivo. Mutation of critical amino acid residues within the baculovirus IAP repeat (BIR) domain or truncation of the N terminus or the C terminus sensitises survivin to proteasome degradation. Together, these results indicate that the ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner and structural changes greatly destabilise the survivin protein.

G(1) phase-dependent expression of bcl-2 mRNA and protein correlates with chemoresistance of human cancer cells.

Recent experiments suggest an interconnection between cell proliferation and programmed cell death (apoptosis), although the detailed molecular mechanisms remain unclear. We have hypothesized that expression of some apoptosis regulators is cell cycle-dependent, which in turn influences tumor cell chemosensitivity in a cell cycle-dependent fashion. To test these hypotheses, we synchronized human leukemia Jurkat T, Neo (using aphidicolin), breast cancer MCF-7, normal fibroblast, and simian virus 40-transformed cells (by aphidicolin or serum starvation), and measured levels of several Bcl-2 family proteins. The highest expression of Bcl-2 protein was found in the G(1) phase of all the five cell lines tested. In contrast, levels of Bax protein remained relatively unchanged in four of the cell lines, and levels of Bcl-X(L), Bcl-X(S), and Bak proteins showed little or no cell cycle-dependent changes in Jurkat T cells. Similar to the changes in Bcl-2 protein levels, its mRNA expression was also G(1) phase-specific, whereas the level of a Bcl-2 cleavage activity remained constitutive. When treated with an anticancer drug (etoposide or cisplatin) or the kinase inhibitor staurosporin, the cells containing a high G(1) population and a high Bcl-2 protein level were much more resistant to the induced apoptosis than the cells containing a high S phase population and a low Bcl-2 protein level. Constitutive overexpression of Bcl-2 protein in Jurkat T cells completely blocked the S phase-associated sensitivity to these apoptosis stimuli. The cell cycle-dependent Bcl-2 protein expression seems to contribute to the regulation of chemosensitivity and apoptotic commitment of human tumor cells.

Regulation of tumor cell apoptotic sensitivity during the cell cycle (Review).

Understanding how current chemotherapeutic modalities induce apoptosis is critical to designing better anti-cancer agents. This review is concerned with how pharmacological agents induce tumor cell apoptosis in a cell cycle-dependent manner. Recent experiments demonstrate that expression of several apoptotic regulatory proteins (such as Bcl-2, Bax, p53, and Survivin) are differentially regulated according to the phases of the cell cycle. This cell cycle-dependent regulation in turn contributes to increased drug sensitivity in different phases of the cell cycle. It is therefore likely that the cell cycle-dependent expression of cell death proteins plays a role in regulating chemosensitivity and apoptotic commitment of human tumor cells.

Involvement of retinoblastoma protein and HBP1 in histone H1(0) gene expression.

The histone H1(0)-encoding gene is expressed in vertebrates in differentiating cells during the arrest of proliferation. In the H1(0) promoter, a specific regulatory element, which we named the H4 box, exhibits features which implicate a role in mediating H1(0) gene expression in response to both differentiation and cell cycle control signals. For instance, within the linker histone gene family, the H4 box is found only in the promoters of differentiation-associated subtypes, suggesting that it is specifically involved in differentiation-dependent expression of these genes. In addition, an element nearly identical to the H4 box is conserved in the promoters of histone H4-encoding genes and is known to be involved in their cell cycle-dependent expression. The transcription factors interacting with the H1(0) H4 box were therefore expected to link differentiation-dependent expression of H1(0) to the cell cycle control machinery. The aim of this work was to identify such transcription factors and to obtain information concerning the regulatory pathway involved. Interestingly, our cloning strategy led to the isolation of a retinoblastoma protein (RB) partner known as HBP1. HBP1, a high-mobility group box transcription factor, interacted specifically with the H1(0) H4 box and moreover was expressed in a differentiation-dependent manner. We also showed that the HBP1-encoding gene is able to produce different forms of HBP1. Finally, we demonstrated that both HBP1 and RB were involved in the activation of H1(0) gene expression. We therefore propose that HBP1 mediates a link between the cell cycle control machinery and cell differentiation signals. Through modulating the expression of specific chromatin-associated proteins such as histone H1(0), HBP1 plays a vital role in chromatin remodeling events during the arrest of cell proliferation in differentiating cells.

Promoter structure and cell cycle dependent expression of the human methylpurine-DNA glycosylase gene

The methylpurine-DNA glycosylase (MPG) gene coding for human 3-methyladenine (3-meAde)-DNA glycosylase functions in the first step of base excision repair (BER) to remove numerous damaged bases including 3-meGua, ethenoadenine, and hypoxanthine (Hx) in addition to 3-meAde. In this report, we identify the length of the minimal MPG promoter region, demonstrate the involvement of several transcription factors in expression of the MPG gene, and determine the point at which transcription initiates. We also demonstrate that control of MPG expression is linked to MPG activity. To initiate studies on how the MPG functions with the ensemble of BER genes to effect repair, we have investigated the cell cycle control of MPG and other BER genes in normal human cells. Steady-state mRNA levels of MPG, human Nth homologue (NTH), and uracil-DNA glycosylase (UDG), DNA glycosylases, and human AP site-specific endonuclease (APE), an endonuclease incising DNA at abasic sites, are cell cycle dependent. In contrast, expression levels of genes coding for human 8-oxoguanine-DNA glycosylase (OGG1) and TDG DNA glycosylases, and O6-methylguanine-DNA methyltransferase (MGMT) gene, and the RPA4 subunit gene do not vary with cell cycle. These observed cell cycle dependent differences might reflect distinct roles of individual BER proteins in mutation avoidance.

Cell cycle-dependent regulation of TIAP/m-survivin expression

TIAP, a murine homologue of human survivin, is a member of the inhibitor of apoptosis (IAP) family and is specifically expressed at G2/M phase of the cell cycle. To elucidate regulatory mechanisms of the cycle-dependent expression, we have analyzed the promoter region of TIAP/mouse survivin (m-survivin). The 5′-flanking region of the TIAP/m-survivin gene contained a TATA-less promoter, two AP2 sites, three NF-kB sites, one Sp1 site, many cell cycle-dependent elements (CDEs) and one cell cycle gene homology region (CHR). Primer extension and 5′-rapid amplification of cDNA ends identified one transcription start site at position −100 upstream of the ATG start site (+1). TIAP/m-survivin promoter–luciferase analysis identified a minimal promoter region within the most proximal −271 bp upstream of the ATG start site, and the region between −410 and −272 was critical for the enhancer activity. The combination between the CHR at −51 and the CDE at −57 is also essential for the cell cycle-dependent expression. Mutation of the CDE/CHR element and the enhancer elements may cause disordered expression of TIAP/m-survivin to affect cell survival and oncogenesis.

Cell cycle-dependent expression of mammalian E2-C regulated by the anaphase-promoting complex/cyclosome.

Progression through mitosis requires the precisely timed ubiquitin-dependent degradation of specific substrates. E2-C is a ubiquitin-conjugating enzyme that plays a critical role with anaphase-promoting complex/cyclosome (APC/C) in progression of and exit from M phase. Here we report that mammalian E2-C is expressed in late G(2)/M phase and is degraded as cells exit from M phase. The mammalian E2-C shows an autoubiquitinating activity leading to covalent conjugation to itself with several ubiquitins. The ubiquitination of E2-C is strongly enhanced by APC/C, resulting in the formation of a polyubiquitin chain. The polyubiquitination of mammalian E2-C occurs only when cells exit from M phase. Furthermore, mammalian E2-C contains two putative destruction boxes that are believed to act as recognition motifs for APC/C. The mutation of this motif reduced the polyubiquitination of mammalian E2-C, resulting in its stabilization. These results suggest that mammalian E2-C is itself a substrate of the APC/C-dependent proteolysis machinery, and that the periodic expression of mammalian E2-C may be a novel autoregulatory system for the control of the APC/C activity and its substrate specificity.

G1 phase-dependent bcl-2 expression correlates with chemoresistance of human cancer cells1

Recent experiments suggest an interconnection between cell proliferation and programmed cell death (apoptosis) although the detailed molecular mechanisms remain unclear. We hypothesized that (i) some apoptosis regulators are periodically expressed during the cell cycle progression, and (ii) if so, cellular response to chemotherapeutic agents should be cell cycle-dependent. To test the first hypothesis, we synchronized multiple human tumor and transformed cell lines and measured protein levels of the cell death inhibitor Bcl-2 and inducer Bax in different phases of the cell cycle. In all the five tested cell lines, levels of Bcl-2 protein were very high in the G1 phase, dramatically decreased in S, G2 and M phase, and increased again in the G1 phase of the next cycle. In contrast, levels of Bax protein remained relatively unchanged in most of the cell lines although S phase-associated Bax expression was detected in human breast carcinoma MCF-7 cells. Similar to its protein levels, Bcl-2 mRNA expression was also G1 phase-specific, whereas the level of the Bcl-2 cleavage activity remained constitutive, indicating that cell cycle-dependent regulation of Bcl-2 is on its mRNA level. To test the second hypothesis, aliquots of the synchronized human tumor or transformed cells were treated with the anticancer drug etoposide or cisplatin, followed by measurement of apoptotic cell death. We demonstrated that the cells containing a high G1 population and a high Bcl-2 protein level were much more resistant to chemotherapy-induced apoptosis than the cells containing a high S phase population and a low Bcl-2 protein level. Furthermore, constitutive overexpression of Bcl-2 protein in human Jurkat T cells partially inhibited the G1 to S transition and completely blocked the S phase-associated chemosensitivity. Therefore, chemosensitivity and apoptotic commitment of human tumor cells are regulated by their cell cycle-dependent Bcl-p2 expression.

HuR regulates cyclin A and cyclin B1 mRNA stability during cell proliferation.

Colorectal carcinoma RKO cells expressing reduced levels of the RNA-binding protein HuR (ASHuR) displayed markedly reduced growth. In synchronous RKO populations, HuR was almost exclusively nuclear during early G(1), increasing in the cytoplasm during late G(1), S and G(2). The expression and half-life of mRNAs encoding cyclins A and B1 similarly increased during S and G(2), then declined, indicating that mRNA stabilization contributed to their cell cycle-regulated expression. In gel-shift assays using radiolabeled cyclin RNA transcripts and RKO protein extracts, only those transcripts corresponding to the 3'-untranslated regions of cyclins A and B1 formed RNA-protein complexes in a cell cycle-dependent fashion. HuR directly bound mRNAs encoding cyclins A and B1, as anti-HuR antibodies supershifted such RNA-protein complexes. Importantly, the expression and half-life of mRNAs encoding cyclins A and B1 were reduced in ASHuR RKO cells. Our results indicate that HuR may play a critical role in cell proliferation, at least in part by mediating cell cycle-dependent stabilization of mRNAs encoding cyclins A and B1.

Hex-Motif-Specific Binding Protein HBP-1b(c38) can Activate Transcription Without Interacting with a Target DNA Sequence.

The type I element (CCACGTCACCAATCCGCG) is a cis-acting element essential for cell cycle-dependent expression of the wheat histone H3 gene (TH012). This element consists of two distinct motifs, Hex (CCACGTCA) and Oct (CGCGGATT). To understand the mechanism of transcriptional regulation via the type I element, we analyzed the function of HBP-1b (c38), a Hex-motif-specific DNA-binding protein of wheat, by using a transient expression system. Cotransfection experiments with effector and reporter plasmids indicated that overexpressed HBP-1b (c38) did not activate expression of the reporter gene with a normal type I element but unexpectedly enhanced the activity of the promoter with a mutated Hex-motif-containing type I element. This activation was Oct-motif-dependent. Basic, leucine zipper, and acidic regions of the HBP-1b (c38) protein were necessary for this activation. These results suggest that HBP-1b (c38) has an ability to activate transcription without interacting with a target DNA sequence.

Nuclear domains enriched in RNA 3'-processing factors associate with coiled bodies and histone genes in a cell cycle-dependent manner.

Nuclear domains, called cleavage bodies, are enriched in the RNA 3'-processing factors CstF 64 kDa and and CPSF 100 kDa. Cleavage bodies have been found either overlapping with or adjacent to coiled bodies. To determine whether the spatial relationship between cleavage bodies and coiled bodies was influenced by the cell cycle, we performed cell synchronization studies. We found that in G1 phase cleavage bodies and coiled bodies were predominantly coincident, whereas in S phase they were mostly adjacent to each other. In G2 cleavage bodies were often less defined or absent, suggesting that they disassemble at this point in the cell cycle. A small number of genetic loci have been reported to be juxtaposed to coiled bodies, including the genes for U1 and U2 small nuclear RNA as well as the two major histone gene clusters. Here we show that cleavage bodies do not overlap with small nuclear RNA genes but do colocalize with the histone genes next to coiled bodies. These findings demonstrate that the association of cleavage bodies and coiled bodies is both dynamic and tightly regulated and suggest that the interaction between these nuclear neighbors is related to the cell cycle-dependent expression of histone genes.

Cell Cycle-dependent Switch of Up- and Down-regulation of Human hsp70 Gene Expression by Interaction between c-Myc and CBF/NF-Y

A CCAAT box-binding protein subunit, CBF-C/NF-YC, was cloned as a protein involved in the c-Myc complex formed on the G(1)-specific enhancer in the human hsp70 gene. CBF-C/NF-YC directly bound to c-Myc in vitro and in vivo in cultured cells. The CBF/NF-Y.c-Myc complex required the HSP-MYC-B element as well as CCAAT in the hsp70 G(1)-enhancer, while the purified CBF subunits recognized only CCAAT even in the presence of c-Myc. Both the HSP-MYC-B and CCAAT elements were also required for the enhancer activity. In transient transfection experiments, the CBF/NF-Y.c-Myc complex, as well as transcription due to the G(1)-enhancer, was increased by the introduction of c-Myc at low doses but decreased at high doses. The repression of both complex formation and transcription by c-Myc at high doses was abrogated by the introduction of CBF/NF-Y in a dose-dependent manner. Furthermore, the CBF/NF-Y.c-Myc complex bound to the G(1)-enhancer appeared in the early G(1) phase of the cell cycle when c-Myc was not higly expressed and gradually disappeared after the c-Myc expression reached its maximum. The results indicate that the cell cycle-dependent expression of the hsp70 gene is regulated by the intracellular amount of c-Myc through the complex formation states between CBF/NF-Y and c-Myc.