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Open access peer-reviewed chapter
To Grow, Stop or Die? – Novel Tumor-Suppressive Mechanism Regulated by the Transcription Factor E2F
By Eiko Ozono, Shoji Yamaoka and Kiyoshi Ohtani
Submitted: May 17th 2012Reviewed: October 18th 2012Published: April 10th 2013
DOI: 10.5772/54510
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chapter and author info
Eiko Ozono
Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Japan
Department of Molecular Virology, Tokyo Medical and Dental University, Japan
Shoji Yamaoka
Kiyoshi Ohtani
*Address all correspondence to:
From the Edited Volume
IntechOpen
Future Aspects of Tumor Suppressor GeneEdited by Yue Cheng
Future Aspects of Tumor Suppressor Gene
Edited by Yue Cheng
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Proliferation of mammalian cells is strictly regulated by growth stimulation. Cell proliferation is stimulated not only by normal growth stimulation but also by abnormal growth stimulation originated from oncogenic changes. Such abnormal growth stimulation leads to tumorigenesis, if not properly guarded by appropriate cellular response. Cells are endowed with intrinsic tumor suppressor pathways to protect cells from tumorigenesis upon such oncogenic threat [1]. The tumor suppressor pathways halt cell proliferation either by restraining cell cycle progression or by inducing apoptosis (programmed cell death) in case of being unable to stop aberrant cell cycle progression. Consequently, the cell-fate, whether to grow, stop growing or die, is dependent on the balance between growth-promoting effects originated from oncogenic changes and growth-suppressive effects mediated by the tumor suppressor pathways upon oncogenic changes (Figure 1). When the tumor suppressor pathways are disabled by further oncogenic changes, the balance of cell-fate determination shifts from growth suppression to proliferation, and cells start deregulated proliferation, leading to tumorigenesis. Among the intrinsic tumor suppressor pathways, two major pathways are the RB pathway and the p53 pathway. Both pathways are important for induction of cell cycle arrest or apoptosis [2]. In addition, accumulating evidence indicates that the tumor suppressor TAp73, a member of the p53 family, also plays crucial roles in tumor suppression by inducing apoptosis independent of p53 [3, 4].
The transcription factor E2F, the main target of the RB pathway, plays crucial roles in cell cycle progression by activating growth-promoting genes [5]. In this regard, E2F is thought to mediate growth-promoting effects originating from normal growth stimulation and oncogenic changes. Supporting this notion, E2F could be an oncoprotein [6]. On the other hand, recent studies indicate that E2F also plays crucial roles in activation of the major intrinsic tumor suppressor pathways by sensing oncogenic changes, halting cell proliferation by inducing cell cycle arrest or apoptosis. Supporting this notion, E2F could also be a tumor suppressor [7]. Taken together, these observations indicate that E2F is located at the center of the balance between cell proliferation and cell cycle arrest or apoptosis, determining the cell-fate upon oncogenic changes (Figure 1). E2F can be regarded as a double-edged sword in cell growth control. In this chapter, we will describe the major intrinsic tumor suppressor pathways and activation of the tumor suppressor pathways by E2F upon oncogenic changes. We will focus on how E2F differentially regulates expression of target genes upon normal growth stimulation and oncogenic changes that have completely opposite roles in cell-fate determination, to grow, stop or die.
To grow, stop or die? The balance between the activity of growth-promoting pathways and tumor suppressor pathways determines the cell fate upon oncogenic changes. E2F is located at the center of the balance (see also Figure 6).
2. Major tumor-suppressor pathways
2.1. The RB pathway (CDK inhibitor–Cyc/CDK–RB)
The retinoblastoma gene (RB1) is the first identified tumor suppressor gene [8]. Individuals with heterozygous deletion or mutation of the RB1 gene are susceptible to retinoblastoma in early life by additional deletion or mutation of the other allele. The RB1 gene product pRB, together with its relatives, p107 and p130, comprises a family of pocket proteins [9, 10] (Hereafter we refer all pocket proteins to RB, and the RB1 gene product to pRB). The main target of RB is the transcription factor E2F, which plays central role in cell proliferation by activating growth-promoting genes including those required for DNA replication and cell cycle progression [9]. In quiescent phase, RB binds to E2F and suppresses transcriptional activity of E2F. Moreover, RB recruits various chromatin-modifying factors, such as histone deacetylases (HDACs) [11] and histone methyl transferase SUV39H1 [12] to actively suppress expression of E2F target genes. Hence the main role of RB in tumor suppression is thought to be suppression of cell proliferation through suppression of E2F target gene expression. Growth stimulation induces expression of cyclins and activates cyclin dependent kinases (CDKs), which are called accelerators and engines in cell cycle progression, respectively. CDKs, in turn, inactivate RB by phosphorylation, leading to expression of E2F target genes by releasing them from suppression by RB during G1 to S phase cell cycle progression (Figure 2) [13]. In contrast, growth-suppressive signals such as contact inhibition and DNA damage induce expression of CDK inhibitors, which are called brakes in cell cycle progression owing to their ability to inhibit activity of CDKs. Suppression of CDKs keeps RB in hypo-phosphorylated form, which binds to and inhibits E2F. Consequently, the activity of E2F to activate growth-promoting genes is controlled by the activity of RB, which is regulated by CDKs and CDK inhibitors. The pathway converging to RB, including CDKs and CDK inhibitors, is referred to the RB pathway.
Whether a cell progresses one round of the cell cycle or not is determined at the restriction point, which is located at late G1 phase of the cell cycle. Once a cell passed through the restriction point, it is programmed that the cell cycle automatically proceeds to the end of M phase. Thus, whether a cell proliferates or not is determined by whether the cell passes through the restriction point or not. Two major determinants whether a cell passes through the restriction point or not are E2F activity and cyclin dependent kinase activity, which is induced by E2F through activation of the Cyclin E (CycE) gene [14]. Since E2F plays essential roles in passing through the restriction point, RB can be regarded as a gatekeeper in cell cycle progression by controlling E2F activity. Hence disruption of the RB pathway and consequent activation of E2F is thought to be an essential event for tumorigenesis [2]. Actually, deletion or mutation of the RB1 gene is observed in about 30% of cancers. Moreover, defects in the RB pathway upstream of RB such as overexpression of CycD and dysfunction of CDK inhibitors such as p16INK4a are frequently observed in other cancers retaining RB1 [2]. It is predicted that all cancers have at least some defect in the RB pathway.
The RB pathway plays essential roles in tumor suppression by inducing cell cycle arrest or cellular senescence through suppression of the activity of E2F. As described below, disruption of the RB pathway reinforces cell cycle arrest by induction of CDK inhibitor p21Cip1 expression through the p53 pathway or p27Kip1 expression. When cells failed to induce cell cycle arrest, apoptosis is triggered through the p53 pathway or TAp73, a p53 family member, which activates various pro-apoptotic genes.
The regulatory mechanism of the transcription factor E2F by the RB pathway.
2.2. The p53 pathway (ARF–p53–cell cycle arrest or apoptosis related effectors)
The tumor suppressor p53 is a transcription factor that is activated by a variety of stress signals, including DNA damage, hypoxia and various oncogenic changes including aberrant activation of E2F [15]. In response to such stress signals, p53 induces either cell cycle arrest or apoptosis. Cell cycle arrest is mainly mediated through activation of the CDK inhibitor p21Cip1 gene [16], whose product suppresses wide range of CDKs. Apoptosis is mainly mediated through activation of the Bax and BH3 only family genes, whose products destabilize mitochondrial membrane to facilitate cytochrome c release, which triggers apoptotic cascades of caspase activation.
Since p53 plays crucial roles in induction of cell cycle arrest or apoptosis, the expression level of p53 is kept low by rapid ubiquitin/proteasome-dependent degradation, mainly caused by Mdm2 (mouse double minute 2, Hdm2 in humans), which is often overexpressed in many cancers [15]. Mdm2 is E3 ubiquitin ligase, which directly binds to p53 and promotes p53 degradation. Mdm2 also inhibits TP53 mRNA translation [17]. The Mdm2 gene is a target of p53, forming a negative-feedback loop to control the level of p53 [18]. Another negative regulator of p53 is MdmX, also known as Mdm4. MdmX has recently emerged as a discrete critical negative regulator of p53 [19]. Though MdmX is not a direct target of p53, structure of MdmX is significantly similar to that of Mdm2. MdmX is reported to enhance p53 ubiquitination by altering the substrate preference of the Mdm2, thereby indirectly regulating p53 [20].
Regarding response to oncogenic stresses, a potent activator of p53 is the tumor suppressor ARF (alternative reading frame, known as p14 in humans and as p19 in rodents) [2]. ARF directly binds to and sequesters Mdm2 to nucleoli, stabilizing p53 that leads to the expression of its target genes [21]. The pathway including upstream and downstream of p53 is referred to the p53 pathway. Of note, the ARF gene is a direct target of E2F [22] and this E2F-ARF interaction connects the RB pathway and the p53 pathway, enabling efficient tumor-suppressive response. When the RB pathway is disrupted by oncogenic changes, E2F is activated to induce ARF gene expression, leading to activation of the p53 pathway, which inhibits oncogenic cell growth by inducing cell cycle arrest or apoptosis (Figure 3). Therefore, disruption of both of the tumor suppressor pathways powerfully shifts the cell-fate determination balance to proliferation (Figure 1) and is thought to be essential to induce deregulated cell proliferation that leads to tumorigenesis. Indeed, about 50% of cancers carry TP53 mutations or deletion and most cancers have defects in the p53 pathway including those in upstream and downstream of p53.
2.3. The TAp73 pathway (E2F–TAp73–pro-apoptotic targets)
The tumor suppressor TAp73 is a homologue of p53 and can induce apoptosis independently of p53 [3, 4, 23]. The TP73 gene encodes two isoforms, TAp73 and DNp73, which are driven by different promoters. DNp73 lacks the transactivation (TA) domain and counteracts TAp73 and p53 [24]. Thus DNp73 is anti-apoptotic. The TAp73 gene is thought to be a tumor suppressor gene and is known to be a target of E2F1 [3, 4, 25]. Similarly to p53, TAp73 is activated by both oncogenic changes and DNA damage [26, 27] and TAp73 target genes partly overlap with those of p53, such as the PUMA and NOXA genes [28, 29] that are crucial for induction of apoptosis. Moreover, TAp73 can induce apoptosis in the absence of p53 [3, 4]. Therefore, TAp73 seems to back-up the important tumor suppressive function of p53.
E2F activates major intrinsic tumor suppressor pathways.
3. The transcription factor E2F
The transcription factor E2F was originally identified as a cellular DNA-binding protein, which mediate E1A-dependent activation of the adenovirus E2 promoter [30]. Members of E2F family are downstream targets of the tumor suppressor RB and make repressor complexes with RB that keep cells in quiescent state [9, 10]. Though E2F plays central roles in cell proliferation by activating growth-promoting genes, certain members of E2F can induce apoptosis [15]. In this paragraph, we will describe three points as follows (1) E2F family members, the role of them in cell cycle progression, cell cycle arrest and apoptosis. (2) Regulatory mechanism of E2F. (3) E2F target genes, to better understand the regulatory mechanism of cell-fate determination mediated through E2F.
3.1. E2F family members
E2F consists of eight family members (E2F1-E2F8). E2F1-E2F5 are bound by their repressor RB family proteins through RB binding domain. E2F1-E2F5 activate transcription when free from RB and repress transcription when bound by RB. E2F1-E2F3a are induced at G1/S boundary and activate transcription free from RB. In contrast, E2F3b-E2F5 are expressed all through the cell cycle and play main roles in transcriptional repression in G0/G1 bound by RB. E2F6-E2F8 repress transcription independently of RB. Hence E2F family members are divided into two groups: activator E2Fs (E2F1-E2F3a) and repressor E2Fs (E2F3b-E2F8). Activator E2Fs play major roles in activation of target genes involved in growth promotion, growth suppression and induction of apoptosis. Repressor E2Fs are roughly divided into two groups; E2F3b-E2F5, which make repressor complex together with RB, and recently identified E2F6-E2F8, which function independently of RB.
Structure of E2F family members. E2F1-E2F6 bind to their target promoter with binding partner DP proteins through dimerization domain. E2F7 and E2F8 have two DNA binding domains and make homodimers or E2F7/E2F8 heterodimer.
A structural characteristic of activator E2Fs (E2F1-E2F3a) is longer N terminal region, which does not exist in repressor E2Fs (E2F3b-E2F6). Expression of E2F1-E2F3a is induced by E2Fs themselves [31]. When growth stimulation inactivates p130 by phosphorylation through activation of CycD/Cdk4 or CycD/Cdk6, repressor E2Fs (E2F4 and E2F5) are released from p130 and activates the E2F1-E2F3a genes. E2F1-E2F3a in turn replace E2F4 and E2F5, and activate growth-promoting genes including E2F1-E2F3a themselves. Each activator E2F has preferential roles in cell cycle progression and induction of apoptosis. p107 preferentially make complexes with E2F4-E2F5 in G1/S to S phases.
E2F1 is generally thought to be the most powerful transcriptional activator of pro-apoptotic genes among activator E2Fs. Overexpression of E2F1 in tissue culture cells alone can induce cell cycle progression in otherwise quiescent fibroblasts [32]. Overexpression of E2F1 can be oncogenic in vitro [33, 34] and in vivo [6]. In contrast, E2F1 is dispensable for cell cycle progression, since E2F1 knockout mice are viable. On the other hand, over expression of E2F1 in cancer cell lines leads to apoptosis [35]. E2F1 also plays a key role in induction of cellular senescence by activating the ARF-p53 pathway, when overexpressed in human normal fibroblasts [36]. Moreover, E2F1 null mice resulted in tumorigenesis [7]. Taken together, E2F1 seems to play the most important roles in tumor suppression among activator E2Fs by activating genes involved in apoptosis and cell cycle arrest.
E2F2 has 46% overall amino acid sequence similarity to E2F1 [37] and is thought to play roles in both cell proliferation and tumor suppression. E2F2 null mice exhibit increased proliferation of hematopoietic cells and frequently develop autoimmunity and tumors [38, 39]. In tumor suppression, E2F2 plays major roles in suppression of Myc-induced T cell lymphomagenesis. Inactivation of neither E2F1 nor E2F3 had no effect on tumor progression in T cells, and only loss of E2F2 accelerated lymphomagenesis [40].
E2F3 is thought to be the most important activator E2F in cell proliferation. Although E2F1 or E2F2 null mice are viable and tumor-prone, E2F3 null mice are typically embryonic lethal in pure background [41] or show partially penetrant embryonic lethality in mixed background [42]. Although mouse embryonic fibroblasts (MEFs) with combined knockout of E2F1 and E2F2 proliferate, those with combined knockout of E2F1, E2F2 and E2F3 fail to proliferate and can not re-enter the cell cycle [5]. The E2F3 locus encodes two isoforms, E2F3a and E2F3b, a truncated variant of E2F3 in its N-terminus [43, 44]. Although both E2F3s partly overlaps their roles in cell cycle progression [45], E2F3a is expressed in G1/S to S phases and is thought to play crucial roles on cell proliferation, while E2F3b is expressed equivalently in quiescent and proliferating cells, and associates with pRB, representing the predominant E2F-pRB complex in quiescent cells [44, 46]. E2F3b also plays roles in myogenic differentiation by promoting gene expression related to differentiation [47].
Roles of activator E2Fs in cell-fate determination. E2F1 plays crucial roles in induction of apoptosis. E2F3 is thought be essential for cell proliferation. It is predicted that the character of E2F2 is in the middle of E2F1 and E2F3.
E2F4 and E2F5 were cloned by their association with p107 and p130, and are significantly detected in quiescent cells [48, 49]. Knockout mice of either E2F4 or E2F5 are viable [50-52]. E2F4 knockout mice are runted and display defects in late stage of maturation. In addition, these mice present reduced thickness of the gut epithelium and developmental craniofacial defects. E2F5 knockout mice develop hydrocephalus after birth apparently due to increased secretion of cerebrospinal fluid by the choroid plexus. E2F4 and E2F5 double knockout mice die before birth because of developmental defects, suggesting that E2F4 and E2F5 have some redundant functions during development [53]. Cells lacking E2F4 and E2F5 are unable to stop cell cycling upon growth-suppressive signals such as TGF-β treatment, suggesting that E2F4 and E2F5 are major repressor E2F in restraining cell cycle progression. These two E2Fs are thought to be important for cell cycle exit and terminal differentiation.
E2F6 does not possess RB binding domain. As predicted from the structure, E2F6 contributes gene silencing independent of RB [54] and is predicted to make repressor complex with polycomb proteins [55]. Consistent with this, E2f6-/- animals display overt homeotic transformations of the axial skeleton that are strikingly similar to the skeletal transformations observed in polycomb deficient mice [56]. E2F6 is reported to bind the same E2F-repressor site as E2F4, suggesting E2F6 may partly overlaps its function as transcriptional repressor with E2F4 in S phase [57].
E2F7 and E2F8 are thought to function as transcriptional repressors independently of RB proteins. An important target of E2F7 and E2F8 is the E2F1 gene. E2F7 and E2F8 knockout mice are embryonic lethal, at least in part, due to apoptosis caused by inability to down regulate expression of E2F1, leading to activation of p53 [58]. Recent report showed that E2F7 and E2F8 promote angiogenesis through transcriptional activation of the VEGFA promoter with hypoxia inducible factor 1 (HIF1) [59].
3.2. Regulatory mechanism of E2F
E2F1 through E2F6 associate with DP family proteins (DP1 or DP2) to form heterodimeric complexes that bind to DNA in a sequence-specific manner (consensus sequence: TTTC/GG/CCGC). E2F7 and E2F8 have two DNA binding domains and do not require DP proteins for binding to DNA. E2F7 and E2F8 make homodimer or E2F7/E2F8 heterodimer to bind to the target [58].
Transcriptional activity of E2F1 through E2F5 is suppressed by binding of RB family proteins. pRB preferentially binds E2F1 through E2F3, whereas p107 and p130 preferentially bind E2F4 and E2F5. However, pRB can also bind E2F4, depending on the cellular circumstances. E2F6 through E2F8 can repress transcription independently of pRB family proteins [10, 60]. In quiescent phase, E2F3b/pRB, E2F4/p130 and E2F5/p130 repress promoters of E2F target genes. Upon growth stimulation, activated CycD/Cdk4 and CycD/Cdk6 phosphorylate pRB and p130, inhibiting their binding to the E2Fs and allowing accumulation of the free E2Fs [31]. This release from repression conferred by the pRB family proteins is the primary activation step for induction of E2F target genes. In this context, E2F3b, E2F4 and E2F5 mainly act as repressors together with the pRB family proteins during G0/G1 phases. Expression of E2F1, E2F2 and E2F3a is induced at the G1/S boundary by E2F itself, and activate the growth related genes, including the CycE gene. CycE activates Cdk2, whose activity is essential for initiating DNA replication, and drive cells into S phase [31]. In late S phase, CycA/Cdk2 complex represses the transcriptional activity of E2F/DP complex by phosphorylation, which releases the complex from the binding element [61].
3.3. E2F target genes
Classical E2F targets are genes involved in DNA replication and cell cycle progression. In addition to these, recent studies with DNA microarray and chromatin immunoprecipitation (ChIP) identified a variety of E2F targets. These include genes involved in DNA repair, checkpoint, differentiation, development, metabolism, micro RNAs, apoptosis, cell cycle arrest and others.
E2F regulates expression of most of the genes involved in initiation of DNA replication: the ORC1 (origin recognition complex1), CDC6, MCM (maintenance of minichromosome) 2-7, ASK and CDC45 genes. These factors are assembled into pre-replication complex, which is activated by CycE/Cdk2 to initiate DNA replication [31]. Cdt1 also plays crucial roles in initiation of DNA replication and is negatively regulated by geminin. The Cdt1 and geminin genes are both E2F targets [62]. Genes, which code for machineries responsible for DNA replication, are classical E2F targets. These include the DHFR (dihydrofolate reductase), DNA polymerase α, thymidine kinase, thymidylate synthase and PCNA (proliferating cell nuclear antigen) [53].
DNA repair, checkpoint
E2F target genes related to DNA repair are the Rad51, MSH2 and MLH1 genes, which are involved in homologous recombination repair and mismatch repair [63]. E2F target genes related to checkpoint are the ATM, Chk1, Mad3, Bub1, Claspin and RanBP1 genes [53, 64-67]. It is expected that E2F regulates these DNA repair and checkpoint genes to prepare machineries to quickly respond in case of emergency.
Cell cycle progression
E2F induces expression of genes, which play major roles in induction of S phase, such as the CycE and activator E2F (E2F1-E2F3a) genes themselves [31]. Also, the upstream negative regulators of pRB, the Emi1 and Skp2 genes are E2F targets [68, 69]. The genes, which encode CycA, Cdc2 (CDK1), CycB and B-myb that are important for S and G2/M phase progression, are also E2F targets [53]. Repressor E2Fs, E2F7 and E2F8 genes are reported to be targets of E2F1 that promote embryonic development by suppressing E2F1-p53 induced apoptosis, forming a negative feedback loop. Although c-Myc is reported as a target of E2F [70], E2F may play a role in suppression of c-Myc expression upon negative growth signals [71].
Development and differentiation
E2F also regulates expression of genes involved in development and differentiation. The Firizzled homologs1-3, Homeobox and TGF genes are shown to be targets of E2F [53]. It is reported that overexpression of E2F1-3 induced various genes involved in development and differentiation [72]. Interestingly, although E2F7 and E2F8 are generally regarded as transcriptional repressors, a recent study reported the roles of E2F7 and E2F8 in transcriptional activation of genes such as VEGFA as written before [59].
Cellular metabolism
Recent work reported that E2F1 and pRB are required for repression of genes implicated in oxidative metabolism [73]. E2F1 repressed key genes that regulated energy homeostasis and mitochondrial functions in muscle and brown adipose tissue, and E2F1 null mice had a marked oxidative phenotype. Their work suggests a metabolic switch from oxidative to glycolytic metabolism that responds to stressful conditions.
Micro RNAs
Accumulating evidence indicates that expression of E2F is regulated by microRNAs and that E2F also induces expression of microRNAs [74]. One of the major microRNAs regulated by E2F is miR-17~92. miR-17~92 is a negative regulator of E2F1-E2F3 and is also a target of E2F1-E2F3, constructing a fail-safe mechanism to regulate E2F activity. E2F seems to be rigorously controlled by many miRNAs, suggesting that E2F activity must be strictly controlled for appropriate cell cycle progression.
Apoptosis and cell cycle arrest
E2F can activate genes involved in apoptosis and cell cycle arrest, which are inconvenient for cell proliferation. Regulatory mechanism of these growth-suppressive genes by E2F is yet to be elucidated, especially regarding regulation of cell growth versus tumor suppression in response to normal growth stimulation and oncogenic changes. The caspase3, caspase7 and Apaf1 genes, which code for apoptotic machineries, are direct targets of E2F [53]. Caspases are expressed as inactive precursors (procaspases), and expression of procaspases and Apaf-1 alone does not necessarily induce apoptosis. These apoptotic machineries require upstream signals to be activated to induce apoptosis. Expression of these two pro-apoptotic E2F targets and all E2F targets mentioned above is induced by growth stimulation. Thus, expression of the pro-apoptotic machinery by growth stimulation is thought to be fail-safe mechanism to induce apoptosis in case of emergency. We refer these E2F target genes, whose expression is induced by growth stimulation, to 'typical E2F targets'. Typical E2F targets are activated by growth stimulation, which physiologically inactivates RB by phosphorylation and activates E2F by release from repression.
In contrast to typical E2F targets, we identified three E2F targets, which are not activated by growth stimulation. These include the tumor suppressor ARF, TAp73 and CDK inhibitor p27Kip1 genes. As described below, these genes are critically important in tumor suppression. We describe the regulatory mechanism of the tumor suppressor ARF, TAp73 and CDK inhibitor p27Kip1 genes by E2F in next paragraph.
E2F activates both growth-promoting genes and growth- suppressive genes, including E2F itself. It is predicted that the expression levels of E2F targets, related to growth- promotion or growth- suppression, decide the balance of cell-fate determination.
4. Deregulated E2F
Since E2F1-E2F3a are activated by growth stimulation, it is generally thought that their target genes are all activated by growth stimulation. However, E2F1-E2F3a also activate genes involved in cell cycle arrest or apoptotic, which are inconvenient for cell growth. It has yet to be elucidated how E2F regulates genes involved in cell cycle arrest or apoptosis, regarding normal cell growth and tumor suppression.
Since all E2F targets are thought to be activated by growth stimulation, it is surprising that our previous work identified three growth suppressive E2F targets, which were not activated by growth stimulation at all in human normal fibroblasts [75-77]. In contrast, these E2F targets were activated by deregulated E2F activity induced by overexpression of E2F1 or forced inactivation of pRB. Overexpression of E2F1 generates exceeding amount of exogenously introduced E2F1, which becomes out control by RB proteins. Forced inactivation of pRB induces endogenous deregulated E2F activity out control by pRB. We refer these growth suppressive E2F targets, which are not activated by growth stimulation but are activated by deregulated E2F, to 'atypical E2F targets'. These atypical E2F targets include the tumor suppressor ARF and TAp73 genes and the CDK inhibitor p27Kip1 gene These three atypical E2F target genes play major roles in tumor suppression. ARF is an upstream activator of the tumor suppressor p53. CDK inhibitor p27Kip1 activates the RB pathway by inhibiting CDKs. TAp73 is the tumor suppressor, which can induce apoptosis in dependently of p53. Our observations suggest that E2F activity induced by RB dysfunction, one of major oncogenic changes, has distinct function from that induced by growth stimulation in activating target genes (Figure 7).
E2F activity induced by RB dysfunction activates both typical and atypical E2F targets. In contrast, E2F activity induced by growth stimulation activates only typical E2F targets and interestingly, not atypical E2F targets. Both of the E2Fs are similar in the sense that they are released from repression by RB. However, it is shown that growth stimulation does not totally inactivate pRB and some portion of activator E2Fs is still in complex with pRB [78]. Moreover, the RB1 gene is an E2F target [79] and expression of pRB is increased in G1/S to S phases [80]. Indeed, the amount of activator E2Fs/pRB complex rather increases in G1/S to S phases, as examined by gel mobility shift assay [78]. This observation indicates that E2F activity induced by growth stimulation is still under control of RB. In contrast, E2F activity induced by dysfunction of RB is thought to be out of control by RB. Here, we refer E2F activated by growth stimulation to 'physiological E2F' and that by dysfunction of RB to 'deregulated E2F'. Our findings indicate that deregulated E2F is functionally different from physiological E2F.
In this paragraph, we describe the regulatory mechanism of atypical E2F targets by deregulated E2F and discuss about the characteristics of deregulated E2F activity regarding its role in tumor suppression.
Atypical E2F target genes are specifically activated by deregulated E2F activity through specific E2F responsive elements.
4.1. Atypical E2F targets
The first atypical E2F target identified was the ARF gene [75]. ARF is the major activator of p53 pathway and links the RB and p53 tumor suppressor pathways [22], playing crucial roles in tumor suppression. Consistent with this notion, deletion, mutation or silencing of the ARF gene is frequently observed in cancers. Moreover, Arf null mice are highly prone to tumorigenesis [81]. Various oncogenic signals are able to elicit the activation of the ARF gene. Overexpression of adenovirus E1a or E2F1 in primary mouse embryonic fibroblasts (MEFs) rapidly induces ARF gene expression and p53-dependent apoptosis [16]. Myc overexpression and oncogenic mutant Ras are also strong activators of the ARF gene and combination of absence of Arf in mice severely impaired the tumor suppressive activity of p53 [82]. Arf promoter seems to monitor these oncogenic signals as shown by ARF promoter-GFP transgenic model, in which GFP expression was observed in tumors induced by Myc or Ras but not in normal growing tissues [83]. Other studies elucidated that ARF also restrains cell growth independently of p53, interacting with other factors [84]. Taken together, the ARF gene plays crucial roles in tumor suppression through p53-dependent and independent pathways.
The CDK inhibitor p27Kip1 is an upstream regulator of the RB pathway and known to contribute to the ability of pRB to induce cell-cycle arrest, differentiation and senescence [85-87]. There is cross regulation between p27Kip1 and pRB. p27Kip1 enhances pRB growth suppressive function by inhibiting Cyc/CDK, keeping pRB in hypo-phosphorylated form. pRB increases the amount of p27Kip1 by sequestrating Skp2, a component of E3 ubiquitin ligase complex, which promotes degradation of p27Kip1 [86]. pRB also known to cooperate with APC/Ccdh1, another E3 ubiquitin ligase, to induces Skp2 degradation, stabilizing p27Kip1 [88]. Taken together, pRB and p27Kip1 seem to keep close relationship in the RB pathway to efficiently suppress aberrant cell proliferation. Indeed, our previous study showed that inactivation of RB by adenovirus E1a increased BrdU (bromodeoxyuridine)-positive cells much earlier in p27Kip1-/- MEFs than in wild type MEFs [66]. These results support the notion that p27Kip1 plays important roles in the RB pathway to suppress cell cycle progression induced by oncogenic changes.
The tumor suppressor TAp73 gene had been identified as a direct target of E2F using cancer cell lines [4]. We found that the TAp73 gene was activated by deregulated E2F but not by physiological E2F in human normal fibroblasts [77]. TAp73 is a p53 family member and plays important roles in tumor suppression with its other family member p53 and p63. All of the three genes express differentially spliced isoforms [89, 90]. Two major isoforms are TA isoforms, which retain transactivation (TA) domain, and delta N (DN) isoforms, which lack TA domain. Since these family members activate their targets as tetramers and DN isoforms lack transactivation (TA) domain, DN isoforms have dominant-negative properties [24]. Although p53 is deleted or mutated in half of all human cancers, deletion or mutations of p73 and p63 occur rarely [91, 92]. Rather over expression of DN form of p73 and p63 are commonly observed in many cancers, such as over expression of DNp73 isoform in gliomas and carcinomas of the breast and the colon [93, 94], and that of DNp63 isoform in bladder carcinomas [95]. Each DN isoform can suppress all three types of TA forms [92, 96, 97]. Therefore, it is expected that tumor-suppressive TA isoforms are suppressed by DN isoforms in many cancers. This could explain why deletion or mutations of the TP73 and TP63 genes are rare. TAp73 can induce apoptosis independently of p53. Moreover, TAp73 knockout mice are tumor prone, infertile, sensitive for carcinogen-induced tumorigenesis and defective for maintenance of genomic stability [98]. The TAp73 gene is activated by various stress signals including deregulated E2F and DNA damage. These observations suggest that TAp73 contributes to tumor suppression in addition to the p53 pathway in response to various oncogenic changes.
Accumulating evidence indicates that the three atypical E2F targets explained above play crucial roles in tumor suppression, by activating the RB pathway, the p53 pathway and the p53 independent pathway. Taken together, deregulated E2F seems to be critically important for activating major intrinsic tumor-suppressor pathways in responding to oncogenic changes to suppress tumorigenesis (Figure 8).
Deregulated E2F plays important roles in activating major tumor suppressor pathways (RB, p53 and TAp73 pathways) by activating the ARF, p27Kip1 and TAp73 genes.
4.2. Distinct transcriptional regulatory mechanism mediated by deregulated E2F
ARF, p27Kip1 and TAp73 exert its effects when expressed unlike pro-apoptotic targets, which are expressed as inactive precursors such as pro-caspases. Thus the regulation of expression of these genes is critically important for tumor suppression. The finding that these genes are specifically activated by deregulated E2F but not by physiologically activated E2F indicates that there is a mechanism to specifically respond to oncogenic changes to suppress tumorigenesis, while allowing normal cell growth upon normal growth stimulation.
We first identified the tumor suppressor ARF gene as an atypical E2F target [75]. In our studies, we used human normal fibroblasts (HFFs or WI-38) to examine the responsiveness of each growth-suppressive E2F target to physiological and deregulated E2F. This is because most of previous studies used cancer cell lines and were unable to examine responsiveness of E2F target genes to normal growth stimulation. To induce physiological E2F, we used serum stimulation, common growth stimulation for fibroblasts. To induce deregulated E2F activity, we used ectopically expressed E2F1 or forced inactivation of RB either by adenovirus E1a, which binds to and inactivates all RB family proteins, or shRNA against RB1 (shRB), which represses the expression of pRB. The latter (adenovirus E1a and shRB) is expected to induce endogenous deregulated E2F activity.
Ectopically expressed E2F1, adenovirus E1a and shRB induced ARF gene expression in RT-PCR, and activated ARF promoter in reporter assay. However, serum stimulation, which physiologically activates E2F, did not induce ARF gene expression or activate ARF promoter under the condition that CDC6 gene (one of typical E2F targets involved in DNA replication) expression was significantly induced and CDC6 promoter was clearly activated. These results indicate that the ARF gene is specifically activated by deregulated E2F but not by physiological E2F. Promoter analyses identified the E2F responsive element of ARF promoter (EREA), which specifically responds to deregulated E2F activity. Interestingly, the sequence of EREA was composed of only GC repeat and lacked T stretch. This is in contrast to that of consensus E2F binding motif, which is composed of T stretch and GC repeat (TTTG/CC/GCGC) in typical E2F targets. In addition, the location of EREA was far upstream from transcription start site compared to that of typical E2F sites, which is within 100 bp from transcription start site in most cases. Moreover, our gel mobility shift assay and ChIP assay showed that EREA specifically binds ectopically expressed E2F1 but not physiological E2F1 induced by serum stimulation, both in vitro and in vivo, respectively. Taken together, these observations suggest that the ARF gene is specifically activated through EREA by deregulated E2F activity, triggered by ectopically expressed E2F1 or forced inactivation of RB, but not by physiological E2F activity, induced by serum stimulation (Figure 7).
A later study showed that ARF promoter lacking EREA was still activated by overexpression of E2F1 [99]. Our further analyses of ARF promoter identified multiples of EREA-like elements in ARF promoter. It seems that, although EREA is the major E2F responsive element in ARF promoter, it is not the sole responsive element and multiple EREA-like elements co-operate to specifically respond to deregulated E2F activity (manuscript in preparation).
Defects in the RB pathway activate E2F out of control by RB, promoting abnormal cell growth. In response to such oncogenic insults, deregulated E2F activates the ARF gene, leading to activation of p53 to protect cell from tumorigenesis. For induction of tumorigenesis, the p53 pathway must be disabled by further oncogenic changes. Indeed, defects in the p53 pathway are observed in almost all cancers. It is expected that the presence of deregulated E2F be tolerated in cancer cells by further inactivation of the p53 pathway. We thus examined the existence of deregulated E2F activity in RB1 deficient cancer cell lines and in normal growing fibroblasts. When we introduced constitutively active form of pRB into RB1 deficient cancer cell lines (5637, Saos-2 and C-33 A) and normal growing fibroblasts (WI-38 and HFF), activity of EREA and ARF promoter were decreased in RB1 deficient cancer cell lines, but not in normal growing fibroblasts. These results showed that deregulated E2F activity specifically exists in RB1 deficient cancer cell lines but not in normal growing fibroblasts. The presence of deregulated E2F activity may serve as a useful marker to discriminate cancer cells from normal growing cells.
Our search for new E2F targets with subtraction method identified the CDK inhibitor p27Kip1 gene as an atypical E2F target [66]. p27Kip1 plays important roles in cell cycle arrest by inhibiting Cyc/CDKs. Using reporter assay, we showed that EREK (E2F responsive element of p27Kip1) was responsible for specifically sensing deregulated E2F activity in human normal fibroblasts and that EREK was specifically activated in the RB1 deficient cancer cell lines [76]. Consistent with EREA, the location of EREK was far upstream compared to typical E2F binding sites. Interestingly, the sequence of EREK contained T in addition to GC repeat and is rather similar to that of typical E2F binging site. However, EREK bound deregulated E2F1 but not physiological E2F1 in ChIP assay, showing that its character was similar to EREA. These results suggest that not only the sequence of E2F responsive elements, but also the sequence around the responsive elements may be important for discriminating deregulated E2F activity from physiological E2F activity. There is also a possibility that structure of the whole promoter might also affect the discrimination.
Third atypical E2F target is the tumor suppressor TAp73 gene [77]. TAp73 promoter specifically responded to deregulated E2F activity through four ERE73s (E2F responsive elements of TAp73), which were specifically activated by deregulated E2F activity. The sequences of ERE73s contained T stretch and were similar to that of typical E2F binding sites. Importantly, our ChIP assay showed that bindings of ectopically expressed 'exogenous' E2F1 and deregulated 'endogenous' E2F1 induced by adenovirus E1a were detected on ERE73s, but not that of physiological E2F1 induced by serum stimulation. Thus, although the sequences of ERE73s were similar to or almost same as that of typical E2F binding sites, the characters of both were completely different. ERE73s were specifically activated by deregulated E2F activity and specifically bound to both 'exogenous' and 'endogenous' deregulated E2F1. These results support the notion that both sequences of the E2F binding site and its flanking region may be important for discriminating deregulated E2F activity from physiological E2F activity. Consistent with EREA and EREK, reporter assay showed that ERE73s were also activated in the RB1 deficient cancer cell lines and not in normal fibroblasts. Moreover, reintroduction of the constitutive active from of pRB by recombinant adenovirus reduced the expression of the TAp73 gene in all the cancer cell lines in RT-PCR, indicating that the cancer cell lines harbor deregulated E2F activity that activates the endogenous TAp73 gene.
Interestingly, our unpublished data suggest that not only the RB1 deficient cancer cell lines but also cancer cell lines retaining pRB harbor deregulated E2F activity. Activity of ERE73s and expression of the TAp73 gene were suppressed by introduction of the constitutive active form of pRB in cancer cell lines retaining pRB. These results suggest the possibility that E2F-mediated transcriptional program can sense defects in the RB pathway, not only pRB itself but also upstream regulators of pRB. Taken together, deregulated E2F activity might become a universal means to discriminate cancer cells (may be regardless of the presence of pRB) from normal growing cells.
4.3. Difference between deregulated E2F and physiologically activated E2F
Deregulated E2F and physiologically activated E2F are similar in a sense that both are 'released from RB'. However, there is a functional difference between deregulated E2F and physiologically activated E2F. Deregulated E2F activates both typical and atypical E2F targets. In contrast, physiologically activated E2F activates typical E2F targets but not atypical E2F targets. Why atypical E2F targets are activated by deregulated E2F and not by physiologically activated E2F? What is the difference between deregulated E2F and physiologically activated E2F? Deregulated E2F is totally 'out of control' by RB due to dysfunction of the RB pathway. In contrast, physiologically activated E2F is temporarily released from RB, predicted to be 'under control' of RB. During normal cell growth, activator E2Fs are induced at G1/S boundary of the cell cycle. At the point, it is generally believed that pRB is phosphorylated and inactivated. However, pRB is not totally inactivated at the point. Previous studies indicate that the RB1 gene is an E2F target [79] and expression of pRB is increased in G1/S to S phases [80]. Moreover, the amount of activator E2Fs/pRB complex rather increases at G1/S boundary as shown by gel mobility shift assay [78], indicating that some portion of pRB is still active and is regulating the activity of activator E2Fs. Thus, physiologically activated E2F is still under control by pRB. It is likely that activity of activator E2Fs is strictly controlled by degree of phosphorylation of pRB dependent on the activity of CDKs, reflecting the strength of growth stimulation. There must be difference between activation of E2F out of control by pRB and activation of E2F under control by pRB. Our studies of regulatory mechanism of atypical target promoters by E2F elucidated the four different points between regulation of typical E2F target promoter and that of atypical E2F target promoter.
Although sequences of EREK and ERE73s resemble that of typical E2F binding sites, sequence of EREA is different from that of typical E2F sites. The sequence of consensus E2F binding motif of typical E2F targets is composed of T stretch and GC repeat (TTTG/CC/GCGC). EREA is composed of only GC repeat and lacks T stretch. Difference in binding sequence suggests the possibility that there may be a difference in factors, which recognize the sequence and bind.
In all three cases, location of E2F responsive elements of atypical E2F targets is far from the transcriptional start site compared to that of typical E2F binding sites in typical E2F targets. In the case of typical E2F targets, location of E2F binding sites is within 100 bp from transcriptional start site in most cases. Typical E2F targets are under repression by E2F/RB complex in quiescent phase and are released from repression upon growth stimulation. Close proximity of E2F binding sites to transcriptional start site may be required for this mode of regulation. In contrast, atypical E2F targets are not under repression by E2F/RB complex and literally activated by deregulated E2F. E2F responsive elements of atypical E2F targets behave as enhancer elements, which can function from distance.
Deregulated E2F1 bound to EREA, EREK and ERE73s and physiologically activated E2F1 did not bind to these elements as shown by ChIP assay. In the case of EREA, it is also shown that repressor type E2F4 does not bind to EREA. This observation is compatible with the observation that these atypical E2F targets are not under repression by E2F/RB. The fact that deregulated E2F bind to atypical E2F targets, while physiological E2F does not bind to atypical E2F targets, suggest that there is difference in binding behavior between deregulated E2F and physiologically activated E2F.
Differences in E2F regulation of target promoters between deregulated E2F and physiological E2F. (1) Sequence of the atypical E2F responsive elements and surrounding regions are different from that of typical E2F targets. (2) E2F responsive elements of the atypical E2F targets locate far upstream from transcriptional start sites compared to that of typical E2F targets. (3) E2F responsive elements of atypical E2F targets specifically bind deregulated E2F and not physiologically activated E2F. (4) Unlike typical E2F targets, promoters of atypical E2F targets are not under repression of RB and are specifically activated by deregulated E2F activity.
Regulatory mechanism of promoters is different between typical E2F targets and atypical E2F targets. Promoters of typical E2F targets are repressed by E2F/RB complex. Growth stimulation inactivates RB and releases promoters from the repression by RB. Thus, so-called activation of typical E2F targets by physiological E2F is 'release from repression by RB'. In contrast, activation of atypical E2F targets by deregulated E2F is literally 'activation'. Mutation of EREA, EREK or ERE73s in corresponding full-length promoter did not enhance basal promoter activities, indicating that these three promoters are not under repression through the E2F responsive elements. This is consistent with the observation that binding of physiological E2F to promoters of atypical E2F targets was not observed in ChIP assay, including repressor type E2F4 (Figure 9).
It is generally accepted that the amount of free E2F is important for differential regulation of E2F targets, which have opposite roles in cell-fate determination, as proposed as threshold model [100]. According to this model, when the amount of free E2F (released from repression of RB) is below the threshold, E2F activates only growth-related target genes. When the amount of free E2F exceeds the threshold, E2F activates not only growth-related targets but also pro-apoptotic targets. However, molecular mechanism of how different amount of free E2F differentially regulates target genes is not yet elucidated. The 'quantitative' difference of free E2F seems not sufficient to explain the four differences between deregulated E2F and physiologically activated E2F. The above-mentioned differences strongly suggest the presence of qualitative difference between physiologically activated E2F by 'temporal release' from RB and deregulated E2F induced by 'dysfunction' of the RB pathway [75-77] (Figure 9). The 'qualitative' difference between both of the E2Fs seems to be a useful cue to elucidate how cells discriminate oncogenic growth stimulation from physiological growth stimulation.
5. Conclusion and further research
Accumulating evidence indicates that E2F plays essential roles in cell-fate determination, to grow, stop or die. Together with G1/S gatekeeper RB, E2F governs control of the restriction point, deciding whether to grow or not. Upon normal growth stimulation, physiologically activated E2F facilitates cell proliferation. Upon dysfunction of the RB pathway, deregulated E2F suppresses cell growth by inducing p27Kip1 to restrain cells from aberrant cell growth. p21Cip1, induced by p53 through activation of the ARF gene, may also contribute to suppression of cell growth. When the arrest mechanism failed to stop the aberrant cell cycle progression, E2F induces apoptosis through activation of p53 and TAp73 to protect cells from tumorigenesis. E2F seems to sense and discriminate between normal growth signals and abnormal growth signals originating from various oncogenic changes, asking cells whether to grow, stop or die.
Deregulated E2F activity specifically exists in cancer cell lines but not in normal growing fibroblasts, suggesting that deregulated E2F activity may be a useful means to discriminate abnormally growing cancer cells from physiologically growing normal cells. Since the generation of deregulated E2F activity is expected to be based on the mechanism of oncogenesis, deregulated E2F activity could be a universal marker to discriminate cancer cells from normal growing cells. Analyses of atypical E2F targets suggest that deregulated E2F might be qualitatively different from physiologically activated E2F. One of the most intriguing issues in the future studies would be the molecular nature of deregulated E2F. By elucidating the molecular nature of deregulated E2F, we might be able to specifically approach cancer cells without affecting normal growing cells. For this purpose, qualitative difference between deregulated E2F and physiological E2F is eager to be elucidated.
This work is supported by a Grant in-Aid for Science Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (21570180 to K. Ohtani and 22 2446 to E. Ozono)
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2.Major tumor-suppressor pathways
3.The transcription factor E2F
4.Deregulated E2F
5.Conclusion and further research
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Eiko Ozono, Shoji Yamaoka and Kiyoshi Ohtani (April 10th 2013). To Grow, Stop or Die? – Novel Tumor-Suppressive Mechanism Regulated by the Transcription Factor E2F, Future Aspects of Tumor Suppressor Gene, Yue Cheng, IntechOpen, DOI: 10.5772/54510. Available from:
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Human Smuggling: Q&A With a Real-Life Coyote on the Border Trade
by Ed Calderon
The coyote is commonly used as a representation of mischief among native peoples in the Americas. The word now has been given to a very different group of characters. Trickery and performing miracles is part of who they are. However, that is about all they have in common with the gods of old.
Coyote is a term commonly used for human smugglers that work in Mexico, a profession that dates back to 1882 and the U.S. Chinese Exclusion Act that cut the flow of cheap labor from Asia into the States. Demand for workers from Mexico filled the void. I managed to convince one of these coyotes to do this interview with questions I sourced from some of my followers on social media.
Human Smuggling in the Eyes of Our Coyote
In his late 30s, soft spoken and a chain smoker, the man I'm about to talk to doesn't look like anyone special. You wouldn't guess that he is one of the most successful coyotes working on the border. Some coyotes just guide people across to specific points along the border; others, like the man I'm about to interview, do so much more.
Two cell phones sit on the table; one is for family and one is for business.
We are sitting outside a seafood place as we talk, and he is understandably nervous as he keeps responding to texts on his phone, ordering his people to do this and that while writing in a little moleskin notebook. I order a few beers to ease the mood, and when they get there, he waves his away, saying with a very serious tone, "I'm working, so no alcohol."
I met this coyote through a contact I have had for years that works providing aid to the migrant communities that live on the border. My contact is a former member of the Catholic Church who now works as an intermediary between people just arriving at the border and people that have been deported. He puts them in touch with the coyote.
This coyote has been in the game for years and he knows it well. His success is a product of word of mouth and recommendations from people who have successfully made it across. He embraced technology early on and is no stranger to things like crypto currency, drone technology and countermeasures against most of the modernized security cameras and monitoring systems now in use on
the border.
After a few days of phone conversations through third parties, he agreed to do this interview.
EC: How did you get into this line of work?
A: I immigrated myself legally years back but found myself in some legal trouble. I was deported because of this. I wanted to get back to my family that I left behind, so I went to a few people (smugglers) that got recommended to me by some of the people that were with me during my deportation process. They told me who to seek out. I tried three times unsuccessfully to get back across. I was basically robbed by some smugglers the second time I tried and left for dead in the desert. They took the last of my money from me.
After that, I figured I'd try my best to do it myself, without any help. I went online and downloaded maps, went to forums to look for people giving out advice about it, studied a few army survival manuals and basically educated myself on how to survive. I figured out fast to look out for larger groups of migrants that would be moving across. The border patrol was stretched out and preferred to go after big groups of migrants instead of individuals. I shadowed a few groups like this as I made my way up. I made it back home to Northern California three days later. The thing with my family didn't work out, as they tend to do when you are gone for a few years. My wife was with someone else when I got back, and well, that didn't really work for me.
My first night back, I got into it with the new boyfriend and the cops were called. I ran and without much to really live for up there, I spent a long night of reflection at a Motel 6.
I was doing the math in my head of how much I had paid in failed crossings and how I could really do better than some of these people. At the border, I went back to the U.S. side and started developing some contacts and relationships with people.
One of the men I met was a guy in his late 60s who gave me a break. He let me help him out with running some safe houses where migrants would be holed up before being moved, once they were across the border and in the U.S. I made it a point to be nice to everyone, and I gave my number out to everyone that passed through that house. After a while, they were calling me to set up crossings for their family members and my business started to grow. The old man who took me in passed away, and his sons were not ready to take on the workload, so, like any good businessman, I took control of the business.
I then made my way back down to Mexico again to manage everything from there, and I have been doing it ever since.
EC: How does this work? If I wanted to hire you now to get me across, what would the process look like?
A: First off, you would never talk to me directly. I don't do work for anyone that knows my face; that is just how I do business. I have people that do that for me. We don't advertise online, and we don't have ads in the paper. We mainly work through word of mouth, and we get recommended by people that we successfully cross into the U.S.
So, you would call a number to ask about our services. One of our guys or girls gets your information, and then we call back and make contact with you after we make sure you are who you say you are. We have one face-to-face meeting and go from there.
Next, there is no such thing as a single flat fee — at least not for us. We charge depending on how individuals want to get across. It depends on the risk they have, their ages and the type of crossing we use to get them in, etc. Some people are harder to move than others. For example, if it's a mother with a small child or an elderly person with special needs, they won't be easy or cheap to get across. We ask for payment up front from people like this. We are not miracle workers, and we don't offer guarantees.
EC: What effects have you felt with the Trump administration's hard stance on immigration and border security?
A: We have raised prices because of it, but it really hasn't become harder to get people across. It is more that people are no longer trying to cross in big numbers, like a few years back, because they no longer feel that they are welcomed in the U.S. We see people looking to Canada now, more and more. We take advantage of the feeling of it being harder to cross. The truth is that we have networks set up that haven't really changed for years. Our halfway houses are still there, and we are still getting people across, and we are still getting paid. Walls are already up, and we have already found ways around them.
EC: What can you tell me about how you get people across?
A: Without giving too much away, we have land, sea and air options for people. People often imagine a line of people crossing a border fence at night in the desert. This is not always the case.
We buy stolen passports and border crossing cards, or we rent them from people, for example.
EC: People rent their passports and IDs?
A: Yes. People have needs. We look for people on both sides of the border that are willing to do this for us, and they get their cut. You would be surprised by the types of people doing it — housewives, young men in the universities — people like that.
You want to cross? We can find you a lookalike in our catalog. We can get you an official ID to match it and even a car with the name attached to it. And you just drive across. We give them something to calm their nerves, and we coach them. And we know and observe the people that work the border crossings. We see what lanes are being manned by agents that are not really paying attention, and we aim our attempts at these lanes. This is just one example of what we do.
EC: Aren't you giving too much away?
A: We know they know. What they don't know, I won't say. There are more levels to it; that's all I can say.
EC: What role does technology play in your world?
A: From communication to payment, it's a big part of what we do. If you don't get wise about technology, it will end you. For example, I never use the same phone for different clients. I have use-and-discard phones, especially for business travel. I never bring anything electronic from work into my personal life. Also, I use public transportation to move away from my regular life and enter my business life. I change clothes and dump everything when I need to go back home. I am careful. Among other things, I worry about corrupt cops here in Mexico trying to extort money from me and about rivals trying to eliminate competition.
Crypto currency is something we now use and drones, as well. We can see the people that want to see us before they see us. We have eyes, too.
EC: Are you worried at all about getting arrested by Mexican police?
A: Human trafficking is a federal crime. If you get caught and have money, you get out. That is really it. So no, I don't really put that fear high on my list. I have already gotten out of it a few times. I pay people to take care of me. As long as I do that, I'm good.
EC: You mean you pay for protection? Pagas piso? (As we say in Mexico.)
A: I can't talk about that.
EC: Do you carry a gun?
A: Yes, when I need to. I don't need to now, but we have means to defend ourselves. Also, I carry cash money. I get out of more problems with my head, a smile and cash money than with bullets. People who learn this early on live longer.
EC: What type of people usually seek you out to cross?
A: I deal in high-paying customers — usually middle-class people that can pay. Some have legal problems in the U.S. or in Mexico that keep them from crossing legally. Some have large families already in the U.S. paying for them. Usually it's men, but women and children are always looking to cross, as well. I try to not deal with that too much.
EC: Are you talking about the kids from Central America that came during the Obama administration?
A: Yes. They were being brought in on buses. It was odd. I don't know exactly what was going on, but there was money behind that whole thing. Someone made a fortune moving all of them up there.
EC: What do you think about the sex-trafficking trade?
A: I don't touch anything like that. That is done by scary people that I don't associate with. But it is a very real business, and it is operating under the noses of most people in big U.S. cities and Mexico. People are delusional if they think this can't affect them. The people that work that side of things are everywhere, and you would never know it by looking at them. They go for girls who are 10 years old on up. It's a nasty business, and you really can't pay your way out of the problems that it brings. Even corrupt cops have lines they won't cross.
EC: Do you think you are exploiting your customers with what you're doing? I mean, not all of them make it, and some of them might be bad people looking to do bad things.
A: Listen, I sell hope. I do miracles. I do things immigration lawyers that went to school for years are unable to do. Some don't make it, but it's the risk they get informed about before they pay. I get prayer cards and gifts on Christmas from people I crossed years back. I get home and I sleep well. If God was against me, he would find a way to make me stop. I feel I am doing what I need to be doing. Can you do what I can do? No, of course not. I'm not angry at the question, I'm just being very clear about how I feel about it.
EC: You mentioned God. Are you a man of faith?
A: Everyone that lives dangerously is, no? I am. I go to Mass and I pay my respects.
EC: Currently in American popular culture there is this recurring depiction of people like you smuggling Muslim terrorists across the border. Is this something that you think could happen? Has it happened already?
A: It's hard to say. I would never do it, knowing that they have these intentions. I don't think anyone would, really. They are bad for business. If you want to be hunted down for letting people like this into the U.S, they will find you. That said, I have heard of and have known people that do this work of moving people from the Middle East through the border. I would never do it, but it has happened.
EC: If you could tell people who don't really know what things are like on the border about one thing to pay attention to, what would it be?
A: The new presidente is probably going to be very good for my business. That should tell you a lot about him.
EC: Thank you for the conversation.
A: Vete con cuidado, cabrón.
Check out our podcast Skillset Live and get in on the conversation with us on IG and Facebook!
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redpajama_set_name: RedPajamaCommonCrawl


I am an advocate of your products and recommend them whenever it's appropriate. Your cookbook sits on the counter in my second home. It's amazing when friends and family come for a visit. I watch them pour over the ailments page as they are asking me for advice on how to cleanse and how I did it. I even had my sister in law do a spit test while she was here for Thanksgiving, sure enough signs of yeast. I weighed in at my last doctors visit and I had dropped 17 lbs. Between the physical appearance and my energy, my friends and family are curious." Anything I can do to help promote your program and products I am happy to.


text:
is a manga illustrator and anime character designer. He has done character designs for the anime Kai Doh Maru, Otogi Zoshi, the survival horror video game Galerians: Ash, and the CGI OVA Galerians: Rion, as well as Kill Bill Chapter 3: The Origin of O-Ren. He also provided character designs and artwork for the Final Fantasy VII novel The Kids Are Alright: A Turks Side Story, some of which later appeared in the video game Final Fantasy VII Remake.

Manga works
 Bitch's Life
 Brothers
 Hunter Dark
 Madara
 Multiple Personality Detective Psycho
 Neo Devilman
 Robot: Super Color Comic

External links/References

 The Ultimate Manga Guide - Tajima Shouu

1966 births
Manga artists from Saitama Prefecture
Japanese illustrators
Living people

ko:나카하라 유우

redpajama_set_name: RedPajamaWikipedia


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Fix typos in a couple words; manager and experiment.
[wiki:Documentation/CGettingStarted Getting started on ORBIT] requires requesting and getting a user account as well as [wiki:Documentation/bAccountManagement/DSSHConf setting up SSH keys] to access to the testbed. To actually use a resource a [wiki:Documentation/bAccountManagement/aScheduler reservation] must be made on the [https://www.orbit-lab.org/loginService/ControlPanel scheduler]. Supplied on this wiki there are a number of [wiki:Documentation/FAQ FAQs] and commonly encountered [wiki:CommonProblems problems] as well as tutorials that will help new users.
There is also a great deal of documentation relating to the [wiki:Hardware hardware] which supports the testbed and is supported by it as well as the [wiki:Software software] which use used to facilitate the running of experiments, such as the [wiki:Software/eAM aggregate manger] and the [wiki:Software/cOMF experement controller (OMF)].
There is also a great deal of documentation relating to the [wiki:Hardware hardware] which supports the testbed and is supported by it as well as the [wiki:Software software] which use used to facilitate the running of experiments, such as the [wiki:Software/eAM aggregate manager] and the [wiki:Software/cOMF experiment controller (OMF)].


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You Can Now Trick or Treat with an Alien Xenomorph Bucket
Tom 4 months ago
Alien enthusiasts can start getting ready for Halloween with this impressive-looking bucket according to Kevin Burwick from MovieWeb, or they can at the very least put it up with their collection if they have one since it'd be hard to pass up on this impressive piece of work. The only thing that would make it better would be if the mouth really opened and shot forth a tinier mouth that could be used to clamp on a piece of candy. Of course given the propensity for misuse that such a thing would garner it seems safer to just keep it as it is. The only thing it seems that would make this stand out any further is if a kid came up dressed as a xenomorph, a predator, or a Marine from the Aliens movie, though it would seem that only two of those costumes might be readily recognizable to a lot of people. It's amazing to say, but this image is what the franchise is known best for since the movies and the books are only followed closely by those that have continued to be interested in the franchise for many years now.
There are quite a few people that know at least something about the xenomorphs, and while Prometheus and a couple of other movies have tried to explain their existence throughout their long history, it's still been kind of muddy since quite honestly they seem as though they were a bio-engineered species since not only are they able to survive in extreme conditions that would kill most organisms, but they're also so extremely deadly that even killing them presents a problem. From sharp claws to powerful jaws to strength that is greater than most any human to tails that can cut people in half, the xenomorphs are pure killing machines that are only susceptible to those that have a serious tendency to get violent and are smart enough to do so from a distance. The predators of course hunt these things as we've seen in the movies such as Alien vs. Predator, but they've been doing so for so long that they've learned how to combat the creatures in a manner that doesn't cause intense personal damage, and they've even made the process into a routine hunt that allows their warriors to prove themselves. Of course there have been times when they've had to nuke everything when the xenomorphs get out of hand, but that's just a part of the story.
The xenomorphs have been part of Halloween and popular culture for a while, though you're likely to see them more as part of the latter than the former since a few of their more curious qualities have etched themselves into the minds of the public thanks to how unusual they are. Acid for blood, a set of interior jaws that can punch through a skull, and of course the manner in which they're 'born'. The chest-bursting scene has been a longstanding favorite moment of many since it was so unexpected and so iconic that it's been referenced throughout the decades in a big way. From Spaceballs to Avengers: Infinity War that simple reference has been used over and over again simply because it's so great and so horrifying that people just can't get away from it. The xenomorphs have been highly popular creatures that are pure nightmare fuel and yet have given rise to a number of copycats throughout the years as well as attempts to replicate their success. Of course if not for having a couple of the greatest enemies to work against the xenomorphs might have faded out a long time ago since as scary and as awesome as they are, they can be wiped out, and as a result they breed in large numbers and tend to swarm their opponents. Only in a few cases in the movies has it been seen that they've taken on an enemy they didn't account for.
One is pretty obvious, the predators. The two races were going at one another in the books and comics before they were seen to brawl on the big screen, but the predators have a long history with the creatures, and tend to use them as well as battle them on various occasions. One could say that humans in general are their next biggest enemy, but that would be a yes and no scenario since a lot of humans have been taken out by the creatures quite easily while some have gone down with a fight. Ellen Ripley is undoubtedly one of the biggest threats that the race has ever faced since she would gladly wipe every last one of them out thanks to her history with the creatures. If a kid could somehow replicate a costume that could be recognized as Ripley then this bucket would seem like a fine addition indeed.
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→ 74 | Flughafen Berlin Brandenburg GmbH Liabilities Details of the remaining terms are shown in the liabilities movement. Liabilities movement in €k Type of liability 1. Loans and overdrafts (previous year) 2. Liabilities from payments received on account (previous year) 3. Trade accounts payable (previous year) 4. Liabilities due to shareholders (previous year) 5. Other liabilities (previous year) thereof for taxes: 1,382 (previous year 2,360) Total (previous year) Remaining term Up to 1 year 1 to 5 years 53,540 (47,812) 529 (400) 34,323 (17,927) 61 (0) 17,531 (43,830) 105,984 (109,969) 1,589,241 (1,433,737) 0 (0) 0 (0) 0 (0) 0 (0) 1,589,241 (1,433,737) Total More than 5 years 31.12.2015 31.12.2014 897,619 2,540,400 (963,992) 2,445,541 0 529 (0) 400 0 34,323 (0) 17,927 132,200 132,261 (0) 0 0 (0) 1,029,819 (963,992) 17,531 2,725,044 43,830 2,507,698 Loans and overdrafts result from the utilisation of the credit lines for the BER long-term financing, of which €2,322,859k is secured by guarantees given by shareholders and €217,541k by mortgages. Deferred income The major components of the deferred income are payments received on account for ground rent and subsidies for investments in buildings or investments for the development of land areas and subsidies for servicing and maintenance obligations. Payments received on account are discounted to the cash value as agreed in the leases and reversed over the duration of the leases. The subsidies are included as operational earnings on the basis of the useful life of the pertinent assets. Deferred taxes Deferred taxes are a consequence of deviations in value measurements of assets, liabilities and deferred expenses and income caused by differences in commercial and tax laws. In the annual accounts per 31 December 2015, the deferred tax liabilities of €20k, which essentially result from differing measurements of tangible assets and other provisions, are offset against deferred tax reimbursements of €7,669k. The option provided under Section 274 (1) second sentence HGB was exercised so that the balance has not been capitalised.
Unsere Zahlen | 75 ← The deferred tax reimbursements are primarily a consequence of the differing valuations of tangible and financials assets, inventories, pension provisions and other provisions. Deferred tax reimbursements arising from the application of tax laws related to accumulated deficits brought forward have not been measured because they cannot be offset within the next five years according to the corporate planning. The deferred taxes are based on a tax rate of 25.5%. No deferred taxes resulted from consolidation measures. Contingent liabilities In connection with the sale of GGB, BFG agreed to a limited guarantee catalogue usual on the market which includes standard guarantees and risks, especially pension provisions, the effectiveness of the retroactive effect of the collective agreement for safeguarding jobs of the GGB and the correctness of disclosed information. A balanced tax exemption obligation, limited to 31 December 2007, was given. The amount of the liability is limited to €2,053k. There are no discernible reasons to expect any claims pursuant to the liability obligation because no claims have been asserted against BFG in the past. FBB has received letters of claim from airlines, service providers, tenants and other companies requesting damages/regress payments as a consequence of the postponement of the operational startup of Berlin Brandenburg Airport originally set for 3 June 2012. The majority of the letters have initially done no more than assert the grounds for the claims; very few of the claim letters include concrete figures or they contain at most estimates. As a consequence, it is not possible at this time to make any definitive statements regarding the total amount of the claims asserted by the various claimants. FBB has reached outof-court settlements with some of the claimants. FBB assumes furthermore that a part of the claims will be clarified in the course of litigation because there are legal issues that will require clarification. Discernible risks have been given due consideration in the annual accounts by the creation of provisions. The possibility of additional risks cannot be completely excluded, but it is not possible at this time to determine their value with any degree of certainty.


text:
Longuita é um distrito peruano localizado na Província de Luya, departamento Amazonas. Sua capital é a cidade de Longuita.

Transporte
O distrito de Longuita é servido pela seguinte rodovia:
 AM-110, que liga o distrito de Tingo à cidade de Chachapoyas

Longuita


text:
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Green light for bilateral trade negotiations between US and EU
The European Council gave green light to the European Commission to enter into formal bilateral trade negotiations with the United States of America. With the mandate to negotiate the Transatlantic Trade and Investment Partnership (TTIP) deal with the US, European Member States set out the objectives the Commission should follow in the negotiations on behalf of the EU.
The 3 main elements of the mandate are:
1) Market Access
Tariffs: Removal of nearly all duties on transatlantic trade in industrial and agricultural products.
Rules of origin: Reconciliation of the EU and US approaches to facilitate trade, while taking into account the interests of the EU producers.
Trade defence measures: The EU wants to establish a regular dialogue on anti-dumping and anti-subsidy measures with the US, without prejudice to its rights to use such measures in the framework of the relevant WTO rules.
Services: Opening of service sectors by both sides towards new sectors, such as the transport sector. Recognition of European professional qualifications on the other side of the Atlantic and that EU companies and their subsidiaries will be able to operate in the US under the same conditions as American domestic companies.
Investment: Achievement of the highest levels of liberalisation and investment protection that both sides have negotiated to date in other trade deals. Guarantees of protection against expropriation, a rule of free transfer of funds, fair and equitable treatment, and a level playing field for the EU companies investing in the US.
Public procurement: Opening up access to government procurement markets at all levels of government without discrimination for European companies and an increase in transparency regarding tendering procedures and getting rid of local content requirements.
2) Regulatory issues and non-tariff barriers towards a more integrated transatlantic marketplace
Reduction of unnecessary costs ('behind-the-border') and delays for companies, while maintaining high levels of health, safety, consumer and environmental protection.
Both sides envisage a framework for a "living agreement" that allows for progressively greater regulatory convergence over time against defined targets and deadlines. This will make it possible not only to eliminate existing barriers, but also to prevent the creation of new ones in the future.
3) Addressing Shared Global Trade Challenges and Opportunities in the 21st century
Maintenance of high intellectual property rights, not necessarily harmonisation, but to identify a number of specific issues where divergences will be addressed. For the EU side, Geographical Indications (GIs) are of particular importance in that context.
Respect of social and environmental aspects of trade and sustainable development.
An economic study (MEMO/13/211) by the Centre for Economic Policy Research, London shows that EU exports to the US would go up by 28%. EU and US trade with the rest of the world would also increase by over €33 billion. If such an ambitious agreement were achieved, it is expected that every year an average European household would gain an extra €545 and the economy would be boosted by 0.5% to up to 1% of GDP, or €119 billion annually, once fully implemented.
Consumer organisations on both continents warn, however, that consumer rights should not be scarified for economic goals. The European Consumer Organisation (BEUC) calls for a transparent process which does not lead to a watering down of consumer standards as official channels allow US industry to give its input throughout the negotiation process. BEUC is concerned that excluding civil society will lead to an uninformed and imbalanced outcome. Monique Goyens, Director General of BEUC, commented: "Trade talks between the EU and US will affect almost every aspect of our life. Data protection, food safety or medical devices are not the sole prerogative of business and politicians."
Gerd Billen, president of the Federation of German Consumer Organisations said: "We expect that the negotiation leads to an upwards harmonisation and not to a further round of downwards deregulation." Delicate issues regarding consumer protection are the labelling of food products in terms of genetic modification as well as treatment of poultry with chlorine and hormone treated meat.
As soon as the US administration is ready, negotiations with the Commission can start. The launch of formal negotiations will probably take place before the summer.
MEMO/13/211 http://europa.eu/rapid/press-release_MEMO-13-211_en.htm
'Reducing Transatlantic Barriers to Trade and Investment: An Economic Assessment', Centre for Economic Policy Research, London http://trade.ec.europa.eu/doclib/docs/2013/march/tradoc_150737.pdf and http://trade.ec.europa.eu/doclib/docs/2013/march/tradoc_150738.pdf
http://ec.europa.eu/trade/policy/in-focus/ttip/
Q&A about the TTIP
http://ec.europa.eu/trade/policy/in-focus/ttip/questions-and-answers/
Source: EU Commission
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The fall brings cooler temps, warmer colours and, for me, rich memories of organizing the Ottawa Browncoats fundraising events for about 6 years with an awesome crew.
Having retired from that a couple of years back, I now choose my volunteer efforts carefully and try to make sure they still touch my heart, without breaking my body!
This year, TAV Creations will be donating to two October events and I'm making artisan pieces with reclaimed textiles for these events.
One will be the 20th annual Witches Gathering, and the other will be the Vixens Victorious Back to the 80s dance party.
This is today's bag- and as I took the photos it reminded me of that moment- if you are a Firefly/Serenity Fan, you know which one I mean….
Previous PostSpeed Demo- Chiffon overskirt with French seamsNext PostFunky bags finished!


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nicole c mullen husband
I remember him often praying and thanking the Lord for what he had been given. I want to explore every avenue available to express this truth.". This article originally appeared on Nashville Tennessean: Nicole C. Mullen describes being physically abused by her first husband in a new book, Like us on Facebook to see similar stories, Live updates on voting on Election Day: USPS misses deadline; misleading robocalls, isolated incidents of glitches, Chipotle struggles with staffing as coronavirus cases rise. They are not the ones who give up easily. ©2020 Growthtrac Ministries. The abuse started before the couple was married, Mullen said. Everybody has a story to tell. Nicole's life path number is 9. Mullen was previously married to CCM singer-songwriter David Mullen before they announced their divorce in 2014. What she's discovered is a closer connection to people, her fans and others. [5][6], Mullen established a mentor group for girls called "The Baby Girls Club" where she mentors youth through the arts. As if to spite and torture me even further because I asked, he would release my neck and deny me my request. At first, they thought they would try and buy the slaves but they discovered the priests would just acquire more slaves to make an even greater profit. After three years, Mullen ran from the house one night while her husband was in the shower. Mullen said she wrote the book to mark the 20th anniversary of her hit, My Redeemer. Nicole C. Mullen has not been previously engaged. She was born and raised in Cincinnati, Ohio. Mom Nicole snapped a photo of the vehicle turned on its side and shared it with her Facebook friends, accompanied by this message: "God spared my Babygirl tonight. Recently she appeared on The Today Show, Marcus and Joni Lamb/ Table Talk on Daystar, several recordings On TBN Praise, (a soon to be aired) broadcast of "Better Together" with Laurie Crouch, Life Today with Shelia Walsh and other media outlets. Mullen said she already has heard from women who've identified with her story, making it worth whatever pain she went through to write it. Though they're economic and cautious, they can also be indecisive. He was a man of easy smiles, few words and a man of faith. He yelped. She told the Tennessean that, through a third party, she offered him forgiveness before he died. Continuing to talk about her clothing line she says, "What is so ironic is I was the one in the family (she grew up in) who couldn't put two things together to save my life. That is the one that should be consistent with everything else that I do. Nicole Started as a Background singer for Amy Grant, Michael W. Smith, Cece Winans, and The Newsboys. "The essence of the whole album is that you take care of the seed. Nicole, now a single mom, is familiar with broken relationships. [3] In 1993, she married singer, songwriter and music producer David Mullen. Bookmark this page and come back for updates. To understand this wonderful lady who despite having numerous chart stopping hit songs and having reached a stature in the music industry that few attain at still a relatively young age, you must understand her heritage. Sources. He beats me today, and I'm going to have to forgive him. ", "...when i wasn't feeling so strong or optimistic, I recall begging him to finish the job as he held my neck against the wall. ... "The Lord has healed me and I pray for my ex-husband's healing as well. Her first solo album, Don't Let Me Go, was released in 1991. Contemporary Christian music artist Nicole C. Mullen gives her first detailed and chilling accounts of the violence she said she suffered in her first marriage in a new book, My Redeemer Lives: It's Personal — A Story of Hope for Our Time. "I thought, hey, God hey god may want me to do the hard thing. After staying with friends, she told her parents and started a life as a touring backup singer and choreographer. Earlier this year, former Host of Regis and Lee and NBC's The Today Show, Kathie Lee Gifford along with Nicole C. Mullen, recorded a song they had written together, entitled "The God Who Sees." Gifford soon became one of Mullen's biggest cheerleaders and personally spent close to a half of a million dollars to add a 60 piece orchestra to the song and shoot a twelve-minute film to their oratorio. Nicole C. Mullen has not been previously engaged. I couldn't believe it, nor had I seen it coming. She worked as a dancer with singer Amy Grant. — Nicole C. Mullen, page 49, My Redeemer Lives. In the past, she frequently graced the stages of Dr. Billy Graham, Franklin Graham, and Women of Faith events. It became the first of two occasions I struck back. These are the stories later expressed by Nicole, before the cameras and Directed by Kathie Lee. The least compatible signs with Capricorn are generally considered to be Aries and Libra. [2], In 2016, it was reported that Mullen had entered into a relationship with minister/singer Donnie McClurkin. As of 2020, Nicole C. Mullen is possibly single. You do the best you can do and leave the results up to God. "There's not an ounce of hate in my life or in my heart for him," Mullen said. Mullen is the only African-American artist to win the Dove Award for Song of the Year, and Songwriter of the Year. Her fourth album, Talk About It, was certified gold for sales in excess of 500,000 copies on April 15, 2008. Network USA. I was in shock. Mullen never saw her first husband again. For a split second, I started to do nothing and just take it, as I was accustomed. They were able to laugh, to cry, to be overjoyed, express emotion and gratitude. It became the first of two occasions I struck back. That is the essence of what happened to me as a child and I want to pass that onto my children now," says Mullen. They are lodged in between the two big well-known generations, the Baby Boomers and the Millennials. It was life changing and I saw the likeness of them in myself. Nicole C. Mullen © 2017 / All Rights Reserved / Designed & Developed by Visuals by Glenn Patrick. As long as one of us is enslaved then we are all slaves. Although they fail to express themselves, they think and work out a solution for problems in relationships. I believe the test is, who brought you through and how it brought you through," she said. This page is updated often with latest details about Nicole C. Mullen. But while he's sorry, he feels nothing. In the book, she detailed him hitting her, and being emotionally and verbally abusive. I had some stinkin' thinkin'," Mullen told the Tennessean. I want them to see the power of Redemption and the relevancy of the Scriptures in our everyday living. Life skills may be as simple as teaching them how to make clothes, soaps and powders or dying fabric, tools that they can use to provide for themselves.", Reflecting upon the heritage of the African American in the United States, Mullen observes, "I often say had there not been men and women of different colors of skin from light to dark, different walks of life, different religious backgrounds who spoke up on my behalf when I was in the loins of my ancestors who were slaves, and spoke up on my behalf for freedom, then I would not be here today singing to you. Certainly earlier this year Mullen took another big step in realizing her goal when she traveled to Ghana in west Africa to take part in ministering spiritually and practically to ex Trokosi slaves. Reach Brad Schmitt at brad@tennessean.com or 615-259-8384 or on Twitter @bradschmitt. Mullen never saw her first husband again. While driving, he raised his fist and punched me in the face. McClurkin has never been married, he is a single father of two. Let's take a look at Nicole C. Mullen past relationships, ex-boyfriends and previous hookups. She was signed to Word Records in 1998 by VP of A&R Brent Bourgeois. Her husband ended up smashing the glass on the floor next to her. Her ministry and travels include France, Belize, England, Jamaica, many nations on the continent of Africa and throughout the USA. "I took my thirteen year old Jasmine and we had a chance firsthand to see what they do. Selected discography. Mullen never reveals her first husband's name, adding he got remarried after their divorce, and that he died from a heart attack in 2009. Is there an opportunity to plant a seed so that God can get the increase later? What she's discovered is a closer connection to people, her fans and others. Mullen has been married twice. They were the type that said, if this is what God has in store for you, He will make a way and He will provide. In the book, Mullen reveals several times she wanted to die during her three-year marriage in the late 1980s to an assistant church pastor in Dallas. They are creative thinkers and appreciate the arts. We were newly engaged, and a dispute broke out between us on a subject that I can't even remember. While studying at Christ for the Nations Institute in Dallas, she joined a vocal group called Living Praise and began pursuing a music career. – When is Nicole C. Mullen's birthday? Fact Check: We strive for accuracy and fairness. She was born on Tuesday, January 3, 1967. In a Facebook post, she cited "Biblical reasons" for the divorce. She has not been previously engaged. [2] Under the name Nicole, she recorded her first solo album, Don't Let Me Go, in 1991 with the independent label Frontline Records, and produced by Tim Miner. He said something I disagreed with and before I knew it, he slapped me and then held me against the wall. Nicole was born in the 1960s. If you do that, it may turn out to be a harvest and one that blesses you later. Is Nicole C. Mullen married or single, and who is she dating now? Nicole C. Mullen spoke to me on the phone from her home in Nashville earlier this year and it was towards the end of our time together that two things emerged, to really define who this wonderfully talented, compassionate and gentle spirited lady is. "God has overshadowed that evil with good.". They didn't remind me that we didn't have the money to do what I felt were my dream in life. The stories shared about its effects are countless, some even miraculous and it further confirms that although Nicole is the sole writer, it's origin is timeless and from above. [citation needed], Learn how and when to remove this template message, Best Pop/Contemporary Gospel Album of the Year, "New couple alert? He yelped. After three years, Mullen ran from the house one night while her husband was in the shower.
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