diminutive
The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells. To execute these
actions, c-Myc is thought to regulate functionally diverse sets of genes that directly govern cellular mass and progression through
critical cell cycle transitions. Several lines of evidence are provided that c-Myc promotes ubiquitin-dependent proteolysis by
directly activating expression of the Cul1 gene, encoding a critical component of the ubiquitin ligase SCFSKP2. The cell cycle
inhibitor p27kip1 is a known target of the SCFSKP2 complex, and Myc-induced Cul1 expression matches well with the kinetics of
declining p27kip1 protein. Enforced Cul1 expression or antisense neutralization of p27kip1 is capable of overcoming the slow-growth phenotype of c-Myc null
primary mouse embryonic fibroblasts (MEFs). In reconstitution assays, the addition of in vitro translated Cul1 protein alone is able to restore p27kip1
ubiquitination and degradation in lysates derived from c-myc/MEFs or density-arrested human fibroblasts. These functional and biochemical
data provide a direct link between c-Myc transcriptional regulation and ubiquitin-mediated proteolysis and together support the view that c-Myc promotes G1
exit in part via Cul1-dependent ubiquitination and degradation of the CDK inhibitor, p27kip1 (O'Hagan, 2000).
Activated lymphocytes must increase in size and duplicate their contents (cell growth) before they can divide. The molecular events that control cell growth in proliferating lymphocytes and other metazoan cells are still unclear. Transgenesis has been utilized to provide evidence suggesting that the basic helix-loop-helix-zipper (bHLHZ) transcriptional repressor Mad1, considered to be an antagonist of Myc function, inhibits lymphocyte expansion, maturation and growth following pre-T-cell receptor (pre-TCR) and TCR stimulation. Furthermore, cDNA microarray technology was used to determine that of the genes repressed by Mad1, the majority (77%) are involved in cell growth, which correlates with a decrease in size of Mad1 transgenic thymocytes. Over 80% of the genes repressed by Mad1 have previously been found to be induced by Myc. These results suggest that a balance between Myc and Mad levels may normally modulate lymphocyte proliferation and development in part by controlling expression of growth-regulating genes (Iritani, 2002).
c-Myc promotes cell growth and transformation by ill-defined mechanisms. cmyc-/- mice die by embryonic day 10.5 (E10.5) with defects in growth and in cardiac and neural development. The lethality of cmyc-/- embryos is also associated with profound defects in vasculogenesis and primitive erythropoiesis. Furthermore, cmyc-/- embryonic stem (ES) and yolk sac cells are compromised in their differentiative and growth potential. These defects are intrinsic to c-Myc, and are in part associated with a requirement for c-Myc for the expression of vascular endothelial growth factor (VEGF), since VEGF can partially rescue these defects. However, c-Myc is also required for the proper expression of other angiogenic factors in ES and yolk sac cells, including angiopoietin-2, and the angiogenic inhibitors thrombospondin-1 and
angiopoietin-1. Finally, cmyc-/- ES cells are dramatically impaired in their ability to form tumors in immune-compromised mice, and the small tumors
that sometimes develop are poorly vascularized. Therefore, c-Myc function is also necessary for the angiogenic switch that is indispensable for the progression and metastasis of tumors. These findings support the model wherein c-Myc promotes cell growth and transformation, as well as vascular and hematopoietic development, by functioning as a master regulator of angiogenic factors (Baudino, 2002).
Hedgehog pathway activation is required for expansion of specific neuronal precursor populations during development and is etiologic in the human cerebellar tumor, medulloblastoma. Sonic hedgehog (Shh) signaling upregulates expression of the proto-oncogene Nmyc in cultured cerebellar granule neuron precursors (CGNPs) in the absence of new protein synthesis. The temporal-spatial expression pattern of Nmyc, but not other Myc family members, precisely coincides with regions of hedgehog proliferative activity in the developing cerebellum and is observed in medulloblastomas of Patched (Ptch) heterozygous mice. Overexpression of Nmyc promotes cell-autonomous G1 cyclin upregulation and CGNP proliferation independent of Shh signaling. Furthermore, Myc antagonism in vitro significantly decreases proliferative effects of Shh in cultured CGNPs. Together, these findings identify Nmyc as a direct target of the Shh pathway that functions to regulate cell cycle progression in cerebellar granule neuron precursors (Kenney, 2003).
The activity of adult stem cells is essential to replenish mature cells constantly lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. Genetic evidence is provided for an unexpected function of the c-Myc protein in the homeostasis of hematopoietic stem cells (HSCs). Conditional elimination of c-Myc activity in the bone marrow (BM) results in severe cytopenia and accumulation of HSCs in situ. Mutant HSCs self-renew and accumulate due to their failure to initiate normal stem cell differentiation. Impaired differentiation of c-Myc-deficient HSCs is linked to their localization in the differentiation preventative BM niche environment, and correlates with up-regulation of N-cadherin and a number of adhesion receptors, suggesting that release of HSCs from the stem cell niche requires c-Myc activity. Accordingly, enforced c-Myc expression in HSCs represses N-cadherin and integrins leading to loss of self-renewal activity at the expense of differentiation. Endogenous c-Myc is differentially expressed and induced upon differentiation of long-term HSCs. Collectively, these data indicate that c-Myc controls the balance between stem cell self-renewal and differentiation, presumably by regulating the interaction between HSCs and their niche (Wilson, 2004).
The stem cell niche is defined as a subset of tissue cells and extracellular substrates that can harbor one or more stem cells controlling their self-renewal and progeny production in vivo. Retention of stem cells in the niche is thought to be accomplished by stem cell niche and stem-cell extracellular matrix (ECM)-ligand interactions. It has been shown in the Drosophila ovary that DE-cadherin-mediated anchoring of germ line and somatic stem cells to the niche is essential for their maintenance. Putative niches have also been identified in vertebrates, including the bulge region in the skin epidermis and the stem cell-bearing base of intestinal crypts. In the BM, HSCs are located at the endosteal lining of the BM cavities, and recent studies show that specialized spindle-shaped N-cadherin+ osteoblasts (SNO) are a key component of the BM stem cell niche. HSCs are thought to be anchored to SNO cells via a homotypic N-cadherin interaction (Wilson, 2004 and references therein).
Although c-myc is the first proto-oncogene described to control stem cell homeostasis, some of its target genes and proteins that collaborate with Myc during tumorigenesis have recently been implicated in stem cell function. For example, the polycomb protein Bmi-1 collaborates with c-Myc during lymphomagenesis, and has been shown to be essential for maintenance of adult HSCs. The CDK inhibitor p21CIP, which is repressed by c-Myc, controls HSC proliferation, and is furthermore required for maintenance of long-term self-renewal. This suggests that part of c-Myc`s effects in HSCs could be mediated by p21 repression. Because c-Myc has been postulated to be an effector of canonical Wnt signaling and also appears to be connected to the Ang-1/Tie2-signaling pathway (referring to angiopoietin and its receptor), this protein is now evolving as a ringmaster in regulating adult stem cell function in vivo. It is thus crucial to elucidate which signaling pathways are responsible for the tight control of c-Myc expression in stem/progenitor cells. Irrespective of what niche signals fine tune c-Myc expression during the constantly changing conditions of BM homeostasis in vivo, it appears that this oncoprotein is a key element that fulfils the function of a homeostat, determining the balance between stem cell self-renewal and differentiation (Wilson, 2004 and references therein).
Myc, development and differentiation To directly assess c-myc function in processes of cellular proliferation, differentiation, and embryogenesis, both heterozygous and homozygous c-myc
mutant ES cell lines were generated using
homologous recombination in embryonic stem cells. The mutation is a null allele at the protein level. Mouse chimeras from seven
heterozygous cell lines transmitted the mutant allele to their offspring. The analysis of embryos from two
clones has shown that the mutation is lethal in homozygotes between 9.5 and 10.5 days of gestation. The
embryos are generally smaller and retarded in development compared with their littermates. Pathologic
abnormalities include the heart, pericardium, neural tube, and delay or failure in the turning of the embryo.
Heterozygous females have reduced fertility owing to embryonic resorption before 9.5 days of gestation in
14% of implanted embryos. c-Myc protein appears to be necessary for embryonic survival beyond 10.5 days of
gestation; however, it appears to be dispensable for cell division both in ES cell lines and in the embryo
before that time (Davis, 1993).
A leaky mutation has been generated in N-myc (the neural myc isoform) by gene targeting in embryonic stem cells. In this allele, the
neo(r) gene was inserted into the first intron of N-myc, in such a way that alternative splicing around this
insertion could result in the generation of a normal N-myc transcript in addition to a mutant transcript. Mice
homozygous for this mutation die immediately after birth owing to their inability to oxygenate blood.
Histological examination reveals a marked underdevelopment in the lung airway epithelium, resulting in a
decreased respiratory surface area. Analysis of N-myc expression in wild-type and homozygous mutant
embryonic lungs suggests that N-myc is required for the proliferation of the lung epithelium in response to
local inductive signals emanating from the lung mesenchyme. Homozygous mutant embryos are slightly
smaller than normal and also had a marked reduction in spleen size, whereas other tissues that normally
express N-myc appeared to be unaffected by the mutation. Molecular analysis reveals that normal N-myc
transcripts are found in tissues from homozygous mutant embryos. Different tissues expressed the normal
N-myc transcript at different levels, relative to those observed in wild-type embryos, with the lowest levels
being observed in the lungs (Moens, 1992).
To investigate liver development in mice, an N-myc mutant mouse line with abnormal liver development was used. N-myc mutant embryos die between 11.5 and 12.5 days postcoitum, most probably from heart failure. At 11.5 days of gestation, extensive apoptosis restricted to the hepatocytes occurs in N-myc mutant liver, when compared to wild-type samples. The number of hematopoietic cells is reduced in the mutant liver. During early liver organogenesis, the N-myc gene is expressed in tissues involved in the induction and the differentiation of the hepatocytes. At 11.5 days of development, both c-myc and N-myc genes are expressed in the liver. While c-myc is expressed at a high level in the organ per se, N-myc expression is mostly confined to the peripheral layer of the liver that will generate the Glisson's capsule. Taken together, the expression pattern of N-myc in the liver and the specific apoptosis of hepatocytes observed in N-myc mutants indicate that N-myc is required for hepatocyte survival and suggest that it is involved in the genetic cascade leading to normal liver development (Giroux, 1998).
The highest expression of the N-myc gene occurs during embryonic organogenesis in the mouse ontogeny,
with the peak of expression around embryonic day 9.5. Homozygous N-myc-deficient mice, produced by
germline transmission of a disrupted allele in ES cells, develop normally to day 10.5, indicating
dispensability of N-myc expression in the earlier period, but later lin their development, they accumulate organogenic abnormalities
and die around day 11.5. The most notable abnormalities are found in the limb bud, visceral organs (lung,
stomach, liver and heart) and the central/peripheral nervous systems; all abnormalities are highly correlated with the
site of N-myc expression. The limb buds and the lungs excised from N-myc-deficient mutant embryos were
placed in culture to allow their continued development to stages beyond the point of death of the embryos. Analyses
indicates that the mutant limbs fail to develop distal structures and the development of bronchi from the
trachea is defective in the lungs. The latter defect is largely corrected by addition of fetal calf serum to
the culture medium, suggesting that an activity missing in the mutant lung is replenished by a component
of the serum. The phenotype of N-myc-deficient mutant embryos indicates requirement of the N-myc
function in many instances of tissue interactions in organogenesis and also in cell-autonomous regulation of
tissue maturation (Sawai, 1993).
Expression of the N-myc gene has also been examined in embryos during
postimplantation development using RNA in situ hybridization. Tissue- and cell-specific patterns of
expression unique to N-myc were observed, as compared with the related c-myc gene. N-myc transcripts
become progressively restricted to specific cell types, primarily to epithelial tissues including those of the
developing nervous system and those in developing organs characterized by epithelio-mesenchymal
interaction. In contrast, c-myc transcripts are confined to the mesenchymal compartments. These data
suggest that c-myc and N-myc proteins may interact with different substrates in performing their function
during embryogenesis and suggest further that there are linked regulatory mechanisms for normal expression
in the embryo. The N-myc locus was mutated via homologous recombination in embryonic stem (ES)
cells and the mutated allele was then introduced into the mouse germ line. Live-born heterozygotes are
under-represented but appear normal. Homozygous mutant embryos die prenatally at approximately 11.5
days of gestation. Histologic examination of homozygous mutant embryos indicates that several developing
organs are affected. These include the central and peripheral nervous systems, mesonephros, lung, and gut.
Thus, N-myc function is required during embryogenesis, and the pathology observed is consistent with the
normal pattern of N-myc expression. Examination of c-myc expression in mutant embryos indicates the
existence of coordinate regulation of myc genes during mouse embryogenesis (Stanton, 1992).
During neural crest development in avian embryos, transcription factor N-myc is initially
expressed in the entire cell population. The expression is then turned off in the period
following colonization in ganglion and nerve cord areas except for the cells undergoing
neuronal differentiation. This is also recapitulated in the culture of Japanese quail neural
crest; the cells expressing N-myc eventually coincided with those expressing
neurofilaments. These findings suggested that N-myc is involved in regulation of neuronal
differentiation in the neural crest cell population. In fact, transient overexpression of N-myc
in the neural crest culture by transfection results in a remarkable promotion of neuronal
differentiation. An experimental procedure was developed to examine the effect of
exogenous N-myc expression in the neural crest cells in embryos. Neural crest cell clusters
still attached to the neural tube were excised from Japanese quail embryos, transfected and
grafted into chicken host embryos. Using this chimera technique,
the consequence of transient high N-myc could be analyzed during the early phase of neural crest migration.
Two effects were demonstrated in the embryos: The epidermis contains two types of proliferative keratinocyte: stem cells, with unlimited self-renewal
capacity, and transit amplifying cells, those daughters of stem cells destined to withdraw from
the cell cycle and terminally differentiate after a few rounds of division. In a search for factors that
regulate exit from the stem cell compartment, c-Myc was constitutively expressed in primary human
keratinocytes by use of wild-type and steroid-activatable constructs. In contrast to its role in other cell
types, activation of c-Myc in keratinocytes causes a progressive reduction in growth rate, without
inducing apoptosis, and a marked stimulation of terminal differentiation. Keratinocytes can be enriched
for stem or transit amplifying cells on the basis of beta1 integrin expression. By the use of this method
to fractionate cells prior to c-Myc activation, it is found that c-Myc acts selectively on stem cells,
driving them into the transit amplifying compartment. As a result, activation of c-Myc in epidermis
reconstituted on a dermal equivalent leads to premature execution of the differentiation program. The
transcriptional regulatory domain of c-Myc is required for these effects because a deletion within
that domain acts as a dominant-negative mutation. These results reveal a novel biological role for c-Myc
and provide new insights into the mechanism regulating epidermal stem cell fate (Gandarillas, 1997).
Mad-Max heterodimers have been shown to antagonize Myc transforming activity by a mechanism
requiring multiple protein-protein and protein-DNA interactions. However, the mechanism by which
Mad functions in differentiation is unknown. Mad functions by an
active repression mechanism to antagonize the growth-promoting function(s) of Myc and bring about a
transition from cellular proliferation to differentiation. Exogenously expressed
c-Myc blocks inducer-mediated differentiation of murine erythroleukemia cells without disrupting the
induction of endogenous Mad; rather, high levels of c-Myc prevent a heterocomplex switch from
growth-promoting Myc-Max to growth-inhibitory Mad-Max. Cotransfection of a constitutive c-myc
with a zinc-inducible mad1 results in clones expressing both genes, whereby a switch from proliferation to differentiation can be modulated. Whereas cells grown in N'N'-hexamethylene bisacetamide in the absence of zinc fail to differentiate, addition of zinc up-regulates Mad expression by severalfold and
differentiation proceeds normally. Coimmunoprecipitation analysis reveals that Mad-Max complexes
are in excess of Myc-Max in these cotransfectants. The Sin-binding, basic
region, and leucine zipper motifs are each required for Mad to function during a molecular switch from
proliferation to differentiation (Cultraro, 1997).
Two novel Mad1- and Mxi1-related proteins, Mad3 and Mad4, interact with both Max and mSin3 and repress transcription from a promoter containing CACGTG binding sites. Both Mad3 and Mad4 inhibit c-Myc dependent cell transformation.
An examination of the expression patterns of all mad genes during murine embryogenesis reveals that mad1, mad3 and mad4 are
expressed primarily in growth-arrested differentiating cells. mxi1 is also expressed in differentiating cells, but is co-expressed with
either c-myc, N-myc, or both in proliferating cells of the developing central nervous system and the epidermis. In the developing
central nervous system and epidermis, downregulation of myc genes occurs concomitant with upregulation of mad family genes.
These expression patterns, together with the demonstrated ability of Mad family proteins to interfere with the proliferation promoting
activities of Myc, suggest that the regulated expression of Myc and Mad family proteins function in a concerted fashion to regulate
cell growth in differentiating tissues (Hurlin, 1995).
The switch from transcriptionally activating MYC-MAX to transcriptionally repressing MAD1-MAX protein heterodimers has been correlated with the initiation of terminal differentiation in many cell types. To investigate the function of MAD1-MAX dimers during differentiation, the Mad1 gene was disrupted by
homologous recombination in mice. Analysis of hematopoietic differentiation in homozygous mutant animals reveals that cell cycle exit of granulocytic precursors is inhibited following the colony-forming cell stage, resulting in increased proliferation and delayed terminal differentiation of low proliferative potential cluster-forming cells. Surprisingly, the numbers of terminally differentiated bone marrow and peripheral blood granulocytes are essentially unchanged in Mad1 null mice. This imbalance between the frequencies of precursor and mature granulocytes is correlated with a compensatory decrease in granulocytic cluster-forming cell survival under apoptosis-inducing conditions. Recovery of the peripheral granulocyte compartment following bone marrow ablation is significantly enhanced in Mad1 knockout mice. Two Mad1-related genes, Mxi1 and Mad3, are expressed ectopically in adult spleen, indicating that functional redundancy and cross-regulation between MAD family members may allow for apparently normal differentiation in the absence of MAD1. These findings demonstrate that MAD1 regulates cell cycle withdrawal during a late stage of granulocyte differentiation, and suggest that the relative levels of MYC versus MAD1 mediate a balance between cell proliferation and terminal differentiation (Foley, 1998).
Members of the myc family of cellular oncogenes have been implicated as transcriptional regulators in pathways that govern cellular
proliferation and death. In addition, N-myc and c-myc are essential for completion of murine embryonic development. However, the
basis for the evolutionary conservation of the myc gene family has remained unclear. To elucidate this issue, mice in which
the endogenous c-myc coding sequences have been replaced with N-myc coding sequences were generated. Strikingly, mice homozygous for this
replacement mutation can survive into adulthood and reproduce. Moreover, when expressed from the c-myc locus, N-myc is similarly
regulated and functionally complementary to c-myc in the context of various cellular growth and differentiation processes. Therefore, the myc gene family must have
evolved, to a large extent, to facilitate differential patterns of expression (Malynn, 2000).
The neural crest, a population of multipotent progenitor cells, is a defining feature of vertebrate embryos. Neural crest precursor cells arise at the neural plate border in response to inductive signals, but much remains to be learned about the molecular mechanisms underlying their induction. The protooncogene c-Myc is an essential early regulator of neural crest cell formation in Xenopus. c-myc is localized at the neural plate border prior to the expression of early neural crest markers, such as the bHLH protein slug. A morpholino-mediated knockdown of c-Myc protein results in the absence of neural crest precursor cells and a resultant loss of neural crest derivatives. These effects are not dependent upon changes in cell proliferation or cell death. Instead, these findings reveal an important and unexpected role for c-Myc in the specification of cell fates in the early ectoderm (Bellmeyer, 2003).
To assess the critical role of Wnt signals in intestinal crypts, transgenic mice were generated ectopically expressing Dickkopf1 (Dkk1), a secreted Wnt inhibitor. Epithelial proliferation is greatly reduced coincidentally with the loss of crypts. Although enterocyte differentiation appears unaffected, secretory cell lineages are largely absent. Disrupted intestinal homeostasis is reflected by an absence of nuclear ß-catenin, inhibition of c-myc expression, and subsequent up-regulation of p21CIP1/WAF1. Thus, these data are the first to establish a direct requirement for Wnt ligands in driving proliferation in the intestinal epithelium, and also define an unexpected role for Wnts in controlling secretory cell differentiation (Pinto, 2003).
The growth inhibitory cytokine TGF-beta enforces homeostasis of epithelia by activating processes such as cell cycle arrest and apoptosis. Id2 expression is often highest in proliferating epithelial cells and declines during differentiation. Recently, Id2 expression has been found to depend on Myc-Max transcriptional complexes. TGF-beta signaling inhibits Id2 expression in human and mouse epithelial cell lines from different tissue origins. Furthermore, the observed Id2 down-regulation by TGF-beta in mouse mammary epithelial cells occurs without a concurrent drop in c-Myc levels. However, sustained Id2 repression in these cells and in human keratinocytes coincides with induction of the Myc antagonistic repressors Mad2 and Mad4, decreased formation of Myc-Max heterodimers and the replacement of Myc-Max complexes with Mad-Max complexes on the Id2 promoter. These results argue that induction of Mad expression and Id2 down-regulation are important events during the TGF-beta cytostatic program in epithelial cells (Siegel, 2003).
The Mnt gene (see Drosophila Mnt) encodes a Mad-family bHLH transcription factor located on human 17p13.3. Mnt is one of 20 genes deleted in a heterozygous fashion in Miller-Dieker syndrome (MDS), a contiguous gene syndrome that consists of severe neuronal migration defects and craniofacial dysmorphic features. Mnt can inhibit Myc-dependent cell transformation and is hypothesized to counterbalance the effects of c-Myc on growth and proliferation in vivo by competing with Myc for binding to Max and by repressing target genes activated by Myc/Max heterodimers. Unlike the related Mad family members, Mnt is expressed ubiquitously and Mnt/Max heterodimers are found in proliferating cells that contain Myc/Max heterodimers, suggesting a unique role for Mnt during proliferation. To examine the role of Mnt in vivo, mice with null (MntKO) and loxP-flanked conditional knock-out (MntCKO) alleles of Mnt were produced. Virtually all MntKO/KO mutants in a mixed (129S6 x NIH Black Swiss) or inbred (129S6) genetic background died perinatally. Mnt-deficient embryos exhibit small size throughout development and show reduced levels of c-Myc and N-Myc. In addition, 37% of the mixed background mutants displayed cleft palate as well as retardation of skull development, a phenotype not observed in the inbred mutants. These results demonstrate an important role for Mnt in embryonic development and survival, and suggest that Mnt may play a role in the craniofacial defects displayed by MDS patients (Toyo-oka, 2004).
Neural crest cells, a population of proliferative, migratory, tissue-invasive stem cells, are a defining feature of vertebrate embryos. These cells arise at the neural plate border during a time in development when precursors of the central nervous system and the epidermis are responding to the extracellular signals that will ultimately dictate their fates. Neural crest progenitors, by contrast, must be maintained in a multipotent state until after neural tube closure. Although the molecular mechanisms governing this process have yet to be fully elucidated, recent work has suggested that Myc functions to prevent premature cell fate decisions in neural crest forming regions of the early ectoderm. The small HLH protein Id3 is a Myc target that plays an essential role in the formation and maintenance of neural crest stem cells. A morpholino-mediated 'knockdown' of Id3 protein results in embryos that lack neural crest. Moreover, forced expression of Id3 maintains the expression of markers of the neural crest progenitor state beyond the time when they would normally be downregulated and blocks the differentiation of neural crest derivatives. These results shed new light on the mechanisms governing the formation and maintenance of a developmentally and clinically important cell population (Light, 2005).
Following their emigration from the neural tube, early migratory neural crest cells initially retain their stem cell-like characteristics, including the potential to contribute to the sensory neuronal, autonomic neuronal, glial, smooth muscle and ectomesenchymal lineages. However, these cells soon begin responding to signals that direct their development into specific neural crest derivatives, as evidenced by the downregulation of pan-neural crest markers expressed by the early progenitor population, and the expression of markers characteristic of specific differentiating lineages. Enforced misexpression of Id3 in the migratory neural crest population maintains the expression of markers characteristic of the progenitor state, and delays or prevents the differentiation of neural crest derivatives. For example, Id3-expressing cells sustain robust expression of Sox10, a factor that has itself been implicated in the maintenance of stem cell identity, and Slug, an important regulatory protein expressed by all neural crest precursor cells, long beyond the time that most neural crest cells on the control side of the embryo have downregulated these factors. Importantly, Id3 expression does not appear to irreversibly alter the potential of these cells. Once their pool of Id3 has turned over, neural crest cells are capable of responding to signals that direct the formation of specific derivatives such as melanocytes. These findings suggest a model in which Id family proteins expressed in the neural crest progenitor pool help control the timing with which these cells respond to differentiative signals during normal development. An alternative explanation of these findings, however, in which Id3 dictates the outcome of neural crest cell fate determination in a dose-dependant fashion, cannot br formally rule out. Future studies might profitably explore whether controlling the timing of release from Id3 activity can lead to excess recruitment of neural crest progenitors to fates other than melanocytes (Light, 2005).
Understanding how lung progenitor cells balance proliferation against differentiation is relevant to clinical disorders such as bronchopulmonary dysplasia of premature babies and lung cancer. Previous studies have established that lung development is severely disrupted in mouse mutants with reduced levels of the proto-oncogene Nmyc, but the precise mechanisms involved have not been explored. Nmyc expression in the embryonic lung is normally restricted to a distal population of undifferentiated epithelial cells, a high proportion of which are in the S phase of the cell cycle. Overexpression of NmycEGFP in the epithelium under the control of surfactant protein C (Sftpc) regulatory elements expands the domain of S phase cells and upregulates numerous genes associated with growth and metabolism, as shown by transcriptional microarray. In addition, there is marked inhibition of differentiation, coupled with an expanded domain of expression of Sox9 protein, which is also normally restricted to the distal epithelial compartment. By contrast, conditional deletion of Nmyc leads to reduced proliferation, epithelial differentiation and high levels of apoptosis in both epithelium and mesenchyme. Unexpectedly, about 50% of embryos in which only one copy of Nmyc is deleted die perinatally, with similarly abnormal lungs. A model is proposed in which Nmyc is essential in the developing lung for maintaining a distal population of undifferentiated, proliferating progenitor cells (Okubo, 2005).
The developing limb serves as a paradigm for studying pattern formation and morphogenetic cell death. Conditional deletion of N-Myc (Mycn) in the developing mouse limb leads to uniformly small skeletal elements and profound soft-tissue syndactyly. The small skeletal elements are associated with decreased proliferation of limb bud mesenchyme and small cartilaginous condensations, and syndactyly is associated with a complete absence of interdigital cell death. Although Myc family proteins have pro-apoptotic activity, N-Myc is not expressed in interdigital cells undergoing programmed cell death. Evidence is provided indicating that the lack of interdigital cell death and associated syndactyly is related to an absence of interdigital cells marked by expression of Fgfr2 and Msx2. Thus, instead of directly regulating interdigital cell death, it is proposed that N-Myc is required for the proper generation of undifferentiated mesenchymal cells that become localized to interdigital regions and trigger digit separation when eliminated by programmed cell death. These results provide new insight into mechanisms that control limb development and suggest that defects in the formation of N-Myc-dependent interdigital tissue may be a root cause of common syndromic forms of syndactyly (Ota, 2007).
Myc family members play crucial roles in regulating cell proliferation, size, differentiation, and survival during development. N-myc is expressed in retinal progenitor cells, where it regulates proliferation in a cell-autonomous manner. In addition, N-myc coordinates the growth of the retina and eye. Specifically, the retinas of N-myc-deficient mice are hypocellular but are precisely proportioned to the size of the eye. N-myc represses the expression of the cyclin-dependent kinase inhibitor p27Kip1 but acts independently of cyclin D1, the major D-type cyclin in the developing mouse retina. Acute inactivation of N-myc leads to increased expression of p27Kip1, and simultaneous inactivation of p27Kip1 and N-myc rescues the hypocellular phenotype in N-myc-deficient retinas. N-myc is not required for retinal cell fate specification, differentiation, or survival. These data represent the first example of a role for a Myc family member in retinal development and the first characterization of a mouse model in which the hypocellular retina is properly proportioned to the other ocular structures. It is proposed that N-myc lies upstream of the cell cycle machinery in the developing mouse retina and thus coordinates the growth of both the retina and eye through extrinsic cues (Martins, 2008).
The transactivation of TCF target genes induced by Wnt pathway mutations constitutes the primary transforming event in colorectal cancer (CRC). Disruption of ß-catenin/TCF-4 activity in CRC cells induces a rapid G1 arrest and blocks a genetic program that is physiologically active in the proliferative compartment of colon crypts. Coincidently, an intestinal differentiation program is induced. The TCF-4 target gene c-MYC plays a central role in this switch by direct repression of the p21CIP1/WAF1 promoter. Following disruption of ß-catenin/TCF-4 activity, the decreased expression of c-MYC releases p21CIP1/WAF1 transcription, which in turn mediates G1 arrest and differentiation. Thus, the ß-catenin/TCF-4 complex constitutes the master switch that controls proliferation versus differentiation in healthy and malignant intestinal epithelial cells (van de Wetering, 2002).
c-MYC plays a central role in the proliferative capacity of many cancers, including CRC. tHE data imply that c-MYC blocks the expression of the cell cycle inhibitor p21CIP1/WAF1. The region responsible for p21CIP1/WAF1 regulation has been mapped to a 200 bp fragment of the proximal promoter.
The presence of MIZ-1 and c-MYC on this promoter suggests that c-MYC-mediated repression of p21CIP1/WAF1 occurs by a mechanism resembling c-MYC control of p15INK4b, i.e., through preventing promoter activation by the transcription factor MIZ-1. Decreased expression of c-MYC would allow MIZ-1 to activate p21CIP1/WAF1 transcription. The complementarity in the expression of c-MYC and p21CIP1/WAF1 in the intestine supports this mechanism (van de Wetering, 2002).
The carboxyl terminus of c-Myc, containing the basic region (B) and helix-loop-helix/leucine zipper
(HLH/LZ) domain, is necessary and sufficient for sequence-specific DNA binding and
heterodimerization with Max. The amino terminus, containing two highly conserved regions
termed Myc box (Mb) I and II, is necessary for transcriptional activation and repression. Both
the transactivation domain (TAD) and the BHLH/LZ domain are necessary for
biological activity. The c-MycS proteins arise from a
leaking scanning mechanism and initiate at two closely spaced downstream AUG codons, yielding
c-Myc proteins lacking ~100 amino-terminal amino acids, including the highly conserved MbI region.
Synthesis of c-MycS increases to levels comparable to c-Myc2 during rapid cell growth, and
constitutively high levels of c-MycS synthesis are found in some tumor cell lines. Transcriptional activation by c-Myc through specific E box elements is thought to be essential for its biological role.
However, c-MycS is unable to activate transcription through these elements and yet retains the ability to stimulate
proliferation, induce anchorage-independent growth, and induce apoptosis. In addition, c-MycS retains the ability to
repress transcription of several specific promoters. Furthermore, c-MycS can rescue the c-myc null phenotype in
fibroblasts with homozygous deletion of c-myc. Taken together, these data argue against the paradigm that all of the
biological functions of c-Myc are mediated by transcriptional activation of specific target genes through E box
elements (Xiao, 1998).
Cyclin E-Cdk2 kinase activation is an essential step in Myc-induced proliferation. It is presumed that this requires sequestration of G1 cell cycle inhibitors p27Kip1 and p21Cip1 (Ckis) via a Myc-induced protein. This sequestration is shown to be mediated by the protein synthesis rate of induction of cyclin D1 and/or cyclin D2. Consistent with this, primary cells from cyclin D1-/- and cyclin D2-/- mouse embryos, unlike wild-type controls, do not respond to Myc with increased proliferation, although they undergo accelerated cell death in the absence of serum. Myc sensitivity of cyclin D1-/- cells can be restored by retroviruses expressing either cyclins D1, D2 or a cyclin D1 mutant that forms kinase-defective, Cki-binding cyclin-cdk complexes. Thus, the sequestration function of D cyclins appears essential for Myc-induced cell cycle progression but dispensable for apoptosis (Perez-Roger, 1999).
The rate of the induction of cyclin D1 and/or cyclin D2 protein synthesis leads to the preferential association of p27Kip1 and p21Cip1 with cyclin D-Cdk complexes. At the same time Myc also induces cyclin E protein synthesis; the rates of induction help to promote a net gain of newly formed Cki-free cyclin E-Cdk2 complexes. These complexes become active concomitant with phosphorylation of the kinase subunit by CAK. Consistent with this model of dynamic equilibrium, cyclin E-Cdk2 kinase activity can be controlled by changes in the rates of cyclin D synthesis. Moreover, as shown with a cyclin D mutant that forms kinase-defective Cki-binding cyclin D-Cdk complexes, this link between cyclins D-Cdk and cyclin E-Cdk2 is independent of cyclin D-Cdk activity, but correlates with the ability of cyclin D-Cdk complexes to bind or sequester Ckis. This is strongly supported by the fact that the deficiency of cyclin D1-/- mouse embryo cells to respond to Myc with increased proliferation is restored by expression of the same cyclin D mutant. Consistent with these findings, transient over-expression of either catalytically inactive cyclin D-Cdk, or cyclin E-Cdk2 complexes can rescue the cell cycle inhibitory effect of a dominant-negative Mad-Myc chimera. It is concluded that due to the nature of physical interactions between cyclin D-Cdks and the cell cycle inhibitors p27Kip1 and p21Cip1, cyclin D-Cdk complexes can fulfil a dual function as cell cycle kinases and as buffers for sequestration or release of cell cycle inhibitors (Perez-Roger, 1999 and references therein).
The ability of c-MycS to repress transcription
suggests that repression of growth inhibitory genes, such as gadd45 and gas1, remains viable as an
alternative model for c-Myc molecular function. Although the transactivation-defective c-MycS protein can function in several
biological assays and can substitute for the full-length c-Myc2 in myc null cells, c-MycS may not be
able to function as full-length c-Myc2 in all assays. For example, c-MycS does not appear to cooperate
with Ras in the transformation of rat embryo fibroblasts (REFs). One explanation for these results is
that the Myc/Ras cotransformation of REF cells requires transactivation of specific myc target genes
through EMS sites that are not required for stimulation of proliferation, apoptosis, or
anchorage-independent growth. Perhaps the ability of c-Myc2 to immortalize, which may be distinct
from its ability to stimulate proliferation or induce apoptosis, is required to render REF cells susceptible
to transformation by Ras, as Ras has been shown to induce senescence.
However, one caveat in the interpretation of these negative results is that in transient transfection
assays c-MycS is expressed severalfold less in REF and other cells compared to c-Myc2. The finding of
new c-Myc target genes and perhaps new DNA-binding sites will also determine whether c-MycS has
any transactivation capabilities. Comparison of c-Myc2 and c-MycS allows the separation of the
transcriptional activation and repression abilities of c-Myc and will allow further insight into the
molecular basis for the complex and diverse biological functions of c-Myc (Xiao, 1998).
Activation of the Ras/Raf/ERK pathway extends the half-life of the Myc protein and thus enhances
the accumulation of Myc activity. Investigated were two N-terminal phosphorylation sites in
Myc, Thr 58 and Ser 62, known to be regulated by mitogen stimulation. Phosphorylation of these
two residues is critical for determining the stability of Myc. Phosphorylation of Ser 62 is required for Ras-induced stabilization of
Myc, likely mediated through the action of ERK. Conversely, phosphorylation of Thr 58, likely mediated by GSK-3 but dependent
on the prior phosphorylation of Ser 62, is associated with degradation of Myc. Further analysis demonstrates that the Ras-dependent PI-3K pathway is also
critical for controlling Myc protein accumulation, likely through the control of GSK-3 activity. These observations thus define a synergistic role for multiple
Ras-mediated phosphorylation pathways in the control of Myc protein accumulation during the initial stage of cell proliferation (Sears, 2000).
The amino acid sequence surrounding Ser 62 represents a consensus ERK recognition sequence, and evidence has been presented that ERK can
mediate the phosphorylation of Myc at Ser 62. Mutation of Ser 62 prevents mitogen- and Ras-induced stabilization of Myc. Moreover, phosphorylation at Ser 62 is enhanced under conditions
where Myc is stabilized. The importance of Ser 62 in the control of Myc stability is seen in the strict requirement
for the stabilization of Myc by Ras, but seen from work that has demonstrated an impaired transforming function when Ser 62 is altered. In contrast, phosphorylation at Thr 58 coincides with a decreased stability of Myc and mutations that prevent Thr 58 phosphorylation lead to stable Myc
protein. Once again, this coincides with work that has shown that alteration of Thr 58 enhances the transforming activity of Myc and that mutations at this site are common in Myc proteins derived from tumors. Various
lines of work suggest that the GSK-3 protein kinase is most likely responsible for the phosphorylation of Myc at Thr 58. Thr 58 lies within an established
consensus, and GSK-3 has been shown to phosphorylate Thr 58 in Myc in vitro. However,
unlike ERK, which is tightly regulated by cell growth, the level of GSK-3 protein is constant and does not fluctuate with cell growth. Nevertheless, despite the
continual presence of GSK-3 protein, the activity of the kinase is regulated during the initial phase of cell proliferation. In particular,
GSK-3 activity is inhibited through the action of PI-3K/AKT. Thus, as Ras initiates the PI-3K/AKT pathway, GSK-3 activity is held
in check, preventing the phosphorylation of Thr 58. Only when AKT activity declines would GSK-3 then have the capacity to phosphorylate Thr 58 to induce
the degradation of Myc. Thus, Ras activation elicits two responses within the cell that can cooperate to enhance Myc stability: a direct effect of ERK and an
indirect effect of AKT (Sears, 2000 and references therein).
Ski is a component of the histone deacetylase complex
required for transcriptional repression by Mad and thyroid
hormone receptor. The basic helix-loop-helix (bHLH) proteins of the Mad family act as transcriptional repressors after
heterodimerization with Max. N-CoR is required for Mad-induced transcriptional repression. The same target sequence of Mad/Max, the so-called E-box, is also recognized by a
heterodimer of Myc/Max that activates transcription. It is believed that transcriptional activation of a
group of target genes by Myc/Max enhances cellular proliferation or transformation, whereas
transcriptional repression of the same target genes by Mad/Max leads to suppression of proliferation or
induction of terminal differentiation in a wide range of cell types.
The N-CoR/SMRT complex containing mSin3 and histone deacetylase (HDAC) mediates transcriptional
repression by nuclear hormone receptors and Mad.
The oncogene v-ski was originally identified in avian Sloan-Kettering viruses, and found to transform
chicken embryo fibroblasts. Overexpression of either c-ski or v-ski induces either
transformation or muscle differentiation of quail embryo fibroblasts, depending on the growth conditions. Furthermore, v-ski transgenic mice
have increased muscle mass caused by hypertrophy of type II fast muscle fibers.
The capacity of ski to induce both transformation (growth) and differentiation, which is usually
associated with the cessation of growth, is an intriguing paradox. The human c-ski proto-oncogene
product (c-Ski) is a 728-amino-acid nuclear protein.
Recombinant c-Ski protein purified from Escherichia coli cannot directly bind to DNA, but c-Ski in
nuclear extracts from mammalian cell cultures binds to DNA, suggesting that c-Ski binds only to DNA
when associated with other proteins. The amino- and carboxy-terminal regions of
c-Ski possess a cysteine-rich and a coiled-coil region, respectively, and both regions contribute
additionally to indirect DNA binding by c-Ski. The v-Ski protein lacks 292 amino acids from the
carboxyl terminus of c-Ski, but still contains the amino-terminal cysteine-rich region. The amino-terminal region is responsible for both the cellular transformation and myogenesis
capacity of ski. The ski gene family comprises two members, ski and sno
(ski-related novel gene) and both have been shown to share clear homology in
their amino- and carboxy-terminal regions. Although it was
speculated that Ski/Sno proteins are involved in transcriptional repression of specific target genes, their function remains unknown (Nomura, 1999 and references).
The proteins encoded by the ski proto-oncogene family
directly bind to N-CoR/SMRT and mSin3A, and form a complex with HDAC. c-Ski and its related gene
product Sno are required for transcriptional repression by Mad and thyroid hormone receptor (TRbeta).
The oncogenic form, v-Ski, which lacks the mSin3A-binding domain, acts in a dominant-negative fashion,
and abrogates transcriptional repression by Mad and TRbeta. In ski-deficient mouse embryos, the ornithine
decarboxylase gene, whose expression is normally repressed by Mad-Max, is expressed ectopically. These
results show that Ski is a component of the HDAC complex and that Ski is required for the transcriptional
repression mediated by this complex. The involvement of c-Ski in the HDAC complex indicates that the
function of the HDAC complex is important for oncogenesis (Nomura, 1999).
Study of the transformation capacity of various forms of c-Ski indicate that the amino-terminal
cysteine-rich region is responsible for cellular transformation, however, the mechanism of transformation
has remained obscure. The results presented here indicate that the amino-terminal region, which is needed for cellular
transformation, is responsible for interaction with N-CoR/SMRT. Furthermore, v-Ski and the
carboxy-truncated form of c-Ski lack the carboxy-terminal mSin3A-binding domain; they abrogate
transcriptional repression by Mad by functioning in a dominant-negative fashion. Transcriptional
activation by Myc causes cell proliferation, whereas transcriptional repression by Mad inhibits cell
proliferation.
Therefore, Mad is thought to act as a tumor suppressor, and in fact, one of the mad-related genes,
mxi1, acts as a tumor suppressor using mutant mice. Therefore, abrogation of Mad-induced transcriptional repression by v-Ski may lead to induction
of Myc target genes and cellular transformation (Nomura, 1999 and references).
To address the role of N-myc in neurogenesis and in nervous system tumors, N-myc expression was conditionally disrupted in neuronal progenitor cells
(NPCs) with a nestin-Cre transgene. Null mice display ataxia, behavioral abnormalities, and tremors that correlate with a twofold
decrease in brain mass that disproportionately affects the cerebellum (sixfold reduced in mass) and the cerebral cortex, both of which
show signs of disorganization. In control mice at E12.5, a domain of high N-Myc protein expression is detected in the rapidly
proliferating cerebellar primordium. Targeted deletion of N-myc results in severely compromised proliferation as shown by a striking
decrease in S phase and mitotic cells as well as in cells expressing the Myc target gene cyclin D2, whereas apoptosis is unaffected. Null progenitor cells also have comparatively high levels of the cdk inhibitors p27Kip1 and p18Ink4c, whereas p15Ink4b, p21Cip1, and p19Ink4d levels are unaffected. Many null progenitors also exhibit altered nuclear morphology and size. In addition, loss of N-myc disrupts neuronal differentiation as evidenced by ectopic staining of the neuron specific marker ßTUBIII in the cerebrum. Furthermore, in progenitor cell cultures derived from null embryonic brain, a dramatic increase is observed in neuronal differentiation compared with controls. Thus, N-myc is essential for normal neurogenesis, regulating NPC proliferation, differentiation, and nuclear size. Its effects on proliferation and differentiation appear due, at least in part, to down-regulation of a specific subset of cyclin-dependent kinase inhibitors (Knoepfler, 2002).
Upon activation, cell surface death receptors, Fas/APO-1/CD95 and tumor necrosis factor receptor-1 (TNFR-1), are attached to cytosolic adaptor proteins, which in turn recruit caspase-8 (MACH/FLICE/Mch5) to activate the interleukin-1 beta-converting enzyme (ICE)/CED-3 family protease (caspase) cascade (see Drosophila Caspase 1). However, it remains unknown whether these apoptotic proteases are generally involved in apoptosis triggered by other stimuli, such as Myc and p53. This study suggests that a death protease cascade consisting of caspases and serine proteases plays an essential role in Myc-mediated apoptosis. When Rat-1 fibroblasts stably expressing either s-Myc or c-Myc are induced to undergo apoptosis by serum deprivation, a caspase-3 (CPP32)-like protease activity that cleaves a specific peptide substrate (Ac-DEVD-MCA) appears in the cell lysates. Induction of s-Myc- and c-Myc-mediated apoptotic cell death is effectively prevented by caspase inhibitors such as Z-Asp-CH2-DCB and Ac-DEVD-CHO. Exposing the cells to a serine protease inhibitor also significantly inhibits s-Myc- and c-Myc-mediated apoptosis and the appearance of the caspase-3-like protease activity in vivo. However, the inhibitor does not directly inhibit caspase-3-like protease activity in the apoptotic cell lysates in vitro. Together, these results indicate that caspase-3-like proteases play a critical role in both s-Myc- and c-Myc-mediated apoptosis and that caspase-3-like proteases function downstream of the protease-sensitive step in the signaling pathway of Myc-mediated apoptosis (Kagaya, 1997).
Induction of apoptosis by oncogenes like c-myc may be important in restraining the emergence of neoplasia. However, the mechanism by which c-myc induces apoptosis is unknown. CD95 (also termed Fas or APO-1) is a cell surface transmembrane receptor of the tumor necrosis factor receptor family that activates an intrinsic apoptotic suicide program in cells upon binding either its ligand CD95L or antibody. c-myc-induced apoptosis is shown to require interaction on the cell surface between CD95 and its ligand. c-Myc acts downstream of the CD95 receptor by sensitizing cells to the CD95 death signal. IGF-I signaling and Bcl-2 suppress c-myc-induced apoptosis by also acting downstream of CD95. These findings link two apoptotic pathways previously thought to be independent and establish the dependency of Myc on CD95 signaling for its killing activity (Hueber, 1997).
Pim-1 oncoprotein is a serine/threonine kinase that can closely cooperate with c-Myc in lymphomagenesis, as does Bcl-2. Although the molecular mechanism of this cooperative transformation remains unknown, it is speculated that (similar to Bcl-2) Pim-1 contributes to transformation by inhibiting apoptosis. In this study, therefore, the effect of Pim-1 expression was examined on c-Myc-mediated apoptosis of Rat-1 fibroblasts triggered by serum deprivation. Rather than inhibiting apoptosis, Pim-1 expression stimulates c-Myc-mediated apoptosis in Rat-1 fibroblasts. Pim-1 stimulates c-Myc-mediated apoptosis through an enhancement of the c-Myc-mediated activation of caspase-3 (CPP32)-like proteases, since the suppression of this activity by a specific caspase inhibitor abolishes the apoptosis stimulation by Pim-1. A kinase-defective Pim-1 mutant fails to stimulate c-Myc-mediated apoptosis; Pim-1 expression alone, in the absence of c-Myc overexpression, does not induce apoptosis of serum-deprived Rat-1 cells, indicating that the kinase activity of Pim-1 and the activated c-Myc signaling pathway are required for apoptosis stimulation by Pim-1. Together, these results suggest that Pim-1 oncoprotein stimulates as a serine/threonine kinase the death signaling elicited by c-Myc at a step upstream of caspase-3-like protease activation in Rat-1 fibroblasts. These results also suggest that Pim-1 kinase might function cooperatively with c-Myc through the phosphorylation of a factor(s) that regulates the common signaling pathway involved in c-Myc-mediated apoptosis and transformation (Mochizuki, 1997).
Nuclear factor kappaB (NF-kappaB) appears to participate in the excitotoxin-induced apoptosis of striatal medium spiny neurons. To elucidate molecular mechanisms by which this transcription factor contributes to NMDA receptor-triggered apoptotic cascades in vivo, rats were given the NMDA receptor agonist quinolinic acid (QA) by intrastriatal infusion, and the role of NF-kappaB in the induction of apoptosis-related genes and gene products was evaluated. QA administration induces time-dependent NF-kappaB nuclear translocation. The nuclear NF-kappaB protein after QA treatment is comprised mainly of p65 and c-Rel subunits as detected by gel supershift assay. Levels of c-Myc and p53 mRNA and protein are markedly increased at the time of QA-induced NF-kappaB nuclear translocation. Immunohistochemical analysis shows that c-Myc and p53 induction occurs in the excitotoxin-sensitive medium-sized striatal neurons. NF-kappaB nuclear translocation is blocked in a dose-dependent manner by the cell-permeable recombinant peptide NF-kappaB SN50, but not by the NF-kappaB SN50 control peptide. NF-kappaB SN50 significantly inhibits the QA-induced elevation in levels of c-Myc and p53 mRNA and protein. Pretreatment or posttreatment with NF-kappaB SN50, but not the control peptide, also substantially reduces the intensity of QA-induced internucleosomal DNA fragmentation. The results suggest that NF-kappaB may promote an apoptotic response in striatal medium-sized neurons to excitotoxic insult through upregulation of c-Myc and p53. This study also provides evidence indicating a unique signaling pathway from the cytoplasm to the nucleus, which regulates p53 and c-Myc levels in these neurons during apoptosis (Qin, 1999).
Human monocytic leukemia U937 cells readily undergo apoptosis when they are treated with TNF-alpha, anti-Fas antibody and anticancer drugs, such as etoposide and Ara-C. To study the mechanism of apoptosis, a novel apoptosis-resistant variant, UC, was developed from U937 cells. The UC cells show resistance to apoptosis induced by TNF-alpha, anti-Fas antibody, etoposide and Ara-C. Somatic cell hybridization between U937 and UC shows that apoptosis-resistance to TNF-alpha in UC is genetically recessive, while resistance to etoposide is dominant, suggesting that UC has at least two different mutations functionally involved in apoptosis. Mechanistic analysis reveals that UC cells express reduced amounts of c-Myc. Transfection of the c-myc gene into UC cells restores the sensitivity of the cells to undergo apoptosis induced by TNF-alpha and anti-Fas, which attributes apoptosis-resistance in this circumstance to the reduced expression of c-Myc. In contrast, c-myc transfection into UC cells can not restore their sensitivity to etoposide- and Ara-C-induced apoptosis, arguing against the role of c-myc in chemotherapy-induced apoptosis. However, treating the parental U937 cells with antisense oligonucleotides designed to reduce c-Myc expression renders the cells resistant to etoposide-induced apoptosis as well as to TNF-alpha-induced apoptosis. These results indicate that the reduced expression of c-Myc in UC is strongly associated with the resistance to etoposide-induced apoptosis. The finding that c-myc transfection into UC cannot restore the sensitivity to etoposide-induced apoptosis, suggests UC could have a second mutation that confers resistance to etoposide-induced apoptosis in a genetically dominant manner. Taken together, these present results indicate that c-Myc plays a role in cellular susceptibility to death receptor-mediated and chemotherapy-induced apoptosis (Dong, 1997).
The INK4a-ARF locus is a common target of deletion and mutation in human cancers, possibly second in frequency only to p53. The INK4a tumor suppressor locus encodes p16INK4a, an inhibitor of cyclin D-dependent kinases, and p19ARF, an alternative reading frame protein that also blocks cell proliferation. Establishment of primary mouse embryo fibroblasts (MEFs) as continuously growing cell lines is normally accompanied by loss of the p53 or p19(ARF) tumor suppressors, which act in a common biochemical pathway. Given their apparent immortalizing functions, it seems paradoxical that myc and E1A are also potent inducers of apoptosis. The sensitivity of rodent fibroblasts to myc- or E1A-induced apoptosis correlates directly with the levels of oncoprotein expression and is greatly potentiated by depriving cells of extracellular survival factors. Both Myc and E1A can induce p53 stabilization and trigger p53-dependent transcription. Several lines of evidence indicate that p53 mediates apoptosis by myc and E1A in primary fibroblasts, with p53 loss rendering cells highly resistant to their deleterious effects. For cells overexpressing myc to grow, programmed cell death must be actively suppressed. Therefore, myc overexpression should provide a strong selective pressure for events that dismantle apoptotic signaling pathways. myc rapidly activates ARF and p53 gene expression in primary MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive myc overexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. MEFs lacking ARF or p53 exhibit an attenuated apoptotic response to myc and rapidly give rise to cell lines that proliferate in chemically defined medium lacking serum. Therefore, ARF regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals (Zindy, 1998).
Overexpression of the MYC protooncogene has been implicated in the genesis of diverse human tumors. Tumorigenesis induced by MYC has been
attributed to sustained effects on proliferation and differentiation. MYC may also contribute to tumorigenesis by destabilizing the cellular
genome. A transient excess of MYC activity increases tumorigenicity of Rat1A cells by at least 50-fold. The increase in tumorigenicity persists for >30 days after the return
of MYC activity to normal levels. The brief surfeit of MYC activity is accompanied by evidence of genomic instability, including karyotypic
abnormalities, gene amplification, and hypersensitivity to DNA-damaging agents. MYC also induced genomic destabilization in normal human fibroblasts,
although these cells do not become tumorigenic. Stimulation of Rat1A cells with MYC accelerates their passage through G1/S. Moreover, MYC can force
normal human fibroblasts to transit G1 and S after treatment with N-(phosphonoacetyl)-L-aspartate (PALA) at concentrations that normally lead to arrest in
S phase by checkpoint mechanisms. Instead, the cells subsequently appear to arrest in G2. It is suggest that the accelerated passage through G1 is
mutagenic but that the effect of MYC permits a checkpoint response only after G2 has been reached. Thus, MYC may contribute to tumorigenesis through
a dominant mutator effect (Felsher, 1999a).
The targeted repair of mutant protooncogenes or the inactivation of their gene products may be a specific and effective therapy for human neoplasia. To examine this
possibility, the tetracycline regulatory system has been used to generate transgenic mice that conditionally express the MYC protooncogene in hematopoietic cells.
Sustained expression of the MYC transgene culminates in the formation of malignant T cell lymphomas and acute myleoid leukemias. The subsequent inactivation of
the transgene causes regression of established tumors. Tumor regression is associated with rapid proliferative arrest, differentiation and apoptosis of tumor cells,
and resumption of normal host hematopoiesis. It is concluded that even though tumorigenesis is a multistep process, remediation of a single genetic lesion may be
sufficient to reverse malignancy (Felsher, 1999b).
The protooncogene c-myc regulates cell growth, differentiation, and apoptosis, and its aberrant expression is frequently observed in human cancer. However, the
consequences of activating c-Myc in an adult tissue, in which these cellular processes are part of normal homeostasis, remain unknown. In order to activate the protein in adult tissue, expression of a switchable form of the c-Myc protein was targeted to the skin epidermis, a well characterized homeostatic tissue. Activation of
c-MycER in adult suprabasal epidermis rapidly triggers proliferation and disrupts differentiation of postmitotic keratinocytes. Sustained activation of c-Myc is
sufficient to induce papillomatosis together with angiogenesis -- changes that resemble hyperplastic actinic keratosis, a commonly observed human precancerous
epithelial lesion. All these premalignant changes spontaneously regress upon deactivation of c-MycER (Pelengaris, 1999).
The bmi-1 and myc oncogenes collaborate strongly in murine lymphomagenesis, but previously, the basis for this collaboration has not been understood. The ink4a-ARF tumor suppressor locus is a critical downstream target of the Polycomb-group transcriptional
repressor Bmi-1. Part of Myc's ability to induce apoptosis depends on induction of p19arf. Down-regulation of ink4a-ARF by Bmi-1 underlies its ability to cooperate with Myc in tumorigenesis. Heterozygosity
for bmi-1 inhibits lymphomagenesis in Eľ-myc mice by enhancing c-Myc-induced apoptosis. Increased apoptosis is observed in bmi-1 -/- lymphoid organs. This apoptosis can be rescued by deletion of ink4a-ARF or overexpression of bcl2. Furthermore, Bmi-1 collaborates with Myc in
enhancing proliferation and transformation of primary embryo fibroblasts (MEFs) in an ink4a-ARF dependent manner, by prohibiting Myc-mediated induction of
p19arf and apoptosis. Strong collaboration is observed between the Eľ-myc transgene and heterozygosity for ink4a-ARF. This heterozygosity is accompanied by loss of the
wild-type ink4a-ARF allele and formation of highly aggressive B-cell lymphomas. Together, these results reinforce the critical role of Bmi-1 as a dose-dependent
regulator of ink4a-ARF, which in its turn acts to prevent tumorigenesis upon activation of oncogenes such as c-myc (Jacobs, 1999).
N-myc is a transcription factor expressed in the developing metanephric kidney and other organs. In mice, complete
disruption of the N-myc gene results in fetal death on the first day of renal organogenesis. In addition to the null N-myc
allele, others have generated a hypomorphic N-myc allele. In this study, combinations of these mutant genes were used to
demonstrate that reduction in N-myc protein levels correlate with fewer developing glomeruli and collecting ducts in
embryonic kidney explants. Histological sections reveal that the mutant kidneys are hypoplastic with normal
developing structures. The data indicate that the hypoplasia is due to a reduction in proliferation rather than an increase in
apoptosis. Thus, N-myc loss causes a decrease in numbers of ureteric bud tips and developing glomeruli in explants and
hypoplastic kidneys in vivo, in a dose-dependent manner (Bates, 2000).
Ids regulate differentiation1 through sequestration of basic helix-loop-helix (bHLH) transcription factors, and the consequent inhibition of their ability to bind DNA.
Although all Id proteins are viewed as positive regulators of cell-cycle progression, this role has been firmly established only for one member of the Id family, Id2. Only Id2, and not the other members of the family, Id1 and Id3, is able to disrupt the antiproliferative effects of tumor suppressor proteins of the Rb family
(the 'pocket' proteins: Rb, p107 and p130), thus allowing cell-cycle progression. This function correlates with the ability of Id2, but not Id1 and Id3, to associate
physically with active, hypophosphorylated forms of the pocket proteins in vitro and in vivo. By inactivating Rb, Id2 is also able to abolish the function of another
growth-inhibitory protein, p16, that operates upstream of Rb (Lasorella, 2000).
The Rb-null phenotype is lethal by embryonic day 14.5 because of widespread proliferation, defective differentiation and apoptosis in the nervous
system and haematopoietic precursors. Since Id2 is expressed in these cell types at the time that Rb-null embryos die1, it is hypothesized that, if Id2 is a natural target
of Rb, manifestation of the Rb-mutant phenotype might require intact Id2 (Lasorella, 2000).
Disruption of the Rb pathway (which also includes cyclin D, cdk4/6 and p16) is a hallmark of cancer and it is widely accepted that normal Rb function must be
removed, one way or another, in all human tumors. Therefore, it was of interest to determine whether tumor cells deregulate Id2 to bypass the Rb pathway. Correct
expression of Id2 is essential to regulate proliferation and differentiation of the neural crest, thus neural crest precursor cells might be sensitive to inappropriate
expression of Id2. In humans, neoplastic transformation of neural crest precursors during embryogenesis causes neuroblastoma. Interestingly, genetic
alterations of Rb, cyclin D, cdk4/6 or p16 are absent in neuroblastoma. The genetic hallmark of neuroblastoma is amplification of the gene for a member of the
Myc family of proto-oncogenes, N-myc. Resembling enforced expression of Id2, Myc overexpression is sufficient to bypass the Rb-p16 growth-inhibitory pathway, in
spite of persistent hypophosphorylated Rb. Consequently, Myc activation may release the pressure to mutate components of the Rb-p16 pathway during
tumorigenesis (Lasorella, 2000).
Id2-Rb double knockout embryos survive to term with minimal
or no defects in neurogenesis and hematopoiesis, but they die at birth from severe reduction of muscle tissue. In neuroblastoma Id2 is overexpressed in cells carrying extra copies of the N-myc gene. In these cells, Id2 is in molar excess of the active
form of Rb. The overexpression of Id2 results from transcriptional activation by oncoproteins of the Myc family. Cell-cycle progression induced by Myc
oncoproteins requires inactivation of Rb by Id2. Thus, a dual connection links Id2 and Rb: during normal cell-cycle, Rb prohibits the action of Id2 on its
natural targets, but oncogenic activation of the Myc-Id2 transcriptional pathway overrides the tumor-suppressor function of Rb (Lasorella, 2000).
Overexpression of the proto-oncogene c-myc has been implicated in the genesis of
diverse human tumors. c-Myc seems to regulate diverse biological processes, but
its role in tumorigenesis and normal physiology remains enigmatic. An allelic series of mice has been generated in which c-myc expression is
incrementally reduced to zero. Fibroblasts from these mice show reduced
proliferation and after complete loss of c-Myc function they exit the cell
cycle. Myc activity is not needed for cellular growth but does
determine the percentage of activated T cells that re-enter the cell cycle. In
vivo, reduction of c-Myc levels results in reduced body mass owing to multiorgan
hypoplasia, in contrast to Drosophila c-myc mutants, which are smaller as a
result of hypotrophy. Drosophila myc substitutes for c-myc in fibroblasts,
indicating they have similar biological activities. This suggests there may be
fundamental differences in the mechanisms by which mammals and insects control
body size. It is proposed that in mammals c-Myc controls the decision to divide or not to divide and thereby functions as a crucial mediator of signals that determine organ and body size (Trumpp, 2001).
Although the data thus appear to suggest that Myc has different
functions in Drosophila and mice, this is contradicted by evidence
that despite their relatively weak sequence homology, both c-Myc
and dMyc proteins have similar biological activities. The results
showing that dmyc expression can at least partially rescue the
proliferation defect in c-myc-deficient mouse fibroblasts
support this view and further suggest that in mouse cells dMyc can
control target genes normally regulated by c-Myc. The difference
between flies and mice may therefore lie in the identity of the target
genes that are controlled by Myc activity in each organism, or the
way in which those target genes are integrated in the genetic
circuitry of that organism. The opposite phenotypes, organ hypotrophy in Drosophila versus hypoplasia in mice, may therefore result
from differences in the way invertebrates and mammals regulate
tissue and body size. Among insect species, organ and body size
differences appear to be a function of cell number and cell size,
whereas among mammalian species they are almost exclusively due
to variations in cell number. This may be due to a tighter
coupling of cell growth and cell division in mammals than in insects. Such a link would maintain average cell size in an expanding population and would thus make tissue size determination in mammals a function of the number of cell divisions and hence a function of Myc activity (Trumpp, 2001).
Myc overexpression is a hallmark of human cancer and promotes transformation by facilitating immortalization. This function has been linked to the ability of c-Myc to induce the expression of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), since ectopic expression of TERT immortalizes some primary human cell types. c-Myc up-regulates telomerase activity in primary mouse embryonic fibroblasts (MEFs) and myeloid cells. Paradoxically, Myc overexpression also triggers the ARF-p53 apoptotic program, which is activated when MEFs undergo replicative crises following culture ex vivo. The rare immortal variants that arise from these cultures generally suffer mutations in p53 or delete Ink4a/ARF, and Myc greatly increases the frequency of these events. Alternative reading frame (ARF)- and p53-null MEFs have increased telomerase activity, as do variant immortal clones that bypass replicative crisis. Similarly, immortal murine NIH-3T3 fibroblasts and myeloid 32D.3 and FDC-P1.2 cells do not express ARF and have robust telomerase activity. However, Myc overexpression in these immortal cells results in remarkably discordant regulation of TERT and telomerase activity. Furthermore, in MEFs and 32D.3 cells, TERT expression and telomerase activity are regulated independently of endogenous c-Myc. Thus, the regulation of TERT and telomerase activity is complex and is also regulated by factors other than Myc, ARF, or p53 (Drissi, 2001).
To explore the role of c-Myc in carcinogenesis, a reversible transgenic model of pancreatic ß cell oncogenesis has been developed using a switchable form of the c-Myc protein. Activation of c-Myc in adult, mature ß cells induces uniform ß cell proliferation but is accompanied by overwhelming apoptosis that rapidly erodes ß cell mass. Thus, the oncogenic potential of c-Myc in ß cells is masked by apoptosis. Upon suppression of c-Myc-induced ß cell apoptosis by coexpression of Bcl-xL, c-Myc triggers rapid and uniform progression into angiogenic, invasive tumors. Subsequent c-Myc deactivation induces rapid regression associated with vascular degeneration and ß cell apoptosis. The data indicate that highly complex neoplastic lesions can be both induced and maintained in vivo by a simple combination of two interlocking molecular lesions. Recent studies with other switchable oncogene transgenic models reinforce the notion that incapacitating the driving oncogenic lesion can lead to expeditious regression of tumors induced in many different tissues by c-Myc, or even T antigens. At least in principle, therefore, the complexity of the tumor phenotype need not be instructed by an equivalent complexity of genetic or epigenetic alteration. Rather, cancers may be underpinned by only a modest number of interdependent, pleiotropic lesions that present themselves as mission-critical targets for effective cancer therapies (Pelengaris, 2002).
In most postmitotic neurons, expression or activation of proteins that stimulate cell cycle progression or DNA replication results in
apoptosis. One potential exception to this generalization is neuroblastoma (NB), a tumor derived from the sympathoadrenal
lineage. NBs often express high levels of N-myc, a proto-oncogene that can potently activate key components of the cell cycle
machinery. In postmitotic sympathetic neurons, N-myc can induce S-phase entry while protecting neurons
from death caused by aberrant cell cycle reentry. Specifically, these experiments demonstrate that expression of N-myc at levels
similar to those in NBs causes sympathetic neurons to reenter S-phase, as monitored by 5-bromo-2-deoxyuridine incorporation and expression of cell cycle
regulatory proteins, and rescues them from apoptosis induced by withdrawal of their obligate survival factor, nerve growth factor. The N-myc-induced cell cycle
entry, but not enhanced survival, is inhibited by coexpression of a constitutively hypophosphorylated form of the retinoblastoma tumor suppressor protein,
suggesting that these two effects of N-myc are mediated by separate pathways. In contrast, N-myc does not cause S-phase entry in postmitotic cortical neurons. Thus,
N-myc both selectively causes sympathetic neurons to reenter the cell cycle and protects them from apoptosis, potentially contributing to their transformation to NBs (Wartiovaara, 2002).
How does N-myc mediate this S-phase entry? The results demonstrating that coexpression of hypophosphorylated pRb rescues the BrdU incorporation argues that
N-myc mediates this effect via pRb. Such an effect could be mediated by direct interactions between N-myc and pRb, and it could also
be indirectly mediated via an N-myc-induced increase in levels of the inhibitory basic helix-loop-helix protein, Id2, which binds to hypophosphorylated pRb and
inhibits its ability to lock cells out of S-phase. An additional, potentially related mechanism involves N-myc-mediated downregulation of the cyclin-dependent kinase inhibitor p27, which in fibroblasts is essential for induction of cyclin E-cdk2 kinase activity, but not for S-phase entry. Although the data
presented here do not distinguish between these alternative explanations, it has been observed that p27 levels are decreased and Id2 levels increased in sympathetic
neurons overexpressing N-myc, suggesting that decreased p27 may collaborate with increased Id2 to trigger S-phase entry (Wartiovaara, 2002).
Results reported here also indicate that N-myc overexpression does not induce S-phase entry in cortical neurons, suggesting that sympathetic and cortical neurons
are locked out of the cell cycle via distinct mechanisms. Such a difference could be predicted by considering the development of these two populations of neurons.
Cortical neurons, like most CNS neurons, induce neuronal gene expression and undergo terminal mitosis at the same time. Perturbation
of this progenitor-to-postmitotic neuron transition, for example, via functional inhibition of the pRb family or via overexpression of Id2, leads to cellular apoptosis; in no conditions yet reported do cortical cells divide while expressing a neuronal phenotype. In contrast, sympathetic neuroblasts transition through a stage in which they express a neuronal phenotype while still dividing, suggesting that the nature of terminal mitosis differs in sympathetic versus CNS neurons. In that regard, findings
may indicate that the mechanisms locking most CNS neurons out of the cell cycle are much more stringent than for sympathetic neurons (Wartiovaara, 2002).
A somewhat surprising finding reported here is that, coincident with S-phase entry, N-myc promotes enhanced survival of sympathetic neurons in the absence of
NGF. This is particularly surprising in light of findings indicating that aberrant cell cycle entry is one of the major mechanisms whereby NGF withdrawal causes
sympathetic neuron apoptosis. In particular, NGF withdrawal causes increased cyclin D1 expression, and inhibition of cdk4 and
-6, both of which phosphorylate and activate pRb, is sufficient to delay NGF withdrawal-induced apoptosis. However, in this regard, NGF withdrawal does not cause enhanced BrdU incorporation and hypophosphorylated pRb is not, by itself, sufficient to rescue
sympathetic neurons from apoptosis. Of themselves, these findings do not necessarily argue against a role for cell cycle dysregulation in NGF withdrawal-induced
apoptosis, although they do demonstrate that this dysregulation does not actually lead to S-phase reentry. Instead, the data suggest that N-myc-induced survival
mechanisms may be 'dominant' to any apoptotic signals deriving from the coincident aberrant reentry into S-phase. Interestingly, data presented here suggest (but do
not definitively establish) that one such N-myc-mediated mechanism may involve downregulation of p75NTR (Wartiovaara, 2002).
N-myc is a true oncogene with overexpression in the sympathetic chain and adrenal medulla of transgenic mice that results, via unknown mechanisms, in malignant
neuroblastoma. The experimental and clinical data showing a strong correlation between N-myc gene amplification and poor outcome in
neuroblastoma suggest that N-myc is involved in the malignant transformation of developing sympathetic precursors or neurons, or both. On the basis of data
showing that N-myc can promote S-phase entry and survival of 'postmitotic' sympathetic neurons, a model is suggested in which N-myc contributes to malignant
neuroblastoma by either stopping sympathetic neuroblasts from exiting the cell cycle or by collaborating with other risk factors to actually transform postmitotic
neurons and cause them to reenter the cell cycle (Wartiovaara, 2002).
Overexpression of c-Myc or E2F1 sensitizes host cells to various types of apoptosis. Overexpressed c-Myc or E2F1 induces accumulation of reactive oxygen species (ROS) and thereby enhances serum-deprived apoptosis in NIH3T3 and Saos-2. During serum deprivation, MnSOD mRNA is induced by NF-kappaB in mock-transfected NIH3T3, while this induction was inhibited in NIH3T3 overexpressing c-Myc or E2F1. In these clones, E2F1 inhibits NF-kappaB activity by binding to its subunit p65 in competition with a heterodimeric partner p50. In addition to overexpressed E2F1, endogenous E2F1 released from Rb is also found to inhibit NF-kappaB activity in a cell cycle-dependent manner by using E2F1+/+ and E2F1-/- murine embryonic fibroblasts. These results indicate that E2F1 promotes apoptosis by inhibiting NF-kappaB activity (Tanaka, 2002).
Oncogene overexpression activates p53 by a mechanism posited to involve uncharacterized hyperproliferative signals. This study was carried out to determine whether such signals produce metabolic perturbations that generate DNA damage, a known p53 inducer. Biochemical, cytological, cell cycle, and global gene expression analyses revealed that brief c-Myc activation can induce DNA damage prior to S phase in normal human fibroblasts. Damage correlates with induction of reactive oxygen species (ROS) without induction of apoptosis. Deregulated c-Myc partially disables the p53-mediated DNA damage response, enabling cells with damaged genomes to enter the cycle, resulting in poor clonogenic survival. An antioxidant reduces ROS, decreases DNA damage and p53 activation, and improves survival. It is proposed that oncogene activation can induce DNA damage and override damage controls, thereby accelerating tumor progression via genetic instability (Vafa, 2002).
The cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p27Kip1 are induced in response to anti-proliferative stimuli and block G1/S-phase progression through the inhibition of CDK2. Although the cyclin E-CDK2 pathway is often deregulated in tumors, the relative contribution of p21Cip1 and p27Kip1 to tumorigenesis is still unclear. The MYC transcription factor is an important regulator of the G1/S transition and its expression is frequently altered in tumors. It has been suggested that p27Kip1 is a crucial G1 target of MYC. In mice, deficiency for p27Kip1 but not p21Cip1 results in decreased survival to retrovirally-induced lymphomagenesis. Importantly, in such p27Kip1 deficient lymphomas an increased frequency of Myc activation is observed. p27Kip1 deficiency also collaborates with MYC overexpression in transgenic lymphoma models. Thus, in vivo, the capacity of MYC to promote tumor growth is fully retained and even enhanced upon p27Kip1 loss. In lymphocytes, MYC overexpression and p27Kip1 deficiency independently stimulate CDK2 activity and augment the fraction of cells in S phase, in support of their distinct roles in tumorigenesis (Martins, 2002).
Myc and E2f1 promote cell cycle progression, but overexpression of either can trigger p53-dependent apoptosis. Mice expressing an Eμ-Myc transgene in B lymphocytes develop lymphomas, the majority of which sustain mutations of either Arf (a tumor suppressor whose product inhibits Mdm2, thereby stabilizing p53) or p53. Eμ-Myc transgenic mice lacking one or both E2f1 alleles exhibit a slower onset of lymphoma development associated with increased expression of the cyclin-dependent kinase inhibitor p27Kip1 and a reduced S phase fraction in precancerous B cells. In contrast, Myc-induced apoptosis and the frequency of Arf and p53 mutations in lymphomas were unaffected by E2f1 loss. Therefore, Myc does not require E2f1 to induce Arf, p53, or apoptosis in B cells, but depends upon E2f1 to accelerate cell cycle progression and downregulate p27Kip1 (Baudino, 2003).
The MYC oncoprotein is a transcription factor that coordinates cell growth and division. MYC overexpression exacerbates genomic instability and sensitizes cells to apoptotic stimuli. MYC directly stimulates transcription of the human Werner syndrome gene, WRN, which encodes a conserved RecQ helicase. Loss-of-function mutations in WRN lead to genomic instability, an elevated cancer risk, and premature cellular senescence. The overexpression of MYC in WRN syndrome fibroblasts or after WRN depletion from control fibroblasts leads to rapid cellular senescence that can not be suppressed by hTERT expression. It is proposed that WRN up-regulation by MYC may promote MYC-driven tumorigenesis by preventing cellular senescence (Grandori, 2003).
Alterations in c-myc oncogene expression have been implicated in the pathogenesis of several human cancers, including Burkitt and diffuse large B-cell lymphomas, breast and prostate cancer, colon cancer, melanoma, and multiple myeloma. The proteins encoded by MYC transcriptional target genes appear to regulate cell-cycle progression and cell growth while sensitizing cells to apoptotic stimuli. MYC may also be able to promote tumorigenesis by up-regulating the expression of genes such as hTERT that play a role in cellular immortalization or the escape from senescence. It was reasoned that MYC might modulate the expression of other genes that control cellular senescence, and thus determined whether the gene encoding the Werner syndrome RecQ helicase protein is a MYC transcriptional target (Grandori, 2003).
Werner syndrome (WRN) is an uncommon, autosomal recessive genetic instability syndrome that results from loss-of-function mutations in the chromosome 8p12-p11.2 WRN gene. The WRN phenotype resembles premature aging, and includes genomic instability, an elevated risk of malignancy, and accelerated cellular senescence. Genetic instability following loss of the 162-kD WRN RecQ helicase protein reflects the physiologic role of WRN in mitotic recombination and repair. Conversely, the elevated levels of WRN observed in immortalized and human tumor cell lines may help insure continuous cell proliferation. In order to delineate potential interactions between MYC and WRN in tumorigenesis, whether WRN expression is modulated by MYC was determined, and cellular responses to MYC overexpression in the absence of WRN were monitored. The results indicate that WRN expression appears to be required to avoid cellular senescence upon MYC up-regulation in hTERT-immortalized fibroblasts (Grandori, 2003).
Mnt is a Max-interacting transcriptional repressor that has been hypothesized to function as a Myc antagonist. To investigate Mnt function the Mnt gene was deleted in mice. Since mice lacking Mnt are born severely runted and typically die within several days of birth, mouse embryo fibroblasts (MEFs) derived from these mice and conditional Mnt knockout mice were used in this study. In the absence of Mnt, MEFs prematurely enter the S phase of the cell cycle and proliferated more rapidly than Mnt+/+ MEFs. Defective cell cycle control in the absence of Mnt is linked to upregulation of Cdk4 and cyclin E and the Cdk4 gene appears to be a direct target of Mnt-Myc antagonism. Like MEFs that overexpress Myc, Mnt-/- MEFs are prone to apoptosis, efficiently escape senescence and can be transformed with oncogenic Ras alone. Consistent with Mnt functioning as a tumor suppressor, conditional inactivation of Mnt in breast epithelium leads to adenocarinomas. These results demonstrate a unique negative regulatory role for Mnt in governing key Myc functions associated with cell proliferation and tumorigenesis (Hurlin, 2003).
Epidemiological findings suggest that the consequences of a given oncogenic stimulus vary depending upon the developmental state of the target tissue at the time of exposure. This is particularly evident in the mammary gland, where both age at exposure to a carcinogenic stimulus and the timing of a first full-term pregnancy can markedly alter the risk of developing breast cancer. Analogous to this, the biological consequences of activating oncogenes, such as MYC, can be influenced by cellular context both in terms of cell lineage and cellular environment. In light of this, it was hypothesized that the consequences of aberrant MYC activation in the mammary gland might be determined by the developmental state of the gland at the time of MYC exposure. To test this hypothesis directly, a doxycycline-inducible transgenic mouse model was used to overexpress MYC during different stages of mammary gland development. Using this model, it was found that the ability of MYC to inhibit postpartum lactation is due entirely to its activation within a specific 72-hour window during mid-pregnancy; by contrast, MYC activation either prior to or following this 72-hour window has little or no effect on postpartum lactation. Surprisingly, it was found that MYC does not block postpartum lactation by inhibiting mammary epithelial differentiation, but rather by promoting differentiation and precocious lactation during pregnancy, which in turn leads to premature involution of the gland. This developmental stage-specific ability of MYC to promote mammary epithelial differentiation is tightly linked to its ability to downregulate caveolin 1 and activate Stat5 in a developmental stage-specific manner. These findings provide unique in vivo molecular evidence for developmental stage-specific effects of oncogene activation, as well as the first evidence linking MYC with activation of the Jak2-Stat5 signaling pathway (Blakely, 2005).
ß-catenin signaling is heavily involved in organogenesis. This study
investigated how pancreas differentiation, growth and homeostasis are affected
following inactivation of an endogenous inhibitor of ß-catenin,
adenomatous polyposis coli (Apc). In adult mice, Apc-deficient pancreata are
enlarged, solely as a result of hyperplasia of acinar cells, which accumulate
ß-catenin, with the sparing of islets. Expression of a target of
ß-catenin, the proto-oncogene c-myc (Myc), is
increased in acinar cells lacking Apc, suggesting that c-myc
expression is essential for hyperplasia. In support of this hypothesis, it was
found that conditional inactivation of c-myc in pancreata lacking Apc
completely reverse the acinar hyperplasia. Apc loss in organs such as the
liver, colon and kidney, as well as experimental misexpression of c-myc in pancreatic acinar cells, lead to tumor formation with high penetrance. Surprisingly, pancreas tumors failed to develop following conditional pancreas Apc inactivation. In Apc-deficient acini of aged mice, these studies revealed a cessation of their exaggerated proliferation and a reduced expression of c-myc, in spite of the persistent accumulation of ß-catenin. In conclusion, this work shows that ß-catenin
modulation of c-myc is an essential regulator of acinar growth
control, and unveils an unprecedented example of Apc requirement in the pancreas that is both temporally restricted and cell-specific. This provides new insights into the mechanisms of tumor pathogenesis and tumor suppression in the pancreas (Strom, 2007).
Inhibition of protein phosphatase 2A (PP2A) activity has been identified as a prerequisite for the transformation of human cells. However, the molecular mechanisms by which PP2A activity is inhibited in human cancers are currently unclear. In this study, a cellular inhibitor of PP2A with oncogenic activity is described. The protein, designated Cancerous Inhibitor of PP2A (CIP2A), interacts directly with the oncogenic transcription factor c-Myc, inhibits PP2A activity toward c-Myc serine 62 (S62), and thereby prevents c-Myc proteolytic degradation. In addition to its function in c-Myc stabilization, CIP2A promotes anchorage-independent cell growth and in vivo tumor formation. The oncogenic activity of CIP2A is demonstrated by transformation of human cells by overexpression of CIP2A. Importantly, CIP2A is overexpressed in two common human malignancies, head and neck squamous cell carcinoma (HNSCC) and colon cancer. Thus, these data show that CIP2A is a human oncoprotein that inhibits PP2A and stabilizes c-Myc in human malignancies (Junttila, 2007).
FoxO transcription factors play critical roles in cell cycle control and cellular stress responses, and abrogation of FoxO function promotes focus formation by Myc in vitro. Stable introduction of a dominant-negative FoxO moiety (dnFoxO) into Eµ-myc transgenic hematopoietic stem cells accelerates lymphoma development in recipient mice by attenuating Myc-induced apoptosis. When expressed in Eµ-myc; p53+/- progenitor cells, dnFoxO alleviates the pressure to inactivate the remaining p53 allele in upcoming lymphomas. Expression of the p53 upstream regulator p19Arf (alternative reading frame of p16INK, also called p14arf in humans and p19arf in mice) is virtually undetectable in most dnFoxO-positive Myc-driven lymphomas. It was found that FoxO proteins bind to a distinct site within the Ink4a/Arf locus and activate Arf expression. Moreover, constitutive Myc signaling induces a marked increase in nuclear FoxO levels and stimulates binding of FoxO proteins to the Arf locus. These data demonstrate that FoxO factors mediate Myc-induced Arf expression and provide direct genetic evidence for their tumor-suppressive capacity (Bouchard, 2007).
The FoxO subclass of forkhead-box transcription factors (consisting of FoxO1 (FKHR), FoxO3a (FKHRL1), FoxO4 (AFX), and FoxO6) regulates numerous cellular functions including proliferation, stress sensitivity, and survival; it has also been implicated in the regulation of organism life span. The members of this family activate gene expression via interaction with a specific DNA sequence, and known targets include the cell cycle regulating Kip1, the proapoptotic Bim, the DNA damage-responsive Gadd45a, and the oxidative stress-protective manganese superoxide dismutase genes. In addition, FoxO proteins can repress several cell cycle promoting genes (e.g., cyclin D1 and cyclin D2) in a manner that might be independent of direct DNA binding (Bouchard, 2007 and references therein).
In response to growth factor signaling and to oxidative stress, FoxO proteins are post-translationally modified by phosphorylation, acetylation, and ubiquitination; collectively, these modifications regulate FoxOs’ subcellular localization, transcriptional activity, and stability. Notably, all FoxO proteins are inhibited by protein kinase B/Akt-mediated phosphorylation that promotes their nuclear export and subsequent proteolytic degradation via ubiquitination by the SCFSkp2 complex. As a consequence, FoxO proteins mediate the induction of p27Kip1 and Bim expression in response to inhibition of the phosphatidylinositol-3-OH (PI3)-kinase/Akt pathway (Bouchard, 2007 and references therein).
Conditional codeletion of the FoxO1, FoxO3, and FoxO4 alleles uncovers a context-dependent cancer-prone phenotype characterized by thymic lymphomas forming in some and hemangiomas developing in most animals after a long latency, suggesting that FoxO proteins exert their tumor-suppressive capability in the presence of additional oncogenic mutations. In support of this view, Akt-mediated phosphorylation of FoxO proteins has been identified as the critical PI3-kinase signaling component that substitutes for oncogenic Ras in Myc-induced proliferation and focus formation in vitro. Furthermore, constitutive Akt signaling cooperates with Myc to accelerate B-cell lymphomagenesis; however, it remains unclear whether Akt-mediated phosphorylation of FoxO proteins contributes to Eµ-myc transgenic lymphoma formation in this setting (Bouchard, 2007).
Proapoptotic Arf/p53 signaling is known as the pivotal Myc-induced tumor-suppressive barrier. Eµ-myc transgenic mice lacking one p53 allele develop lymphomas that inactivate the remaining wild-type allele. Likewise, Eµ-myc; Arf+/- or Eµ-myc; Ink4a/Arf+/- mice produce tumors that lack expression of p19Arf. Primary Arf deletions protect cells from acquiring p53 mutations during lymphoma development. Similarly, introduction of strictly anti-apoptotic genes such as bcl2 or a dominant-negative form of caspase 9 into Eµ-myc; p53+/- hematopoietic stem cells alleviates the pressure to inactivate p53, thereby underscoring apoptosis as the critical p53-governed tumor suppressor function in Myc-driven lymphomagenesis (Bouchard, 2007).
Previous work has shown that p53 and FoxO3a share target genes and that FoxO3a can activate transcription via p53 sites, suggesting a potential collaboration of FoxO3a and p53 in tumor suppression. Although a direct interaction between FoxO3a and p53 proteins has been demonstrated under conditions of overexpression, the observed collaboration would be consistent with an as-yet-unidentified FoxO target acting upstream of p53. This study reports that FoxO factors elicit their tumor-suppressive potential as critical inducers of Arf during Myc-driven lymphomagenesis, providing further evidence for a close link between the FoxO and p53 tumor suppressor pathways (Bouchard, 2007).
Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. This study demonstrates
induction of pluripotent stem cells from mouse embryonic or adult tail tip fibroblasts (TTFs) by introducing four factors, Oct3/4, Sox2, c-Myc,
and Klf4, under ES cell culture conditions. Unexpectedly, Nanog was dispensable. These
cells, which have been designated iPS (induced pluripotent
stem) cells, exhibit the morphology and growth properties of ES cells and express ES
cell marker genes. Subcutaneous transplantation
of iPS cells into nude mice resulted in tumors containing a variety of tissues from all
three germ layers. Following injection into blastocysts,
iPS cells contributed to mouse embryonic development. These data demonstrate
that pluripotent stem cells can be directly generated
from fibroblast cultures by the addition of only a few defined factors (Takahashi, 2007).
Oct3/4, Sox2, and Nanog have been shown to function
as core transcription factors in maintaining pluripotency. Among the three,
it was found that Oct3/4 and Sox2 are essential for the generation
of iPS cells. c-Myc and Klf4 were also identified as essential factors.
These two tumor-related factors could not be replaced by
other oncogenes including E-Ras, Tcl1, β-catenin, and Stat3 (Takahashi, 2007).
The c-Myc protein has many downstream targets that
enhance proliferation and transformation, many of which may have roles in the generation
of iPS cells. Of note, c-Myc associates with histone
acetyltransferase (HAT) complexes, including TRRAP,
which is a core subunit of the TIP60 and GCN5 HAT complexes, CREB binding protein
(CBP), and p300. Within the mammalian genome, there may be up to 25,000 c-Myc binding
sites, many more than the predicted number of Oct3/4 and Sox2 binding sites. c-Myc protein may induce global histone acetylation, thus allowing Oct3/4 and Sox2 to bind to their specific target loci. Klf4 has been shown to repress p53 directly, and p53 protein has been shown to suppress Nanog during ES cell differentiation. iPS cells showed levels of p53 protein lower than those in MEFs. Thus, Klf4 might contribute
to activation of Nanog and other ES cell-specific genes
through p53 repression. Alternatively, Klf4 might function
as an inhibitor of Myc-induced apoptosis through the repression
of p53 in this system. In contrast, Klf4 activates p21CIP1, thereby suppressing
cell proliferation. This antiproliferation function of Klf4 might be inhibited by c-Myc, which suppresses the expression of p21CIP1. The balance between c-Myc and Klf4 may be important for the generation of iPS cells (Takahashi, 2007).
One question that remains concerns the origin of the iPS cells. With the retroviral expression system, it is estimated that only a small portion of cells expressing the
four factors become iPS cells. The low frequency suggests that rare tissue stem/progenitor cells that coexisted in the fibroblast cultures might have given
rise to the iPS cells. Indeed, multipotent stem cells have
been isolated from skin. These studies showed that ~0.067% of
mouse skin cells are stem cells. One explanation for the
low frequency of iPS cell derivation is that the four factors
transform tissue stem cells. However, it was found that the
four factors induced iPS cells with comparably low efficiency
even from bone marrow stroma, which should be more enriched in mesenchymal stem cells and other multipotent cells. Furthermore, cells induced by the three factors were nullipotent. DNA microarray analyses suggested that iPS-MEF4 cells and iPS-MEF3 cells have the same origin. These results do not favor multipotent tissue stem cells as the origin of iPS cells (Takahashi, 2007).
There are several other possibilities for the low frequency
of iPS cell derivation. First, the levels of the four
factors required for generation of pluripotent cells may
have narrow ranges, and only a small portion of cells expressing
all four of the factors at the right levels can acquire
ES cell-like properties. Consistent with this idea, a mere 50% increase or decrease in Oct3/4 proteins induces differentiation of ES cells. iPS clones
overexpressed the four factors when RNA levels were analyzed,
but their protein levels were comparable to those in
ES cells, suggesting that the iPS clones possess a mechanism (or mechanisms) that
tightly regulates the protein levels of the four factors. It is
speculated that high amounts of the four factors are required
in the initial stage of iPS cell generation, but, once
they acquire ES cell-like status, too much of the factors
are detrimental for self-renewal. Only a small portion of
transduced cells show such appropriate transgene expression. Second, generation of pluripotent cells may require additional chromosomal alterations, which take place spontaneously during culture or are induced by some of the four factors. Although the iPS-TTFgfp4 clones had largely normal karyotypes, the existence of minor chromosomal alterations cannot be ruled out. Site-specific retroviral insertion may also play a role. Southern blot analyses showed that each iPS clone has ~20 retroviral
integrations. Some of these may have caused
silencing or fusion with endogenous genes. Further studies
will be required to determine the origin of iPS cells (Takahashi, 2007).
Another unsolved question is whether the four factors
identified play roles in reprogramming induced by fusion
with ES cells or nuclear transfer into oocytes. Since the four factors are expressed in ES cells at high levels, it is reasonable to speculate that they are involved in the
reprogramming machinery that exists in ES cells. These result
is also consistent with the finding that the reprogramming
activity resides in the nucleus, but not in the cytoplasm,
of ES cells. However, iPS cells were not identical to ES cells, as shown by the global
gene-expression patterns and DNA methylation status. It is possible that additional important factors have been missed. One such candidate is ECAT1, although its forced
expression in iPS cells did not consistently upregulate ES cell marker genes (Takahashi, 2007).
More obscure are the roles of the four factors, especially Klf4 and c-Myc, in the reprogramming observed in oocytes. Both Klf4 and c-Myc are dispensable for preimplantation mouse development. Furthermore, c-myc is not detected in oocytes. In contrast, L-myc is expressed maternally in oocytes. Klf17 and Klf7, but not Klf4, are found in expressed sequence-tag libraries derived from unfertilized mouse eggs. Klf4 and c-Myc might be compensated by these related proteins. It is highly likely that other factors are also required to induce complete reprogramming and totipotency in oocytes (Takahashi, 2007).
Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism. Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation. Recently, murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Using these same factors, iPS cells were derived from fetal, neonatal and adult human primary cells, including dermal fibroblasts isolated from a skin biopsy of a healthy research subject. Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogramme human cells to pluripotency, and establish a method whereby patient-specific cells might be established in culture (Park, 2008).
Jasmonates (JA) are important regulators of plant defense responses that activate expression of many wound-induced genes including the tomato proteinase inhibitor II (pin2) and leucine aminopeptidase (LAP) genes. Elements required for JA induction of the LAP gene are all present in the -317 to -78 proximal promoter region. Using yeast one-hybrid screening, the bHLH-leu zipper JAMYC2 and JAMYC10 proteins were identified, specifically recognizing a T/G-box AACGTG motif in this promoter fragment. Mutation of the G-box element decreases JA-responsive LAP promoter expression. Expression of JAMYC2 and JAMYC10 is induced by JA, with a kinetics that precedes that of the LAP or pin2 transcripts. JAMYC overexpression enhanced JA-induced expression of these defense genes in potato, but did not result in constitutive transcript accumulation. Using footprinting assays, an additional protected element was identified, located directly adjacent to the T/G-box motif. Mutation of this element abolishes JA response, showing that recognition of this duplicated element is also required for gene expression. Knockout mutants in the AtMYC2 homolog gene of Arabidopsis are insensitive to JA and exhibit a decreased activation of the JA-responsive genes AtVSP and JR1. Activation of the PDF1.2 and b-CHI, ethylene/JA-responsive genes, is, however, increased in these mutants. These results show that the JAMYC/AtMYC2 transcription factors function as members of a MYC-based regulatory system conserved in dicotyledonous plants with a key role in JA-induced defense gene activation (Boter, 2004).
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Thus, N-myc is involved in regulation of the neural
crest fate in two different aspects: ventral migration and neuronal differentiation (Wakamatsu, 1997).
Society for Developmental Biology's Web server.