Inhibiting MYC binding to the E-box DNA motif by ME47 decreases tumour xenograft growth.

Developing therapeutics to effectively inhibit the MYC oncoprotein would mark a key advance towards cancer patient care as MYC is deregulated in over 50% of human cancers. MYC deregulation is correlated with aggressive disease and poor patient outcome. Despite strong evidence in mouse models that inhibiting MYC would significantly impact tumour cell growth and patient survival, traditional approaches have not yet yielded the urgently needed therapeutic agents that directly target MYC. MYC functions through its interaction with MAX to regulate gene transcription by binding to E-box DNA response elements of MYC target genes. Here we used a structure-based strategy to design ME47, a small minimalist hybrid protein (MHP) able to disrupt the MAX:E-box interaction/binding and block transcriptional MYC activity. We show that inducing ME47 expression in established tumour xenografts inhibits tumour growth and decreases cellular proliferation. Mechanistically, we show by chromatin immunoprecipitation that ME47 binds to E-box binding sites of MYC target genes. Moreover, ME47 occupancy decreases MYC:DNA interaction at its cognate E-box binding sites. Taken together, ME47 is a prototypic MHP inhibitor that antagonizes tumour cell growth in vitro and in vivo and inhibits the interaction of MYC with DNA E-box elements. These results support ME47's role as a MYC inhibitor and suggest that MHPs provide an alternative therapeutic targeting system that can be used to target transcription factors important in human diseases, including cancer.

Lysine-52 stabilizes the MYC oncoprotein through an SCFFbxw7-independent mechanism.

The oncogenic transcription factor c-MYC (MYC) is deregulated and often overexpressed in more than 50% of cancers. MYC deregulation is associated with poor prognosis and aggressive disease, suggesting that the development of therapeutic inhibitors targeting MYC would markedly impact patient outcome. MYC is highly regulated, with a protein and mRNA half-life of ~30 min. The most extensively studied pathway regulating MYC protein stability involves ubiquitylation and proteasomal degradation mediated by the E3-ligase, SCFFbxw7. Here we provide evidence for an SCFFbxw7-independent regulatory mechanism centred on the highly conserved lysine-52 (K52) within MYC Box I. This residue has been shown to be post-translationally modified by both ubiquitylation and SUMOylation, hinting at the interplay of post-translational modifications at this site and the importance of this residue. We demonstrate that mutation of K52 to arginine (R) renders the MYC protein more labile. Mechanistically, we show that the degradation pathway regulated by K52 is independent of the Cullin-RING ligase family of E3-ligases, which includes not only the canonical SCFFbxw7 but also other known MYC-targeting E3-ligases, such as SCFSkp2, SCFßTCRP, SCFFbxo28 and DCXTRUSS. Taken together, our data identify a novel regulatory pathway centred on K52 that may be exploited for the development of anti-MYC therapeutics.

MYC activity is negatively regulated by a C-terminal lysine cluster.

The MYC oncogene is not only deregulated in cancer through abnormally high levels of expression, but also through oncogenic lesions in upstream signalling cascades. Modelling MYC deregulation using signalling mutants is a productive research strategy. For example, the MYC threonine-58 to alanine substitution mutant (T58A) within MYC-homology box 1 is more transforming than wild-type (WT) MYC, because of decreased apoptosis and increased protein stability. Understanding the regulatory mechanisms controlling T58 phosphorylation has led to new approaches for the development of MYC inhibitors. In this manuscript, we have extensively characterized a MYC signalling mutant in which six lysine residues near the highly conserved MYC homology box IV and basic region have been substituted to arginines (6KR). Previous literature suggests these lysines can undergo both ubiquitylation and acetylation. We show MYC 6KR is able to fully rescue the slow growth phenotype of HO15.19 MYC-null fibroblasts, and promote cell cycle entry of serum-starved MCF10A cells. Remarkably, 6KR increased anchorage-independent colony growth compared with WT MYC in both SH-EP and MCF10A cells. Moreover, it was also more potent in promoting xenograft tumour growth of Rat1A and SH-EP cells. Combined, our data identify this region and these six lysines as important residues for the negative regulation of MYC-induced transformation. Mechanistically, we demonstrate that, unlike T58A, the increased transformation is not a result of increased protein stability or a reduced capacity for 6KR to induce apoptosis. Through expression analysis and luciferase reporter assays, we show that 6KR has increased transcriptional activity compared with WT MYC. Combined, through a comprehensive evaluation across multiple cell types, we identify an important regulatory region within MYC. A better understanding of the full scope of signalling through these residues will provide further insights into the mechanisms contributing to MYC-induced tumorigenesis and may unveil novel therapeutic strategies to target Myc in cancer.

Targeting tumor cell metabolism with statins.

The mevalonate pathway is a core biochemical process, crucial for the generation of cholesterol and other key metabolic end products. The rate-limiting enzyme of the mevalonate pathway, hydroxymethylglutaryl coenzyme A reductase (HMGCR), is safely and effectively targeted by the statin family of inhibitors to treat hypercholesterolemia. The anticancer activity of statins has also been widely reported, yet the tumor-selective mechanisms that mediate these antiproliferative effects remain largely unclear. The importance of altered metabolism in the context of tumorigenesis has received renewed attention as metabolic changes entwined with the molecular hallmarks of cancer have been elucidated. Although several metabolic pathways have been linked to cancer progression and etiology, it was only recently that HMGCR and the mevalonate pathway were also shown to have a distinct role in cellular transformation. In this review, we chart the historical progression of statins from cholesterol-lowering blockbusters to anticancer agents with imminent potential, and further discuss an emerging role for HMGCR and the mevalonate pathway in the metabolic reprogramming of cancer.

New model systems provide insights into Myc-induced transformation.

The ability of Myc to promote cellular transformation is well established; however, a better understanding of the mechanisms through which Myc mediates tumorigenesis is essential for the development of therapeutic approaches to target this potent oncoprotein. Structure-function studies in rodent fibroblast cells have provided the basis for much of our current understanding of these mechanisms. To build on these approaches, we have characterized three novel human cell line models of Myc-dependent transformation: MCF10A, SH-EP Tet21/N-Myc, and LF1/TERT/LT/ST cells. We have also evaluated Myc family proteins (c-Myc and L-Myc), a naturally occurring isoform of Myc (MycS), and a set of N-terminal domain mutants (ΔMBII, W135E, T58A) for their ability to promote anchorage-independent growth in these models. Taken together, these results provide the field with three new human cell-based models to study Myc activity, highlight the importance of cellular context, and challenge the paradigm that the ability of Myc to promote tumorigenesis is exclusively MBII-dependent.

Repression of the human immunodeficiency virus type-1 long terminal repeat by the c-Myc oncoprotein.

The effect of trans-acting factors on cis-acting DNA elements on the HIV-1 promoter are the principal determinant regulating transcriptional activation and repression. Host factors that limit viral replication can contribute to the emergence and maintenance of proviral reservoirs. The current paradigm is that this sub-population of latently infected cells confers a biological advantage to the virus by facilitating evasion of immunologic responses and therapeutic strategies resulting in life-long and persistent infection. In this report, we show that ectopic expression of the nuclear phosphoprotein, c-Myc can inhibit HIV-1 gene expression and virus production in CD4+ T-lymphocytes. The effect exerted does not appear to involve other known functions of c-Myc such as proliferation, or apoptosis. The mechanism does implicate c-Myc in a direct role. We have found evidence that c-Myc can specifically recognize the HIV-1 initiator element surrounding the start site of transcription and linker scanning mutagenesis experiments confirmed a loss of c-Myc-mediated repression in the absence of this region. Moreover, we show that c-Myc can interact with the initiator binding proteins YY-1 and LBP-1 and can cooperate with these factors to synergistically repress HIV-1 LTR transcription. Taken together, these results indicate that c-Myc is an important regulator of HIV-1 transcription that potentially contributes to the latent proviral state.

HMG-CoA reductase inhibitors and the malignant cell: the statin family of drugs as triggers of tumor-specific apoptosis.

The statin family of drugs target HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway, and have been used successfully in the treatment of hypercholesterolemia for the past 15 years. Experimental evidence suggests this key biochemical pathway holds an important role in the carcinogenic process. Moreover, statin administration in vivo can provide an oncoprotective effect. Indeed, in vitro studies have shown the statins can trigger cells of certain tumor types, such as acute myelogenous leukemia, to undergo apoptosis in a sensitive and specific manner. Mechanistic studies show bcl-2 expression is down-regulated in transformed cells undergoing apoptosis in response to statin exposure. In addition, the apoptotic response is in part due to the depletion of the downstream product geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate or other products of the mevalonate pathway including cholesterol. Clinically, preliminary phase I clinical trials have shown the achievable plasma concentration corresponds to the dose range that can trigger apoptosis of tumor types in vitro. Moreover, little toxicity was evident in vivo even at high concentrations. Clearly, additional clinical trials are warranted to further assess the safety and efficacy of statins as novel and immediately available anti-cancer agents. In this article, the experimental evidence supporting a role for the statin family of drugs to this new application will be reviewed.

Apoptosis modulators as cancer therapeutics.

In the past ten years a wealth of fundamental knowledge delineating the molecular mechanism(s) of apoptosis has emerged, and can now be exploited to identify novel apoptotic modulators for the treatment of cancer. Two distinct yet complimentary classes of non-genotoxic agonists that can selectively kill tumor cells are discussed; agents that target 'classical' and 'atypical' apoptotic signaling pathways. The goal of agents targeting classical apoptosis and survival pathways is to directly modulate key apoptotic regulators such as Bcl-2, Akt/PKB, and p53. The aim of agents targeting atypical apoptotic pathways is to target signaling cascades whose inhibition remains non-lethal in normal cells, yet is suicidal in tumor cells. Such compounds presently under development include inhibitors of heat shock protein 90, histone deacetylases and HMG-CoA reductase. Both classes of apoptotic modulators have merit and identification of additional agonists of this nature will provide the many diverse cytotoxic agents that are required to combat the many diseases we call cancer.

Blocking protein geranylgeranylation is essential for lovastatin-induced apoptosis of human acute myeloid leukemia cells.

Lovastatin is an inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the major regulatory enzyme of the mevalonate pathway. We have previously reported that lovastatin induces a significant apoptotic response in human acute myeloid leukemia (AML) cells. To identify the critical biochemical mechanism(s) essential for lovastatin-induced apoptosis, add-back experiments were conducted to determine which downstream product(s) of the mevalonate pathway could suppress this apoptotic response. Apoptosis induced by lovastatin was abrogated by mevalonate (MVA) and geranylgeranyl pyrophosphate (GGPP), and was partially inhibited by farnesyl pyrophosphate (FPP). Other products of the mevalonate pathway including cholesterol, squalene, lanosterol, desmosterol, dolichol, dolichol phosphate, ubiquinone, and isopentenyladenine did not affect lovastatin-induced apoptosis in AML cells. Our results suggest that inhibiting geranylgeranylation of target proteins is the predominant mechanism of lovastatin-induced apoptosis in AML cells. In support of this hypothesis, the geranylgeranyl transferase inhibitor (GGTI-298) mimicked the effect of lovastatin, whereas the farnesyl transferase inhibitor (FTI-277) was much less effective at triggering apoptosis in AML cells. Inhibition of geranylgeranylation was monitored and associated with the apoptotic response induced by lovastatin and GGTI-298 in the AML cells. We conclude that blockage of the mevalonate pathway, particularly inhibition of protein geranylgeranylation holds a critical role in the mechanism of lovastatin-induced apoptosis in AML cells.

Cerivastatin triggers tumor-specific apoptosis with higher efficacy than lovastatin.

The statin family of drugs inhibits 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme of the mevalonate pathway, and is used clinically as a safe and effective approach in the control of hypercholesterolemia. We have shown previously (Dimitroulakos, J., Nohynek, D., Backway, K. L., Hedley, D. W., Yeger, H., Freedman, M. H., Minden, M D., and Penn, L. Z. Increased sensitivity of acute myelogenous leukemias to lovastatin-induced apoptosis: a potential therapeutic approach. Blood, 93: 1308-1318, 1999) that lovastatin, a prototypic member of the statin family, can induce apoptosis of human acute myeloid leukemia (AML) cells in a sensitive and specific manner. In the present study, we evaluated the relative potency and mechanism of action of the newer synthetic statins, fluvastatin, atorvastatin, and cerivastatin, to trigger tumor-specific apoptosis. Cerivastatin is at least 10 times more potent than the other statins at inducing apoptosis in AML cell lines. Cerivastatin-induced apoptosis is reversible with the addition of the immediate product of the HMG-CoA reductase reaction, mevalonate, or with a distal product of the pathway, geranylgeranyl pyrophosphate. This suggests protein geranylgeranylation is an essential downstream component of the mevalonate pathway for cerivastatin similar to lovastatin-induced apoptosis. The enhanced potency of cerivastatin expands the number of AML patient samples as well as the types of malignancies, which respond to statin-induced apoptosis with acute sensitivity. Cells derived from acute lymphocytic leukemia are only weakly sensitive to lovastatin cytotoxicity but show robust response to cerivastatin. Importantly, cerivastatin is not cytotoxic to nontransformed human bone marrow progenitors. These results strongly support the further testing of cerivastatin as a novel anticancer therapeutic alone and in combination with other agents in vivo.

Lovastatin induced control of blast cell growth in an elderly patient with acute myeloblastic leukemia.

We recently reported that AML cells derived either from cell lines or from patients undergo apoptosis in response to lovastatin, an agent used extensively in the treatment of hypercholesterolemia. The concentration of lovastatin required to achieve this in culture varies from patient to patient, however, the in vitro concentrations required to kill AML cells, can be attained clinically. While in vitro studies assessing responsiveness of leukemic cells to lovastatin were being performed, a 72 year old female presented with relapsed AML. The patient did not desire any further induction therapy. As the patient's cells proved to be sensitive in culture to lovastatin, the patient was offered this drug. In this brief report we describe a case in which there was apparent control of the patient's leukemic blast cells by lovastatin at a dose double the usual recommended dose for hypercholesterolemia. This case illustrates the potential for lovastatin to provide a novel means of controlling leukemic cell growth in AML patients.

Myc potentiates apoptosis by stimulating Bax activity at the mitochondria.

The ability of the c-Myc oncoprotein to potentiate apoptosis has been well documented; however, the mechanism of action remains ill defined. We have previously identified spatially distinct apoptotic pathways within the same cell that are differentially inhibited by Bcl-2 targeted to either the mitochondria (Bcl-acta) or the endoplasmic reticulum (Bcl-cb5). We show here that in Rat1 cells expressing an exogenous c-myc allele, distinct apoptotic pathways can be inhibited by Bcl-2 or Bcl-acta yet be distinguished by their sensitivity to Bcl-cb5 as either susceptible (serum withdrawal, taxol, and ceramide) or refractory (etoposide and doxorubicin). Myc expression and apoptosis were universally associated with Bcl-acta and not Bcl-cb5, suggesting that Myc acts downstream at a point common to these distinct apoptotic signaling cascades. Analysis of Rat1 c-myc null cells shows these same death stimuli induce apoptosis with characteristic features of nuclear condensation, membrane blebbing, poly (ADP-ribose) polymerase cleavage, and DNA fragmentation; however, this Myc-independent apoptosis is not inhibited by Bcl-2. During apoptosis, Bax translocation to the mitochondria occurs in the presence or absence of Myc expression. Moreover, Bax mRNA and protein expression remain unchanged in the presence or absence of Myc. However, in the absence of Myc, Bax is not activated and cytochrome c is not released into the cytoplasm. Reintroduction of Myc into the c-myc null cells restores Bax activation, cytochrome c release, and inhibition of apoptosis by Bcl-2. These results demonstrate a role for Myc in the regulation of Bax activation during apoptosis. Moreover, apoptosis that can be triggered in the absence of Myc provides evidence that signaling pathways exist which circumvent Bax activation and cytochrome c release to trigger caspase activation. Thus, Myc increases the cellular competence to die by enhancing disparate apoptotic signals at a common mitochondrial amplification step involving Bax activation and cytochrome c release.

Endoplasmic reticulum localized Bcl-2 prevents apoptosis when redistribution of cytochrome c is a late event.

The disruption of mitochondrial function is a key component of apoptosis in most cell types. Localization of Bcl-2 to the outer mitochondrial and endoplasmic reticulum membranes is consistent with a role in the inhibition of many forms of apoptosis. In Rat-1 cells, a Bcl-2 mutant targeted exclusively to the endoplasmic reticulum (Bcl-cb5) was effective at inhibiting apoptosis induced by serum starvation/myc, or ceramide but not apoptosis induced by etoposide. The former conditions cause a decrease in mitochondrial transmembrane potential (Deltapsi(m)) as an early event that precedes the release of cytochrome c from mitochondria. By contrast, when cells are exposed to etoposide, a situation in which cytochrome c release and membrane localization of the pro-apoptotic protein Bax precede loss of Deltapsi(m), wild type Bcl-2 but not Bcl-cb5 prevents apoptosis. Therefore, Bcl-2 functions in spatially distinct pathways of apoptosis distinguished by the order of cytochrome c release and loss of Deltapsi(m).

Mechanism for the transcriptional repression by c-Myc on PDGF beta-receptor.

c-Myc plays a key role in the cell cycle dependent control of the PDGF beta-receptor mRNA. The mouse platelet-derived growth factor (PDGF) beta-receptor promoter contains a CCAAT motif, and NF-Y plays an essential role in its transcription. Coexpression of c-Myc represses PDGF beta-receptor luciferase reporter activity, and the CCAAT motif in the promoter is indispensable for this repression. Here we show that c-Myc binds NF-Y subunits, YB and YC, by immunoprecipitation from cotransfected COS-1 cells. The in vitro-translated c-Myc also binds the glutathione S-transferase (GST)-NF-YB fusion protein and GST-NF-YC, but not GST-NF-YA. The most C-terminal region of HAP domains of NF-YB and NF-YC, and the Myc homology boxes, but not the C-terminal bHLHZip domain, are indispensable for the coimmunoprecipitation, and also for the repression of PDGF beta-receptor. c-Myc binds NF-Y complex without affecting the efficiency of NF-Y binding to DNA. However, the expression of Myc represses the transcriptional activation of NF-YC when fused to the GAL4 DNA binding domain. Furthermore, this repression was seen only when Myc homology boxes are present, and NF-YC contains the c-Myc binding region.

Differential sensitivity of various pediatric cancers and squamous cell carcinomas to lovastatin-induced apoptosis: therapeutic implications.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase is the rate-limiting enzyme of the mevalonate pathway, the diverse array of end products of which are vital for a variety of cellular functions, including cholesterol synthesis and cell cycle progression. We showed previously that this enzyme holds a critical role in regulating tumor cell fate, including cell death, as its expression is down-regulated in response to retinoic acid, a potent anticancer therapeutic. Indeed, direct inhibition of HMG-CoA reductase with lovastatin, a competitive inhibitor of this enzyme, induced a pronounced apoptotic response in neuroblastoma and acute myeloid leukemic cells. We have now extended this work and evaluated a wide variety and large number of tumor-derived cell lines for their sensitivity to lovastatin-induced apoptosis. These cell lines were exposed to a wide range (0-100 microM) of lovastatin for 2 days and assayed for cell viability using the 3,4,5-dimethyl thiazlyl-2,2,5-diphenyltetrazolium bromide assay and the induction of apoptosis by flow cytometric and ultrastructural analyses. Lovastatin induced a pronounced apoptotic response in cells derived from juvenile monomyelocytic leukemia, pediatric solid malignancies (rhabdomyosarcoma and medulloblastoma), and squamous cell carcinoma of the cervix and of the head and neck. Interestingly, the subset of malignancies that are particularly sensitive to lovastatin-induced apoptosis correspond to those tumor subtypes that are sensitive to the biological and antiproliferative effects of retinoids in vitro. The nature of the biologically active form of lovastatin has been challenged recently as the growth-inhibitory effects of this drug were attributed to its prodrug lactone form that does not inhibit HMG-CoA reductase function. In this report, we demonstrate that the apoptotic properties of lovastatin are triggered by the open ring acid form that is a potent inhibitor of HMG-CoA reductase activity. Thus, we have identified a subset of tumors that are sensitive to lovastatin-induced apoptosis and show HMG-CoA reductase as a potential therapeutic target of these cancers.

Lysophosphatidic acid prevents apoptosis in fibroblasts via G(i)-protein-mediated activation of mitogen-activated protein kinase.

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid with multiple biological functions. In the present study, we demonstrate that, besides its mitogenic activity, LPA is a potent survival factor, preventing serum-deprivation-induced apoptosis in fibroblasts and other cell types. Both the proliferative effect and survival activity of LPA are sensitive to the action of pertussis toxin (PTX), indicating that both processes are mediated by G(i) protein(s). We therefore focused on the role of G(i)-protein-mediated signalling events in the promotion of cell survival by LPA. In addition to activation of mitogen-activated protein kinase (MAPK), LPA stimulates a modest PTX-sensitive phosphorylation/activation of the serine/threonine kinase Akt, a survival mediator downstream of phosphoinositide 3-kinase (PI3K). Inhibition of PI3K with LY 294002 or wortmannin resulted in a marked inhibition of LPA-induced DNA synthesis, and yet the survival activity of LPA decreased by only 20-30%, suggesting a limited input of the PI3K-Akt cascade in LPA-induced cell survival. In contrast, inhibition of MAPK activation by the MEK-1 inhibitor, PD 98059, blocked both the proliferative and survival effects of LPA. These results indicate that LPA promotes cell survival largely via G(i)-protein-mediated activation of ERK1/ERK2, or other PD 98059-sensitive member(s) of the MAPK family.

Myc is an essential negative regulator of platelet-derived growth factor beta receptor expression.

Platelet-derived growth factor BB (PDGF BB) is a potent mitogen for fibroblasts as well as many other cell types. Interaction of PDGF BB with the PDGF beta receptor (PDGF-betaR) activates numerous signaling pathways and leads to a decrease in receptor expression on the cell surface. PDGF-betaR downregulation is effected at two levels, the immediate internalization of ligand-receptor complexes and the reduction in pdgf-betar mRNA expression. Our studies show that pdgf-betar mRNA suppression is regulated by the c-myc proto-oncogene. Both constitutive and inducible ectopic Myc protein can suppress pdgf-betar mRNA and protein. Suppression of pdgf-betar mRNA in response to Myc is specific, since expression of the related receptor pdgf-alphar is not affected. We further show that Myc suppresses pdgf-betar mRNA expression by a mechanism which is distinguishable from Myc autosuppression. Analysis of c-Myc-null fibroblasts demonstrates that Myc is required for the repression of pdgf-betar mRNA expression in quiescent fibroblasts following mitogen stimulation. In addition, it is evident that the Myc-mediated repression of pdgf-betar mRNA levels plays an important role in the regulation of basal pdgf-betar expression in proliferating cells. Thus, our studies suggest an essential role for Myc in a negative-feedback loop regulating the expression of the PDGF-betaR.

Novel synthetic organosulfur compounds induce apoptosis of human leukemic cells.

It has been well documented that natural organosulfur compounds (OSCs) derived from plants such as garlic, onions and mahogany trees possess antiproliferative properties; however, the essential chemical features of the active OSC compounds remain unclear. To investigate the association between OSC structure and growth inhibitory activity, we synthesized novel relatives of dysoxysulfone, a natural OSC derived from the Fijian medicinal plant, Dysoxylum richii. In this study, we have examined the antiproliferative effects of these novel OSCs on a model human leukemic cell system and show that the compounds segregate into three groups. Group I, consisting of compounds A, B, G and J, did not affect either cell proliferation or the cell cycle profile of the leukemic cell lines. Group II, consisting of compounds F and H, induced the cells to undergo apoptosis from the G2/M phase of the cell cycle. Group III, consisting of compounds C, D, E and I, decreased cell proliferation and induced apoptosis throughout the cell cycle. The apoptotic agonists of Group II and III shared a common disulfide moiety, essential for leukemic cell cytotoxicity. Interestingly, Group II compounds did not affect cell viability of normal human diploid cells, suggesting the regions flanking the disulfide group contributes to the specificity of cell killing. Thus, we provide evidence that structure-activity analysis of natural products can identify novel compounds for the development of new therapeutics that can trigger apoptosis in a tumor-specific manner.

Promotion of growth and apoptosis in c-myc nullizygous fibroblasts by other members of the myc oncoprotein family.

c-myc nullizygous fibroblasts (KO cells) were used to compare the abilities of c-myc, N-myc and L-myc oncoproteins to accelerate growth, promote apoptosis, revert morphology, and regulate the expression of previously described c-myc target genes. All three myc oncoproteins were expressed following retroviral transduction of KO cells. The proteins all enhanced the growth rate of KO cells and significantly shortened the cell cycle transition time. They also accelerated apoptosis following serum deprivation, reverted the abnormal KO cell morphology, and modulated the expression of previously described c-myc target genes. In most cases, L-myc was equivalent to c-myc and N-myc in restoring all of the c-myc-dependent activities. These findings contrast with the previously reported weak transforming and transactivating properties of L-myc. Myc oncoproteins may thus impart both highly similar as well as dissimilar signals to the cells in which they are expressed.