c-Myc target genes involved in cell growth, apoptosis, and metabolism.
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Animals↗
Biomedical subjects
C V Dang
Publications and source records attributed to C V Dang.
c-Myc overexpression uncouples DNA replication from mitosis.
c-myc has been shown to regulate G(1)/S transition, but a role for c-myc in other phases of the cell cycle has not been identified. Exposure of cells to colcemid activates the mitotic spindle checkpoint and arrests cells transiently in metaphase. After prolonged colcemid exposure, the cells withdraw from mitosis and enter a G(1)-like state. In contrast to cells in G(1), colcemid-arrested cells have decreased G(1) cyclin-dependent kinase activity and show hypophosphorylation of the retinoblastoma protein. We have found that overexpression of c-myc causes colcemid-treated human and rodent cells to become either apoptotic or polyploid by replicating DNA without chromosomal segregation. Although c-myc-induced polyploidy is not inhibited by wild-type p53 in immortalized murine fibroblasts, overexpression of c-myc in primary fibroblasts resulted in massive apoptosis of colcemid-treated cells. We surmise that additional genes are altered in immortalized cells to suppress the apoptotic pathway and allow c-myc-overexpressing cells to progress forward in the presence of colcemid. Our results also suggest that c-myc induces DNA rereplication in this G(1)-like state by activating CDK2 activity. These observations indicate that activation of c-myc may contribute to the genomic instability commonly found in human cancers.
Animals↗
Neoplastic transformation of RK3E by mutant beta-catenin requires deregulation of Tcf/Lef transcription but not activation of c-myc expression.
Current models predict that beta-catenin (beta-cat) functions in Wnt signaling via activation of Tcf/Lef target genes and that its abundance is regulated by the adenomatous polyposis coli (APC) and glycogen synthase kinase 3beta (GSK3beta) proteins. In colon and other cancers, mutations in APC or presumptive GSK3beta phosphorylation sites of beta-cat are associated with constitutive activation of Tcf/Lef transcription. In spite of assumptions about its oncogenic potential, prior efforts to demonstrate that mutated beta-cat will induce neoplastic transformation have yielded equivocal results. We report here that mutated, but not wild-type, beta-cat proteins induced neoplastic transformation of RK3E, an adenovirus E1A-immortalized epithelial cell line. Analysis of the properties of mutant beta-cat proteins and studies with a dominant negative Tcf-4 mutant indicated that the ability of beta-cat to bind and activate Tcf/Lef factors is crucial for transformation. c-myc has recently been implicated as a critical Tcf-regulated target gene. However, c-myc was not consistently activated in beta-cat-transformed RK3E cells, and a dominant negative c-Myc mutant protein failed to inhibit beta-cat transformation. Our findings underscore the role of beta-cat mutations and Tcf/Lef activation in cancer and illustrate a useful system for defining critical factors in beta-cat transformation.
Adenoviridae↗
Gene therapy and translational cancer research.
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Forecasting↗
Immunoablative high-dose cyclophosphamide without stem-cell rescue for refractory, severe autoimmune disease.
BACKGROUND: Immunoablative high-dose cyclophosphamide without stem-cell rescue induces durable, complete remission in most patients with aplastic anemia. OBJECTIVE: To determine the efficacy of high-dose cyclophosphamide in various refractory, severe autoimmune diseases. DESIGN: Prospective phase II study. SETTING: Johns Hopkins University (Baltimore, Maryland) and Hahnemann University (Philadelphia, Pennsylvania). PATIENTS: Eight patients with refractory, severe autoimmune disease. INTERVENTION: Immunoablative high-dose cyclophosphamide (50 mg/kg of body weight per day) for 4 consecutive days. MEASUREMENTS: Clinical and laboratory variables of autoimmune disease. RESULTS: Seven patients improved markedly: Five achieved complete remission and two achieved partial remission. Four patients have remained in continuous complete remission for 3 to 21 months, and two patients in partial remission continue to improve after 14 and 19 months of follow-up. High-dose cyclophosphamide was well tolerated; median times to a neutrophil count of 0.5 x 10(9) cells/L and platelet transfusion independence were 17 and 16 days, respectively. CONCLUSIONS: Immunoablative high-dose cyclophosphamide without stem-cell rescue can induce complete remission in patients with refractory, severe autoimmune disease. Reemergence of marrow function is similar to that seen after autologous transplantation and does not carry the risk for reinfusion of autoaggressive lymphocytes with the autograft.
Adult↗
Association of the decreased expression of alpha3beta1 integrin with the altered cell: environmental interactions and enhanced soft agar cloning ability of c-myc-overexpressing small cell lung cancer cells.
Small cell lung cancer (SCLC) is a highly invasive and metastatic tumor, and the decreased expression of alpha3beta1 integrin may contribute to its virulence. Alpha3beta1 is a critical integrin for pulmonary development and epithelial integrity, and its reduced expression has been linked to the increased malignancy and invasion of other cancers. The amplification of the c-myc oncogene is seen frequently in relapsed SCLC tumors and is associated with a worsened prognosis. In the present study using a model of SCLC tumor progression, overexpression of c-myc in a classic SCLC cell line, NCI H209, enhanced in vitro features of tumorigenesis, altered the relationships between cell and environment, and markedly down-regulated the expression of the alpha3 integrin subunit at both the transcript and protein levels. This inverse relationship between the expression of the alpha3 integrin subunit and c-myc is mimicked by other c-myc-overexpressing SCLC cell lines. Restoring alpha3 expression in the myc-transfected 209 cells reversed the effects of c-myc: alpha3 transfection increased cell:cell adhesion and reduced soft agar cloning without affecting the in vitro doubling time. The diminished soft agar cloning produced by alpha3 transfection was reversed by an antibody that specifically engages alpha3beta1 integrins, P1B5. These results suggest first, that alpha3beta1 integrin mediates homotypic adhesion of SCLC cells, and second, that unengaged alpha3beta1 integrin suppresses the growth of disaggregated SCLC cells. Thus, the down-regulation of the alpha3 integrin subunit may contribute to the enhanced tumorigenicity of c-myc-overexpressing SCLCs by allowing the growth of tumor cells that have reduced contact with ligand-expressing substratum or cells, a condition that occurs during the growth of the primary tumor, tumor invasion, and metastasis.
Agar↗
Transient global amnesia after sex.
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Aged↗
Tumor growth inhibition induced in a murine model of human Burkitt's lymphoma by a proteasome inhibitor.
Cell growth and viability are dependent on the function of the multicatalytic proteinase complex (proteasome), a multisubunit particle that affects progression through the mitotic cycle by degradation of cyclins. Exposure of rodent fibroblasts and human lymphoblasts in culture to benzyloxycarbonyl-leucyl-leucyl-phenylalaninal (Z-LLF-CHO), a cell-permeable peptidyl aldehyde inhibitor of the chymotrypsin-like activity of the proteasome, resulted in the induction of apoptosis in a rapid, dose-dependent fashion. Fibroblasts transformed with ras and myc, lymphoblasts transformed by c-myc alone, and a Burkitt's lymphoma (BL) cell line that overexpresses c-Myc were up to 40-fold more susceptible to apoptosis than were either primary rodent fibroblasts or immortalized nontransformed human lymphoblasts, respectively. To determine whether such preferential apoptosis could impact upon tumor growth in vivo, toxicological studies were performed in mice with severe combined immunodeficiency and showed that mice tolerated single interscapular doses of Z-LLF-CHO without unacceptable toxicity. Severe combined immunodeficient mice bearing s.c. BL tumors in the flank were treated interscapularly with Z-LLF-CHO or a comparable dose of the peptidyl alcohol (Z-LLF-OH), which does not induce proteasome inhibition or apoptosis. Single doses of Z-LLF-CHO induced statistically significant (P < 0.0001) early tumor regression and a significant (P < 0.0001) delay in tumor progression. Analysis of tumor specimens revealed increased apoptosis in BL tumors from mice treated with Z-LLF-CHO. These results, showing a 42% tumor growth delay, indicate that proteasome inhibitors have the potential of curbing the growth of a c-myc-related tumor.
Animals↗
Elevated extracellular calcium can prevent apoptosis via the calcium-sensing receptor.
The calcium-sensing receptor (CaR) is a membrane-bound, G-protein-coupled receptor present on parathyroid cells which monitors the level of extracellular calcium (Ca2+o) and transduces signals involved in serum calcium regulation. Expression of CaR protein in tissues with functions unrelated to systemic calcium homeostasis, including the brain, suggests that extracellular calcium (Ca2+o) may act as a first messenger to regulate diverse cellular functions. To test this hypothesis, we examined the effect of increasing Ca2+o on apoptosis induced by Sindbis Virus in AT-3 prostate carcinoma cells. We found a steep increase in cell survival with between 5 and 7 mM added Ca2+o (EC50 = 6.1 mM). Magnesium, a less potent agonist of the calcium sensing receptor, was also protective (EC50 = 23.4 mM). Northern and immunocytochemical analyses confirmed the presence of the CaR message and protein in AT-3 prostate carcinoma cells. Enforced expression of CaR protein by stable transfection in human embryonic kidney (HEK)-293 cells, which normally don't express the receptor, resulted in resistance to SV-induced apoptosis in the presence of elevated Ca2+o. In addition to preventing SV-induced death, elevated Ca2+o also abrogated apoptosis induced by c-Myc overexpression/serum deprivation in rat 1A fibroblasts, and these fibroblasts were shown to express CaR message and protein. Altogether, these observations suggest that Ca2+o can act with the CaR to prevent apoptosis and define a novel mechanism by which calcium ions can regulate cell survival.
Animals↗
Identification of a large Myc-binding protein that contains RCC1-like repeats.
The protooncogene MYC plays an important role in the regulation of cellular proliferation, differentiation, and apoptosis and has been implicated in a variety of human tumors. MYC and the closely related MYCN encode highly conserved nuclear phosphoproteins (Myc and NMyc) that apparently function as transcription factors in the cell. We have identified a large and highly conserved nuclear protein that interacts directly with the transcriptional activating domain of Myc (designated "protein associated with Myc" or Pam). Pam contains an extended amino acid sequence with similarities to a protein known as regulator of chromosome condensation (RCC1), which may play a role in the function of chromatin. The gene encoding Pam (PAM) is expressed in all of the human tissue examined, but expression is exceptionally abundant in brain and thymus. Pam binds specifically to Myc, but not NMyc. The region in Myc required for binding to Pam includes a domain that is essential for the function of Myc and that is frequently mutated in Burkitt's lymphomas. PAM is located within a 300-kb region on chromosome 13q22.
Adaptor Proteins, Signal Transducing↗
Pancytopenia secondary to oxalosis in a 23-year-old woman.
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Adult↗
A unique glucose-dependent apoptotic pathway induced by c-Myc.
The lactate dehydrogenase A (LDH-A) gene, whose product participates in normal anaerobic glycolysis and is frequently increased in human cancers, has been identified as a c-Myc-responsive gene. It was of interest, therefore, to compare the effect of glucose deprivation in c-Myc-transformed and nontransformed cells. We observed that glucose deprivation or treatment with the glucose antimetabolite 2-deoxyglucose caused nontransformed cells to arrest in the G0/G1 phase of the cell cycle. In contrast, c-Myc-transformed fibroblasts, lymphoblastoid, or lung carcinoma cells underwent extensive apoptosis. Ectopic expression of LDH-A alone in Rat1a fibroblasts was sufficient to induce apoptosis with glucose deprivation but not with serum withdrawal, suggesting that LDH-A mediates the unique apoptotic effect of c-Myc when glycolysis is blocked. The apoptosis caused by glucose deprivation was blocked by Bcl-2 expression but appeared to be independent of wild-type p53 activity. These studies provide insights on the coupling of glucose metabolism and the cell cycle in c-Myc-transformed cells and may in the future be exploited for cancer therapeutics.
Animals↗
Mammalian BUB1 protein kinases: map positions and in vivo expression.
The spindle assembly checkpoint modulates the timing of anaphase initiation in mitotic cells containing improperly aligned chromosomes and increases the probability of successful delivery of a euploid chromosome set to each daughter cell. We have characterized cDNA sequences from several organisms with highly significant predicted protein sequence homologies to Saccharomyces cerevisiae Bub1p, a protein required for function of the spindle assembly checkpoint in budding yeast. The localization of mouse and human orthologs is in agreement with known conservation of synteny. Mouse backcross mapping data indicate that the murine gene resides on chromosome 2 near IL1A, 73 cM from the mouse centromere. Radiation hybrid mapping data indicate that the human locus exhibits linkage to microsatellite marker D2S176, which is located within 10 cM of human IL1A. Multiple-tissue Northern analysis indicates conservation of expression pattern in mouse and human with markedly high mRNA levels in testis. Northern analysis of two different spindle assembly checkpoint protein gene products from human, BUB1 and MAD2, reveals an expression pattern with common tissue distribution consistent with roles in a common pathway. In addition, we demonstrate that an mRNA found to accumulate in a rat fibroblast cell transformation system encodes rat BUB1, and we find that rat BUB1 mRNA accumulation correlates with the proliferation status of cells in culture.
Amino Acid Sequence↗
MXI1, a putative tumor suppressor gene, suppresses growth of human glioblastoma cells.
The Mxi1 protein functions in a regulatory network with members of the c-Myc family, in which c-Myc activates transcription and stimulates cell proliferation, and Mxi1 negatively regulates these actions. Inactivation of the MXI1 gene could, therefore, inhibit differentiation and enhance proliferation in the presence of normal levels of c-Myc, and thus MXI1 is a potential tumor suppressor gene. We and others have previously mapped the MXI1 gene to the distal portion of chromosome 10q, a region that is rearranged or affected by allelic loss in many astrocytic brain tumors. Using a newly described polymorphic CA microsatellite repeat in the third MXI1 intron, we show that 7 of 11 informative glioblastomas demonstrated MXI1 allelic loss. Sequence analysis revealed no somatic mutations in any of the six MXI1 coding exons, similar to findings in prostate tumors with MXI1 allelic loss. To determine whether MXI1 can indeed function as a suppressor of growth, we have introduced a steroid-inducible MXI1 expression vector into the U87MG cell line, a glioblastoma cell line lacking endogenous MXI1 expression. Induction of MXI1 expression resulted in a decreased growth rate and distinct morphological changes. Furthermore, cell cycle analysis demonstrated that induction of MXI1 results in accumulation of cells in the G2-M phase. Thus, these studies support the notion that MXI1 normally functions to suppress cell growth and suggest that loss of MXI1 function may play a role in human glioblastoma development.
Basic Helix-Loop-Helix Proteins↗
17beta-estradiol inhibits apoptosis of endothelial cells.
Endothelial cells provide an antithrombotic and anti-inflammatory barrier for the normal vessel wall. Dysfunction of endothelial cells has been shown to promote atherosclerosis, and normalization of previously dysfunctional endothelial cells can inhibit the genesis of atheroma. In normal arteries, endothelial cells are remarkably quiescent. Acceleration of the turnover rate of endothelial cells can lead to their dysfunction. Apoptosis is a physiological process that contributes to vessel homeostasis, by eliminating damaged cells from the vessel wall. However, increased endothelial cell turnover mediated through accelerated apoptosis may alter the function of the endothelium and therefore, promote atherosclerosis. Apoptotic endothelial cells can be detected on the luminal surface of atherosclerotic coronary vessels, but not in normal vessels. This finding links endothelial cell apoptosis and the process of atherosclerosis, although a causative role for apoptosis in this process remains hypothetical. Estrogen metabolites have been shown to be among the most potent anti-atherogenic agents available to date for post-menopausal women. The mechanism of estrogen's protective effect is currently incompletely characterized. Here we show that 17beta-estradiol, a key estrogen metabolite, inhibits apoptosis in cultured endothelial cells. Our data support the hypothesis that 17beta-estradiol's anti-apoptotic effect may be mediated via improved endothelial cell interaction with the substratum, increased tyrosine phosphorylation of pp125 focal adhesion kinase, and a subsequent reduction in programmed cell death of endothelial cells. Inhibition of apoptosis by estrogens may account for some of the anti-atherogenic properties of these compounds.
Animals↗
Nuclear punctate distribution of ALL-1 is conferred by distinct elements at the N terminus of the protein.
The ALL-1 gene positioned at 11q23 is directly involved in human acute leukemia either through a variety of chromosome translocations or by partial tandem duplications. ALL-1 is the human homologue of Drosophila trithorax which plays a critical role in maintaining proper spatial and temporal expression of the Antennapedia-bithorax homeotic genes determining the fruit fly's body pattern. Utilizing specific antibodies, we found that the ALL-1 protein distributes in cultured cells in a nuclear punctate pattern. Several chimeric ALL-1 proteins encoded by products of the chromosome translocations and expressed in transfected cells showed similar speckles. Dissection of the ALL-1 protein identified within its approximately 1,100 N-terminal residues three polypeptides directing nuclear localization and at least two main domains conferring distribution in dots. The latter spanned two short sequences conserved with TRITHORAX. Enforced nuclear expression of other domains of ALL-1, such as the PHD (zinc) fingers and the SET motif, resulted in uniform nonpunctate patterns. This indicates that positioning of the ALL-1 protein in subnuclear structures is mediated via interactions of ALL-1 N-terminal elements. We suggest that the speckles represent protein complexes which contain multiple copies of the ALL-1 protein and are positioned at ALL-1 target sites on the chromatin. Therefore, the role of the N-terminal portion of ALL-1 is to direct the protein to its target genes.
Amino Acid Sequence↗
c-Myc transactivation of LDH-A: implications for tumor metabolism and growth.
Cancer cells are able to overproduce lactic acid aerobically, whereas normal cells undergo anaerobic glycolysis only when deprived of oxygen. Tumor aerobic glycolysis was recognized about seven decades ago; however, its molecular basis has remained elusive. The lactate dehydrogenase-A gene (LDH-A), whose product participates in normal anaerobic glycolysis and is frequently increased in human cancers, was identified as a c-Myc-responsive gene. Stably transfected Rat1a fibroblasts that overexpress LDH-A alone or those transformed by c-Myc overproduce lactic acid. LDH-A overexpression is required for c-Myc-mediated transformation because lowering its level through antisense LDH-A expression reduces soft agar clonogenicity of c-Myc-transformed Rat1a fibroblasts, c-Myc-transformed human lymphoblastoid cells, and Burkitt lymphoma cells. Although antisense expression of LDH-A did not affect the growth of c-Myc-transformed fibroblasts adherent to culture dishes under normoxic conditions, the growth of these adherent cells in hypoxia was reduced. These observations suggest that an increased LDH-A level is required for the growth of a transformed spheroid cell mass, which has a hypoxic internal microenvironment. Our studies have linked c-Myc to the induction of LDH-A, whose expression increases lactate production and is necessary for c-Myc-mediated transformation.
Animals↗
Max and inhibitory c-Myc mutants induce erythroid differentiation and resistance to apoptosis in human myeloid leukemia cells.
We have used the human leukemia cell line K562 as a model to study the role of c-myc in differentiation and apoptosis. We have generated stable transfectants of K562 constitutively expressing two c-Myc inhibitory mutants: D106-143, that carries a deletion in the transactivation domain of the protein, and In373, that carries an insertion in the DNA-interacting region. We show here that In373 is able to compete with c-Myc for Max binding and to inhibit the transformation activity of c-Myc. K562 cells can differentiate towards erythroid or myelomonocytic lineages. K562 transfected with c-myc mutants showed a higher expression of erythroid differentiation markers, without any detectable effects in the myelomonocytic differentiation. We also transfected K562 cells with a zinc-inducible max gene. Ectopic Max overexpression resulted in an increased erythroid differentiation, thus reproducing the effects of c-myc inhibitory mutants. We also studied the role of c-myc mutants and max in apoptosis of K562 induced by okadaic acid, a protein phosphatases inhibitor. The expression of D106-143 and In373 c-myc mutants and the overexpression of max reduced the apoptosis mediated by okadaic acid. The common biochemical activity of D106-143 and In373 is to bind Max and hence to titrate out c-Myc to form non-functional Myc/Max dimers. Similarly, Max overexpression would decrease the relative levels of c-Myc/Max with respect to Max/Max. The results support a model where a threshold of functional c-Myc/Max is required to maintain K562 cells in an undifferentiated state and to undergo drug-mediated apoptosis.
Apoptosis↗