RESUMO
Metastasis suppression by high-dose, multi-drug targeting is unsuccessful due to network heterogeneity and compensatory network activation. Here, we show that targeting driver network signaling capacity by limited inhibition of core pathways is a more effective anti-metastatic strategy. This principle underlies the action of a physiological metastasis suppressor, Raf Kinase Inhibitory Protein (RKIP), that moderately decreases stress-regulated MAP kinase network activity, reducing output to transcription factors such as pro-metastastic BACH1 and motility-related target genes. We developed a low-dose four-drug mimic that blocks metastatic colonization in mouse breast cancer models and increases survival. Experiments and network flow modeling show limited inhibition of multiple pathways is required to overcome variation in MAPK network topology and suppress signaling output across heterogeneous tumor cells. Restricting inhibition of individual kinases dissipates surplus signal, preventing threshold activation of compensatory kinase networks. This low-dose multi-drug approach to decrease signaling capacity of driver networks represents a transformative, clinically relevant strategy for anti-metastatic treatment.
Assuntos
Redes e Vias Metabólicas/efeitos dos fármacos , Metástase Neoplásica/prevenção & controle , Proteína de Ligação a Fosfatidiletanolamina/genética , Transdução de Sinais/efeitos dos fármacos , Animais , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular , Combinação de Medicamentos , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos NusRESUMO
Although the translational function of tRNA has long been established, extra translational functions of tRNA are still being discovered. We previously developed a computational method to systematically predict new tRNA-protein complexes and experimentally validated six candidate proteins, including the mitogen-activated protein kinase kinase 2 (MEK2), that interact with tRNA in HEK293T cells. However, consequences of the interaction between tRNA and these proteins remain to be elucidated. Here we tested the consequence of the interaction between tRNA and MEK2 in pancreatic cancer cell lines. We also generated disease and drug resistance-derived MEK2 mutants (Q60P, P128Q, S154F, E207K) to evaluate the function of the tRNA-MEK2 interaction. Our results demonstrate that tRNA interacts with the wild-type and mutant MEK2 in pancreatic cancer cells; furthermore, the MEK2 inhibitor U0126 significantly reduces the tRNA-MEK2 interaction. In addition, tRNA affects the catalytic activity of the wild type and mutant MEK2 proteins in different ways. Overall, our findings demonstrate the interaction of tRNA with MEK2 in pancreatic cancer cells and suggest that tRNA may impact MEK2 activity in cancer cells.
Assuntos
MAP Quinase Quinase 2/metabolismo , Neoplasias Pancreáticas/metabolismo , RNA de Transferência/metabolismo , Butadienos/farmacologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/genética , Mutação , Nitrilas/farmacologia , Neoplasias Pancreáticas/patologiaRESUMO
The ten-eleven translocation (TET) family of methylcytosine dioxygenases initiates demethylation of DNA and is associated with tumorigenesis in many cancers; however, the mechanism is mostly unknown. Here we identify upstream activators and downstream effectors of TET1 in breast cancer using human breast cancer cells and a genetically engineered mouse model. We show that depleting the architectural transcription factor high mobility group AT-hook 2 (HMGA2) induces TET1. TET1 binds and demethylates its own promoter and the promoter of homeobox A (HOXA) genes, enhancing its own expression and stimulating expression of HOXA genes including HOXA7 and HOXA9. Both TET1 and HOXA9 suppress breast tumor growth and metastasis in mouse xenografts. The genes comprising the HMGA2-TET1-HOXA9 pathway are coordinately regulated in breast cancer and together encompass a prognostic signature for patient survival. These results implicate the HMGA2-TET1-HOX signaling pathway in the epigenetic regulation of human breast cancer and highlight the importance of targeting methylation in specific subpopulations as a potential therapeutic strategy.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Proteína HMGA2/genética , Proteínas de Homeodomínio/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Proteína HMGA2/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Immunoblotting , Estimativa de Kaplan-Meier , Masculino , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Oxigenases de Função Mista , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMO
Cervical cancer is one of the most common cancers in women worldwide, being high-risk group the HPV infected, the leading etiological factor. The raf kinase inhibitory protein (RKIP) has been associated with tumor progression and metastasis in several human neoplasms, however its role on cervical cancer is unclear. In the present study, 259 uterine cervix tissues, including cervicitis, cervical intraepithelial lesions and carcinomas, were analyzed for RKIP expression by immunohistochemistry. We found that RKIP expression was significantly decreased during malignant progression, being highly expressed in non-neoplastic tissues (54% of the samples; 73/135), and expressed at low levels in the cervix invasive carcinomas (â¼15% (19/124). Following in vitro downregulation of RKIP, we observed a viability and proliferative advantage of RKIP-inhibited cells over time, which was associated with an altered cell cycle distribution and higher colony number in a colony formation assay. An in vitro wound healing assay showed that RKIP abrogation is associated with increased migratory capability. RKIP downregulation was also associated with an increased vascularization of the tumors in vivo using a CAM assay. Furthermore, RKIP inhibition induced cervical cancer cells apoptotic resistance to cisplatin treatment. In conclusion, we described that RKIP protein is significantly depleted during the malignant progression of cervical tumors. Despite the lack of association with patient clinical outcome, we demonstrate, in vitro and in vivo, that loss of RKIP expression can be one of the factors that are behind the aggressiveness, malignant progression and chemotherapy resistance of cervical cancer.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Adulto , Idoso , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Pessoa de Meia-Idade , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/genética , Resultado do Tratamento , Neoplasias do Colo do Útero/tratamento farmacológicoRESUMO
Transfer RNAs (tRNAs) are typically considered housekeeping products with little regulatory function. However, several studies over the past 10 years have linked tRNA misregulation to cancer. We have previously reported that tRNA levels are significantly elevated in breast cancer and multiple myeloma cells. To further investigate the cellular and physiological effects of tRNA overexpression, we overexpressed tRNAi(Met) in two human breast epithelial cell lines. We then determined tRNA abundance changes and performed phenotypic characterization. Overexpression of tRNAi(Met) significantly altered the global tRNA expression profile and resulted in increased cell metabolic activity and cell proliferation. Our results extend the relevance of tRNA overexpression in human cells and underscore the complexity of cellular regulation of tRNA expression.
Assuntos
Neoplasias da Mama/metabolismo , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , Aminoacil-RNA de Transferência/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , RNA de Transferência/genética , TranscriptomaRESUMO
Malignant gliomas are highly infiltrative and invasive tumors, which precludes the few treatment options available. Therefore, there is an urgent need to elucidate the molecular mechanisms underlying gliomas aggressive phenotype and poor prognosis. The Raf Kinase Inhibitory protein (RKIP), besides regulating important intracellular signaling cascades, was described to be associated with progression, metastasis and prognosis in several human neoplasms. Its role in the prognosis and tumourigenesis of gliomas remains unclear. In the present study, we found that RKIP protein is absent in a low frequency (10%, 20/193) of glioma tumors. Nevertheless, the absence of RKIP expression was an independent prognostic marker in glioma. Additionally, by in vitro downregulation of RKIP, we found that RKIP inhibition induces a higher viability and migration of the cells, having no effect on cellular proliferation and angiogenesis, as assessed by in vivo CAM assay. In conclusion, this is the largest series studied so far evaluating the expression levels of this important cancer suppressor protein in glioma tumors. Our results suggest that in a subset of tumors, the absence of RKIP associates with highly malignant behavior and poor survival of patients, which may be a useful biomarker for tailored treatment of glioma patients.
Assuntos
Neoplasias Encefálicas/diagnóstico , Glioma/diagnóstico , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Adolescente , Adulto , Idoso , Animais , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/fisiologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Células Cultivadas , Embrião de Galinha , Criança , Pré-Escolar , Progressão da Doença , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/genética , Glioma/metabolismo , Glioma/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Fisiológica/efeitos dos fármacos , Proteína de Ligação a Fosfatidiletanolamina/antagonistas & inibidores , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/fisiologia , Prognóstico , RNA Interferente Pequeno/farmacologia , Transfecção , Adulto JovemRESUMO
BACKGROUND: Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized. METHODS/FINDINGS: In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity. CONCLUSIONS/SIGNIFICANCE: This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.
Assuntos
Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Bibliotecas de Moléculas Pequenas/análise , Animais , Sítios de Ligação , Bioensaio , Sobrevivência Celular/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Células HeLa , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Ligantes , Espectroscopia de Ressonância Magnética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/química , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ratos , Bibliotecas de Moléculas Pequenas/metabolismo , Quinases raf/metabolismoRESUMO
Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 to 100,000 couplings. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.
Assuntos
Imunidade Inata , Metionina/metabolismo , Estresse Oxidativo/fisiologia , Aminoacilação de RNA de Transferência/fisiologia , Adenoviridae/fisiologia , Animais , Código Genético , Células HeLa , Humanos , Ligantes , Metionina/genética , Camundongos , Modelos Genéticos , NADPH Oxidases/metabolismo , Orthomyxoviridae/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Especificidade por Substrato , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Aminoacilação de RNA de Transferência/efeitos dos fármacosRESUMO
Ultraviolet (UV) exerts its biological activities by activating downstream effectors, including NF-kappaB, JNK, and caspases. Activation of JNK is required for UV-induced apoptosis. It is unknown whether any crosstalk occurs between NF-kappaB and JNK in response to UV and, if so, how it affects UV killing. Here we report that NF-kappaB promotes UV-induced JNK activation, thereby contributing to UV-induced apoptosis. UV-induced JNK activation is impaired in RelA/NF-kappaB null murine embryonic fibroblasts. In resting cells, the preexisting nuclear RelA has already been recruited to PKCdelta promoter and is essential for its expression. UV-induced rapid and robust activation of JNK requires PKCdelta, which augments JNK phosphorylation-activation by its upstream kinases. The RelA/NF-kappaB-PKCdelta-JNK pathway is critical for UV-induced apoptosis, as it induces the immediate expression of the proapoptotic Fas ligand. Thus, our results demonstrate that RelA/NF-kappaB via PKCdelta positively regulates UV-induced JNK activation and provide a mechanism by which NF-kappaB promotes UV-induced apoptosis.
Assuntos
Apoptose/efeitos da radiação , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteína Quinase C-delta/metabolismo , Animais , Células Cultivadas , Ativação Enzimática , Proteína Ligante Fas , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fatores de Necrose Tumoral/metabolismo , Raios UltravioletaRESUMO
Proteins like Raf kinase inhibitory protein (RKIP) that serve as modulators of signaling pathways, either by promoting or inhibiting the formation of productive signaling complexes through protein-protein interactions, have been demonstrated to play an increasingly important role in a number of cell types and organisms. These proteins have been implicated in development as well as the progression of cancer. RKIP is a particularly interesting regulator, as it is a highly conserved, ubiquitously expressed protein that has been shown to play a role in growth and differentiation in a number of organisms and can regulate multiple signaling pathways. RKIP is also the first MAP kinase signaling modulator to be identified as playing a role in cancer metastasis, and identification of the mechanism by which it regulates Raf-1 activation provides new targets for therapeutic intervention.
Assuntos
Sistema de Sinalização das MAP Quinases , Quinases raf/metabolismo , Animais , Bovinos , Diferenciação Celular , Proliferação de Células , Progressão da Doença , Humanos , Camundongos , Metástase Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Neurônios/metabolismo , Proteína Quinase C/metabolismo , Ratos , Transdução de SinaisRESUMO
We developed clonal cell lines of human bronchial smooth muscle origin by retroviral transduction of temperature-sensitive simian virus 40 large tumor (T) antigen. These cells show increased growth potential at 33 degrees C, but on shift to the nonpermissive temperature (39 degrees C), they show diminished or arrested growth. In addition to the expected reduction in the level of large T antigen, cells shifted to 39 degrees C show increased expression of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1), characteristic of cells arrested in G1 of the cell cycle. Shifted cells undergo a process of cell hypertrophy, as demonstrated by increased time of flight and forward scatter, as well as increased expression of the contractile proteins alpha-smooth muscle actin, myosin light chain kinase, and SM22. Changes in contractile protein expression were regulated primarily in a posttranscriptional manner. Phosphatidylinositol 3-kinase activity was increased in shifted cells, and chemical inhibition of phosphatidylinositol 3-kinase attenuated alpha-actin and myosin light-chain kinase expression. We have developed clonal cell lines of human bronchial smooth muscle origin that may be useful for the study of airway smooth muscle biology. Furthermore, we demonstrate that arrest of airway smooth muscle cell cycle traversal can induce cellular hypertrophy, which parallels changes observed in the airways of patients with severe asthma.
Assuntos
Brônquios/citologia , Brônquios/fisiologia , Linhagem Celular Transformada/fisiologia , Células Clonais/fisiologia , Miócitos de Músculo Liso/fisiologia , Antígenos Virais de Tumores , Asma/genética , Técnicas de Cultura de Células , Divisão Celular/fisiologia , Transformação Celular Viral , Humanos , Hipertrofia/genética , Oncogenes , Fenótipo , Vírus 40 dos Símios/imunologia , TemperaturaRESUMO
Generation of oxidative stress/reactive oxygen species (ROS) is one of the causes of neuronal apoptosis. We have examined the effects of ROS at the transcriptional level in an immortalized hippocampal neuronal cell line (H19-7) and in rat primary hippocampal neurons. Treatment of H19-7 cells with hydrogen peroxide (150 micro m) resulted in a 40% decrease in Bcl-2 protein and a parallel decrease in bcl-2 mRNA levels. H19-7 cells overexpressing bcl-2 were found to be resistant to ROS-induced apoptosis. We had previously shown that bcl-2 promoter activity is positively regulated by the transcription factor cyclic AMP response element binding protein (CREB) in neurons. In the present study, we demonstrate that ROS decreases the activity of luciferase reporter gene driven by a cyclic AMP response element site containing bcl-2 promoter. Exposure of neurons to ROS for 6 h resulted in basal and fibroblast growth factor-2-stimulated phosphorylation/activation of CREB. Chronic 24 h treatment with ROS led to a significant (p < 0.01) decrease in CREB protein and CREB mRNA levels. Adenoviral overexpression of wild type CREB in H19-7 cells resulted in significant (p < 0.01) protection against ROS-induced apoptosis through up-regulation of Bcl-2 expression whereas dominant negative CREB exaggerated the injury. These findings demonstrate that loss of CREB function contributes to oxidative stress-induced neuronal dysfunction.
Assuntos
Regulação para Baixo/fisiologia , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Regiões Promotoras Genéticas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Glucose Oxidase , Hipocampo/citologia , Neurônios/citologia , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) regulates immune responses, inflammation, and programmed cell death (apoptosis). TNF-alpha exerts its biological activities by activating multiple signaling pathways, including IkappaB kinase (IKK), c-Jun N-terminal protein kinase (JNK), and caspases. IKK activation inhibits apoptosis through the transcription factor NF-kappaB, whose target genes include those that encode inhibitors of both caspases and JNK. Despite activation of the antiapoptotic IKK/NF-kappaB pathway, TNF-alpha is able to induce apoptosis in cells sensitive to it, such as human breast carcinoma MCF-7 and mouse fibroblast LM cells. The molecular mechanism underlying TNF-alpha-induced apoptosis is incompletely understood. Here we report that in TNF-alpha-sensitive cells activation of the IKK/NF-kappaB pathway fails to block TNF-alpha-induced apoptosis, although its inactivation still promotes TNF-alpha-induced apoptosis. Interestingly, TNF-alpha-induced apoptosis is suppressed by inhibition of the JNK pathway but promoted by its activation. Furthermore, activation of JNK by TNF-alpha was transient in TNF-alpha-insensitive cells but prolonged in sensitive cells. Conversion of JNK activation from prolonged to transient suppressed TNF-alpha-induced apoptosis. Thus, absence of NF-kappaB-mediated inhibition of JNK activation contributes to TNF-alpha-induced apoptosis.
Assuntos
Apoptose/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Interleucina-1/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Inibidor de NF-kappaB alfa , NF-kappa B/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Acetato de Tetradecanoilforbol/metabolismoRESUMO
The precise mechanism by which protein kinase C-delta (PKCdelta) inhibits cell cycle progression is not known. We investigated the regulation of cyclin D1 transcription by PKCdelta in primary bovine airway smooth muscle cells. Overexpression of the active catalytic subunit of PKCdelta attenuated platelet-derived growth factor (PDGF)-mediated transcription from the cyclin D1 promoter, whereas overexpression of a dominant-negative PKCdelta increased promoter activity. A PKCdelta-specific pseudosubstrate increased cyclin D1 protein abundance. To determine the transcriptional mechanism by which PKCdelta negatively regulates cyclin D1 expression, we transiently transfected cells with cDNAs encoding cyclin D1 promoter 5' deletions and site mutations in the context of a -66 promoter fragment. We found that the -57 to -52 CRE/ATF2 site functions as a basal level and PDGF enhancer, whereas the -39 to -30 nuclear factor-kappaB site functions as a basal level suppressor. Further, PDGF and PKCdelta responsiveness of the cyclin D1 promoter was maintained following 5' deletion to the Ets-containing -22 minimal promoter. Finally, using electrophoretic mobility gel shift and reporter assays, we determined that PKCdelta inhibits CRE/ATF2 binding and transactivation, activates nuclear factor-kappaB binding and transactivation, and attenuates Ets transactivation. These data suggest that PKCdelta attenuates cyclin D1 promoter activity via the regulation of three distinct cis-acting regulatory elements.