RESUMO
Multiple myeloma (MM) is an incurable malignancy with an unmet need for innovative treatment options. Histone deacetylase inhibitors (HDACi) are a new class of anticancer agent that have demonstrated activity in hematological malignancies. Here, we investigated the efficacy and safety of HDACi (vorinostat, panobinostat, romidepsin) and novel combination therapies using in vitro human MM cell lines and in vivo preclinical screening utilizing syngeneic transplanted Vk*MYC MM. HDACi were combined with ABT-737, which targets the intrinsic apoptosis pathway, recombinant human tumour necrosis factor-related apoptosis-inducing ligand (rhTRAIL/MD5-1), that activates the extrinsic apoptosis pathway or the DNA methyl transferase inhibitor 5-azacytidine. We demonstrate that in vitro cell line-based studies provide some insight into drug activity and combination therapies that synergistically kill MM cells; however, they do not always predict in vivo preclinical efficacy or toxicity. Importantly, utilizing transplanted Vk*MYC MM, we report that panobinostat and 5-azacytidine synergize to prolong the survival of tumor-bearing mice. In contrast, combined HDACi/rhTRAIL-based strategies, while efficacious, demonstrated on-target dose-limiting toxicities that precluded prolonged treatment. Taken together, our studies provide evidence that the transplanted Vk*MYC model of MM is a useful screening tool for anti-MM drugs and should aid in the prioritization of novel drug testing in the clinic.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Azacitidina/uso terapêutico , Compostos de Bifenilo/uso terapêutico , Inibidores de Histona Desacetilases/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Nitrofenóis/uso terapêutico , Sulfonamidas/uso terapêutico , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Azacitidina/farmacologia , Compostos de Bifenilo/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Nitrofenóis/farmacologia , Panobinostat , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores de Morte Celular/metabolismo , Proteínas Recombinantes , Sulfonamidas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
We explored the activity of SIRT1 activators (SRT501 and SRT2183) alone and in combination with panobinostat in a panel of malignant lymphoid cell lines in terms of biological and gene expression responses. SRT501 and SRT2183 induced growth arrest and apoptosis, concomitant with deacetylation of STAT3 and NF-κB, and reduction of c-Myc protein levels. PCR arrays revealed that SRT2183 leads to increased mRNA levels of pro-apoptosis and DNA-damage-response genes, accompanied by accumulation of phospho-H2A.X levels. Next, ChIP assays revealed that SRT2183 reduces the DNA-binding activity of both NF-κB and STAT3 to the promoter of GADD45G, which is one of the most upregulated genes following SRT2183 treatment. Combination of SRT2183 with panobinostat enhanced the anti-growth and anti-survival effects mediated by either compound alone. Quantitative-PCR confirmed that the panobinostat in combination with SRT2183, SRT501 or resveratrol leads to greater upregulation of GADD45G than any of the single agents. Panobinostat plus SRT2183 in combination showed greater inhibition of c-Myc protein levels and phosphorylation of H2A.X, and increased acetylation of p53. Furthermore, EMSA revealed that NF-κB binds directly to the GADD45G promoter, while STAT3 binds indirectly in complexes with NF-κB. In addition, the binding of NF-κB/STAT3 complexes to the GADD45G promoter is inhibited following panobinostat, SRT501 or resveratrol treatment. Moreover, the combination of panobinostat with SRT2183, SRT501 or resveratrol induces a greater binding repression than either agent alone. These data suggest that STAT3 is a corepressor with NF-κB of the GADD45G gene and provides in vitro proof-of-concept for the combination of HDACi with SIRT1 activators as a potential new therapeutic strategy in lymphoid malignancies.
Assuntos
Histona Desacetilases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição STAT3/metabolismo , Sirtuína 1/metabolismo , Acetilação , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Linfoma/metabolismo , Linfoma/patologia , NF-kappa B/genética , Panobinostat , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Resveratrol , Fator de Transcrição STAT3/genética , Sirtuína 1/química , Estilbenos/farmacologia , Regulação para Cima/efeitos dos fármacos , Proteínas GADD45RESUMO
Panobinostat is a potent oral pandeacetylase inhibitor that leads to acetylation of intracellular proteins, inhibits cellular proliferation and induces apoptosis in leukemic cell lines. A phase Ia/II study was designed to determine the maximum-tolerated dose (MTD) of daily panobinostat, administered on two schedules: three times a week every week or every other week on a 28-day treatment cycle in patients with advanced hematologic malignancies. The criteria for hematologic dose-limiting toxicities differed between patients with indications associated with severe cytopenias at baseline (leukemia and myeloid disorders) and those less commonly associated with baseline cytopenias (lymphoma and myeloma). In patients with leukemia and myeloid disorders, 60 mg was the MTD for weekly as well as biweekly panobinostat. In patients with lymphoma and myeloma, 40 mg was the recommended dose for phase II evaluation (formal MTD not determined) of weekly panobinostat, and 60 mg was the MTD for biweekly panobinostat. Overall, panobinostat-related grade 3-4 adverse events included thrombocytopenia (41.5%), fatigue (21%) and neutropenia (21%). Single-agent activity was observed in several indications, including Hodgkin lymphoma and myelofibrosis. This phase Ia/II study provided a broad analysis of the safety profile and efficacy of single-agent panobinostat in patients with hematologic malignancies.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Hematológicas/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Indóis/uso terapêutico , Acetilação , Administração Oral , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Esquema de Medicação , Feminino , Neoplasias Hematológicas/diagnóstico , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/efeitos adversos , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Ácidos Hidroxâmicos/efeitos adversos , Indóis/administração & dosagem , Indóis/efeitos adversos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Panobinostat , Resultado do Tratamento , Adulto JovemRESUMO
Acute myeloid leukemia (AML) is a clonal disorder characterized by the accumulation of myeloid blasts in the bone marrow. Here, we report the effects of the novel histone deacetylase inhibitor panobinostat (LBH589) in combination with doxorubicin on AML cells. Panobinostat exhibited potent anti-AML activity in all AML cell lines tested and in primary AML cells from patients (IC(50)<20 nM). In addition, panobinostat potentiated the action of several standard-of-care anti-AML compounds, particularly, doxorubicin. The molecular effects induced by panobinostat and doxorubicin treatment were investigated by analyzing gene expression, cell cycle, apoptosis and signaling pathways. Analyses of gene expression profiles identified 588 genes whose expression was exclusively affected by the combination of panobinostat and doxorubicin. The combination induced AML cell death by an increase in the mitochondrial outer membrane permeability and release of cytochrome c from the mitochondria, resulting in caspase-dependent apoptosis and accompanied by the upregulation of Bax, Bak and, particularly, Bad. The drug combination provoked a strong activation of a DNA damage response, indicating that this combination may trigger cell death by a mechanism that induced DNA double-strand breaks. These data indicate that the combination of panobinostat and doxorubicin may be an effective therapy for the treatment of AML.
Assuntos
Reparo do DNA/efeitos dos fármacos , Doxorrubicina/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Antibióticos Antineoplásicos , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Humanos , Indóis , Mitocôndrias/efeitos dos fármacos , Panobinostat , Células Tumorais CultivadasRESUMO
NVP-LAQ824 is a novel potent hydroxamic acid-derived histone deacetylase inhibitor that induces apoptosis in nanomolar concentrations in myeloid leukemia cell lines and patient samples. Here we show the activity of NVP-LAQ824 in acute myeloid leukemia cells and BCR/ABL-expressing cells of mouse and human origin, both sensitive and resistant to imatinib mesylate (Gleevec, STI-571). Whereas imatinib inhibited overall cellular tyrosine phosphorylation in Ba/F3.p210 cells, NVP-LAQ824 did not inhibit tyrosine phosphorylation, and did not affect BCR/ABL or ABL protein expression. Neither compound was able to inhibit cellular tyrosine phosphorylation in the imatinib-resistant Ba/F3.p210-T315I cell line. These data taken together suggest that BCR/ABL kinase activity is not a direct target of NVP-LAQ824. Synergy between NVP-LAQ824 and imatinib was demonstrated against BCR/ABL-expressing K562 myeloid leukemia cell lines. In addition, we show that NVP-LAQ824 was well tolerated in vivo in a pre-clinical murine leukemia model, with antileukemia activity resulting in significant prolongation of the survival of mice when treated with NVP-LAQ824 compared to control mice. Taken together, these findings provide the framework for NVP-LAQ824 as a novel therapeutic in myeloid malignancies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Leucemia Mieloide/tratamento farmacológico , Acetilação , Doença Aguda , Animais , Apoptose/efeitos dos fármacos , Benzamidas , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Ciclo Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Mesilato de Imatinib , Técnicas In Vitro , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos , Piperazinas/administração & dosagem , Pirimidinas/administração & dosagem , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
P-glycoprotein (P-gp) is a transmembrane glycoprotein that confers multidrug resistance (MDR). It has been demonstrated that the Gly185 residue within the cytoplasmic loop between predicted transmembrane portions 2 and 3 plays an important role in substrate specificity of human P-gp. Derivatives of cyclosporin interact with and reverse the ability of P-gp to act as a drug efflux pump. To determine if the Gly185 residue of human P-gp is also important for the interaction of P-gp with closely related cyclosporin derivatives, we examined the effect of PSC-833 and CsA on P-gp in KB3-1 cells transfected with human wild-type P-gp (GSV-2) or with the mutant P-gp (VSV-1) that habored the Gly185-->Val substitution. While the ability of CsA to sensitize VSV-1 cells to anticancer agents was enhanced, no changes in the potency of PSC-833 against cells transfected with either the wild-type or mutant P-gp were observed. In addition, VSV-1 transfected cells were more sensitive to CsA inhibition of verapamil-stimulated ATPase activity than cells transfected with wild-type P-gp. Furthermore, the intracellular accumulation of CsA was low in GSV-2 P-gp-expressing cells, compared with its accumulation in VSV-1 cells and it was found to be as high as in non-P-gp expressing KB3-1 cells. These results indicated an enhanced sensitivity of Val185-P-gp expressing cells to CsA that correlated with increased intracellular accumulation in these cells. In contrast, no significant difference in the accumulation of PSC-833 was observed among the parental, wild-type or resistant cells. Since PSC-833 was found to be more potent than CsA, these studies provided insight into the effects of the structure of MDR modulators in mediating sensitivity to anticancer drugs.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Substituição de Aminoácidos , Antineoplásicos/metabolismo , Ciclosporina/farmacologia , Ciclosporinas/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Proteínas de Neoplasias/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Adenosina Trifosfatases/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Carcinoma de Células Escamosas/patologia , Colchicina/metabolismo , Daunorrubicina/metabolismo , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/fisiologia , Paclitaxel/metabolismo , Mutação Puntual , Relação Estrutura-Atividade , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Verapamil/farmacologia , Vimblastina/metabolismoRESUMO
The expression of drug efflux mechanisms by cancer cells during chemotherapy leads to multidrug resistance (MDR) and constitutes a major obstacle in the effective treatment of cancer. The most widely characterized drug effluxes pump is P-glycoprotein (P-gp) and efforts are being directed towards identifying agents that reverse P-gp mediated drug resistance. PSC-833 is a non-immunosuppressive cyclosporin derivative that potently and specifically inhibits P-gp. The current review focuses on the elucidation of the mechanism of action of PSC-833 as a potential MDR reversing agent, using syngeneic multidrug resistant sublines of MDA435 human breast adenocarcinoma cell line that express increasing levels of P-gp. In vitro experiments indicate that PSC-833 interacts directly with P-gp with high affinity and probably interferes with the ATPase activity of P-gp. Studies in multidrug resistant tumor models confirm P-gp as the in vivo target of PSC-833 and demonstrate the ability of PSC-833 to reverse MDR leukemias and solid tumors in mice. Presently, PSC-833 is being evaluated in the clinic.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Ciclosporinas/farmacologia , Ciclosporinas/uso terapêutico , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Animais , Humanos , Camundongos , Neoplasias Experimentais/tratamento farmacológico , RatosRESUMO
Human diploid fibroblasts (HDFs) undergo a limited number of population doublings in vitro and are widely used as a model of cellular aging. Despite growing evidence that cellular aging occurs as a result of altered gene expression, little is known about the activity of transcription factors that regulate gene expression in aging cells. Here we survey the relevant literature regarding altered gene expression and the role of transcription factors during cellular aging, focusing upon the serum response factor (SRF). SRF is hyperphosphorylated in senescent HDFs and fails to bind to the serum-response element in the c-fos promoter. Differential phosphorylation during replicative aging may contribute, at least in part, to the altered activity of SRF and possibly other transcription factors and to subsequent changes in the expression of serum-regulated genes in senescent HDFs.
Assuntos
Fenômenos Fisiológicos Celulares , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Senescência Celular/fisiologia , Fibroblastos/fisiologia , Genes Supressores de Tumor , HumanosRESUMO
The balance between the activities of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) is an important control point in tissue remodeling. Previous studies have demonstrated elevated expression of the MMPs collagenase and stromelysin-1 by aged human diploid fibroblasts compared to early-passage cultures. We show here that aging cells display an altered response to transforming growth factor-beta 1 (TGF beta 1) that selectively affects MMP mRNA expression. In both young and old cells, phorbol myristoyl-13 acetate (PMA) induced the expression of transcripts of collagenase, stromelysin-1, gelatinase-B, TIMP-1, and TIMP-3. In young cells, TGF beta 1 reciprocally modulated PMA-induced MMP and TIMP gene expression leading to reduced levels of transcripts for the MMPs and augmented accumulation of TIMP-1 and TIMP-3 mRNAs. However, repressing effects of TGF beta 1 on collagenase, stromelysin-1, and gelatinase-B RNA expression were not apparent in old cells, though induction of the TIMP genes was unimpaired. By electrophoretic mobility shift analysis the nuclear transcription factors AP1 and serum response factor (SRF) showed reduced levels of DNA binding activities in old fibroblasts compared to young cells. A probe for the TGF beta-inhibitory element (TIE) gave equivalent levels of complexes with nuclear extracts from both types of cells, though of different mobilities. We conclude that the effects of TGF beta 1 on MMP and TIMP gene expression involve different cellular intermediaries, and suggest that altered composition or modification of TIE binding factors in aging cells may underlie the failure of TGF beta 1-mediated transcription repression. This mechanism may contribute to elevated constitutive expression of MMPs in old cells and to the connective tissue deterioration that accompanies the aging process.
Assuntos
Senescência Celular , Metaloendopeptidases/análise , Inibidores de Proteases/análise , Fator de Crescimento Transformador beta/farmacologia , Células Cultivadas , Colagenases/análise , Colagenases/genética , Fibroblastos , Glicoproteínas/análise , Glicoproteínas/genética , Humanos , Metaloproteinase 3 da Matriz , Metaloproteinase 9 da Matriz , Metaloendopeptidases/genética , Proteínas/análise , Proteínas/genética , Acetato de Tetradecanoilforbol/farmacologia , Inibidor Tecidual de Metaloproteinase-2 , Inibidor Tecidual de Metaloproteinase-3 , Inibidores Teciduais de MetaloproteinasesRESUMO
Several lines of evidence suggest that the limited replication potential of normal human cells is due to the presence of an intrinsic genetic programme. This "senescence programme" is believed to reduce the incidence of cancer by limiting the growth of most of the transformed cells arising in vivo, although some cells do escape senescence becoming both immortalized and transformed. Here we review the literature that describes the senescence process in terms of gene expression and the regulation of gene expression by a variety of mechanisms affecting transcription factor activity. We focus on regulation of the c-fos gene through posttranslational modification of the serum response factor (SRF) as an example of altered gene expression during cellular aging.
Assuntos
Senescência Celular , Regulação da Expressão Gênica , Genes fos , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteína do Retinoblastoma/metabolismo , Fator de Resposta Sérica , Proteína Supressora de Tumor p53/metabolismoRESUMO
The p53 tumor-suppressor protein binds DNA and activates the expression of a 21-kDa protein that inhibits both the activity of cyclin-dependent kinases and the function of proliferating cell nuclear antigen. Since p21 expression has been reported to increase 10- to 20-fold as human diploid fibroblasts lose the ability to replicate, we examined the expression and activity of p53 during replicative aging. Similar levels of total p53 mRNA and protein were expressed in low-passage (young) and high-passage (old) cells but both DNA binding activity in vitro and transcriptional activity of p53 in vivo were increased severalfold in high-passage cells. While the basis of increased p53 activity is presently unclear, it is not correlated with differential phosphorylation or changes in p53-mouse double minute 2 gene product interactions. These results provide evidence for the activation of a protein involved in the control of cell cycle checkpoints during cellular aging, in the absence of increased expression.
Assuntos
Senescência Celular/fisiologia , Proteínas Nucleares , Proteína Supressora de Tumor p53/fisiologia , Adulto , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA/metabolismo , Fibroblastos/citologia , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Human cyclin D1 forms complexes with several Cdks, with proliferating cell nuclear antigen, and with a recently discovered 21-kDa inhibitor of Cdk activity. Substrates for cyclin D1/Cdks have not been identified in vivo, but it has been proposed that the D class of cyclins might play a role in regulating the activity of the retinoblastoma gene product p105Rb. Here we report that normal human diploid fibroblasts that endogenously or ectopically express high levels of cyclin D1 are unable to enter S phase in response to normally mitogenic stimuli. Fibroblasts that have reached the end of their in vitro life span (senescent cells) express five-fold higher levels of cyclin D1 protein than low-passage cells and individual cells in mass culture that fail to initiate DNA synthesis in response to serum addition have severalfold higher levels of this cyclin than proliferation-competent cells. These observations provide evidence that cyclin D1 is involved with the regulation of cell proliferation by more than one mechanism.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Divisão Celular/fisiologia , Ciclinas/fisiologia , Fibroblastos/fisiologia , Proteínas Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas , Sequência de Bases , Sangue , Linhagem Celular , Linhagem Celular Transformada , Senescência Celular/fisiologia , Clonagem Molecular , Ciclina D1 , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/biossíntese , Ciclinas/genética , DNA/biossíntese , Primers do DNA , Diploide , Fibroblastos/enzimologia , Humanos , Mitose/fisiologia , Dados de Sequência Molecular , Proteínas Oncogênicas/biossíntese , Proteínas Oncogênicas/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/fisiologiaRESUMO
Human diploid fibroblasts undergo a limited number of population doublings in vitro and are used widely as a model of cellular aging. Despite growing evidence that cellular aging occurs as a consequence of altered gene expression, little is known about the activity of transcription factors in aging cells. Here, we report a dramatic reduction in the ability of proteins extracted from the nuclei of near-senescent fibroblasts to bind the serum response element which is necessary for serum-induced transcription of the c-fos gene. In contrast, the activities of proteins binding to the RNA polymerase core element, TATA, as well as to the cyclic AMP response element were maintained during cellular aging. While no major differences in the expression of the serum response factor (SRF) that binds the serum response element were seen between early-passage and late-passage cells, hyperphosphorylation of SRF was observed in near-senescent cells. Furthermore, removal of phosphatase inhibitors during the isolation of endogenous nuclear proteins restored the ability of SRF isolated from old cells to bind the SRE. These data, therefore, indicate that hyperphosphorylation of SRF plays a role in altering the ability of this protein to bind to DNA and regulate gene expression in senescent cells.