RESUMO
Exposure to hypoxia-induced replication arrest initiates a DNA damage response that includes both ATR- and ATM-mediated signaling. DNA fiber analysis was used to show that these conditions lead to a replication arrest during both the initiation and elongation phases, and that this correlated with decreased levels of nucleotides. The DNA damage response induced by hypoxia is distinct from the classical pathways induced by damaging agents, primarily due to the lack of detectable DNA damage, but also due to the coincident repression of DNA repair in hypoxic conditions. The principle aims of the hypoxia-induced DNA damage response seem to be the induction of p53-dependent apoptosis or the preservation of replication fork integrity. The latter is of particular importance should reoxygenation occur. Tumor reoxygenation occurs as a result of spontaneous changes in blood flow and also therapy. Cells experiencing hypoxia and/or reoxygenation are, therefore, sensitive to loss or inhibition of components of the DNA damage response, including Chk1, ATM, ATR, and poly(ADP-ribose) polymerase (PARP). In addition, restoration of hypoxia-induced p53-mediated signaling may well be effective in the targeting of hypoxic cells. The DNA damage response is also induced in endothelial cells at moderate levels of hypoxia, which do not induce replication arrest. In this situation, phosphorylation of H2AX has been shown to be required for proliferation and angiogenesis and is, therefore, an attractive potential therapeutic target.
Assuntos
Dano ao DNA/fisiologia , Enzimas Reparadoras do DNA/antagonistas & inibidores , Terapia de Alvo Molecular/métodos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Hipóxia Celular/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Humanos , Modelos Biológicos , Oxigênio/farmacologia , Transdução de SinaisRESUMO
Chronic lymphocytic leukemia (CLL) cells are susceptible to oxidative stress. The expanded porphyrin, motexafin gadolinium (MGd), reacts with intracellular reducing metabolites and protein thiols to generate reactive oxygen species (ROS). A phase II trial administered MGd 5 mg/kg/day IV for 5 days every 3 weeks until disease progression to patients with previously treated CLL and small lymphocytic lymphoma. Thirteen patients (median age 66 years) with a median of four prior therapies (range 2-9) were enrolled. Modest anti-tumor activity was seen in three patients, with improvement in lymphocytosis, lymphadenopathy and/or splenomegaly, but no patient achieved a partial or complete response by NCI 96 criteria. Flow cytometry confirmed tumor uptake of MGd. Serial increase in AKT phosphorylation in patient samples following MGd treatment was not observed, suggesting intracellular generation of ROS was not optimal. Therefore, this schedule of administration achieved MGd uptake into primary tumor cells in vivo, but clinical activity was modest.
Assuntos
Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Metaloporfirinas/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Deleção Cromossômica , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 17/genética , Esquema de Medicação , Resistencia a Medicamentos Antineoplásicos , Feminino , Citometria de Fluxo , Humanos , Immunoblotting , Hibridização in Situ Fluorescente , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Metaloporfirinas/efeitos adversos , Metaloporfirinas/farmacocinética , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resultado do TratamentoRESUMO
The ATM kinase has previously been shown to respond to the DNA damage induced by reoxygenation following hypoxia by initiating a Chk 2-dependent cell cycle arrest in the G(2) phase. Here we show that ATM is both phosphorylated and active during exposure to hypoxia in the absence of DNA damage, detectable by either comet assay or 53BP1 focus formation. Hypoxia-induced activation of ATM correlates with oxygen concentrations low enough to cause a replication arrest and is entirely independent of hypoxia-inducible factor 1 status. In contrast to damage-activated ATM, hypoxia-activated ATM does not form nuclear foci but is instead diffuse throughout the nucleus. The hypoxia-induced activity of both ATM and the related kinase ATR is independent of NBS1 and MRE11, indicating that the MRN complex does not mediate the DNA damage response to hypoxia. However, the mediator MDC1 is required for efficient activation of Kap1 by hypoxia-induced ATM, indicating that similarly to the DNA damage response, there is a requirement for MDC1 to amplify the ATM response to hypoxia. However, under hypoxic conditions, MDC1 does not recruit BRCA1/53BP1 or RNF8 activity. Our findings clearly demonstrate that there are alternate mechanisms for activating ATM that are both stress-specific and independent of the presence of DNA breaks.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Hipóxia Celular , Proteínas de Ligação a DNA/metabolismo , Oxigênio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: Sapphyrin analogues and related porphyrin-like species have attracted attention as anticancer agents due to their selective localization in various cancers, including hematologic malignancies, relative to surrounding tissues. Sapphyrins are electron affinic compounds that generate high yields of singlet oxygen formation. Although initially explored in the context of photodynamic therapy, sapphyrins have intrinsic anticancer activity that is independent of their photosensitizing properties. However, the mechanisms for their anticancer activity have not been fully elucidated. RESULTS: We have prepared a series of hydrophilic sapphyrins and evaluated their effect on proliferation, uptake, and cell death in adherent human lung (A549) and prostate (PC3) cancer cell lines and in an A549 xenograft tumor model. PCI-2050, the sapphyrin derivative with the highest in vitro growth inhibitory activity, significantly lowered 5-bromo-2'-deoxyuridine incorporation in S-phase A549 cells by 60% within eight hours and increased levels of reactive oxygen species within four hours. The growth inhibition pattern of PCI-2050 in the National Cancer Institute 60 cell line screen correlated most closely using the COMPARE algorithm with known transcriptional or translational inhibitors. Gene expression analyses conducted on A549 plateau phase cultures treated with PCI-2050 uncovered wide-spread decreases in mRNA levels, which especially affected short-lived transcripts. Intriguingly, PCI-2050 increased the levels of transcripts involved in RNA processing and trafficking, transcriptional regulation, and chromatin remodeling. We propose that these changes reflect the activation of cellular processes aimed at countering the observed wide-spread reductions in transcript levels. In our A549 xenograft model, the two lead compounds, PCI-2050 and PCI-2022, showed similar tumor distributions despite differences in plasma and kidney level profiles. This provides a possible explanation for the better tolerance of PCI-2022 relative to PCI-2050. CONCLUSION: Hydrophilic sapphyrins were found to display promise as novel agents that localize to tumors, generate oxidative stress, and inhibit gene expression.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Análise de Sequência com Séries de Oligonucleotídeos , Porfirinas/farmacocinética , Porfirinas/toxicidade , Linhagem Celular Tumoral , Proliferação de Células , Análise por Conglomerados , Perfilação da Expressão Gênica , Humanos , Estrutura Molecular , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
There is an emerging appreciation of the importance of zinc in regulating cancer cell growth and proliferation. Recently, we showed that the anticancer agent motexafin gadolinium (MGd) disrupted zinc metabolism in A549 lung cancer cells, leading, in the presence of exogenous zinc, to cell death. Here, we report the effect of MGd and exogenous zinc on intracellular levels of free zinc, oxidative stress, proliferation, and cell death in exponential phase human B-cell lymphoma and other hematologic cell lines. We find that increased levels of oxidative stress and intracellular free zinc precede and correlate with cell cycle arrest and apoptosis. To better understand the molecular basis of these cellular responses, gene expression profiling analyses were conducted on Ramos cell cultures treated with MGd and/or zinc acetate. Cultures treated with MGd or zinc acetate alone elicited transcriptional responses characterized by induction of metal response element-binding transcription factor-1 (MTF-1)-regulated and hypoxia-inducible transcription factor-1 (HIF-1)-regulated genes. Cultures cotreated with MGd and zinc acetate displayed further increases in the levels of MTF-1- and HIF-1-regulated transcripts as well as additional transcripts regulated by NF-E2-related transcription factor 2. These data provide insights into the molecular changes that accompany the disruption of intracellular zinc homeostasis and support a role for MGd in treatment of B-cell hematologic malignancies.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linfoma de Células B/tratamento farmacológico , Metaloporfirinas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Zinco/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Elementos de Resposta , Fase S/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Fator MTF-1 de TranscriçãoRESUMO
BACKGROUND: Previous studies showed that primary cultures of normal and malignant human prostatic epithelial cells are defective in their ability to upregulate the tumor suppressor protein p53 in response to DNA damage. This dysfunctional regulation of p53 may be relevant to both the high incidence of prostate cancer and its resistance to chemotherapy. Leptomycin B (LMB) has recently been found to increase the protein level and transcriptional activity of p53 by interfering with nucleocytoplasmic export and subsequent degradation by the proteasome. We investigated the ability of LMB to activate p53 in prostatic epithelial cells. METHODS: Primary cultures and the cell lines LNCaP and DU 145 were treated with LMB. p53 protein was evaluated in Western blots and by immunocytochemistry. Induction of downstream targets of p53 was evaluated in Western and Northern blots. Growth inhibition, cell cycle arrest, and apoptosis in response to LMB were measured in clonal growth assays, by flow cytometry, and by Hoescht/propidium iodide staining, respectively. RESULTS: Treatment of prostatic epithelial cells with LMB led to post-translational stabilization of p53, activation of downstream target genes, and induction of cell cycle arrest in primary cultures and apoptosis in LNCaP (with wild-type p53) but not DU 145 (with mutant p53) cells. CONCLUSIONS: p53 in primary cultures of normal and malignant prostate cells, although dysfunctional in that it is not responsive to DNA damage, is activated by LMB. The ability of LMB to stabilize p53 and induce expression of p53-responsive growth inhibitory genes may be a useful lead in the development of chemopreventive or therapeutic small molecules that can modulate p53 function in prostatic epithelial cells.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Próstata/citologia , Neoplasias da Próstata , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ciclo Celular/genética , Divisão Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Clonais , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/genética , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/análise , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Interest in exploiting traditional medicines for prevention or treatment of cancer is increasing. Extracts from the herb Tripterygium wilfordii hook F have been used in China for centuries to treat immune-related disorders. Recently it was reported that triptolide (PG490), a purified compound from Tripterygium, possessed antitumor properties and induced apoptosis by p53-independent mechanisms in a variety of malignant cell lines. This property of triptolide attracted our attention because we have found that primary cultures of human prostatic epithelial cells derived from normal tissues and adenocarcinomas are in general extremely resistant to apoptosis. Furthermore, the function of wild-type p53 is impaired in these cells such that drugs that require p53 activity to induce cell death are ineffective. Therefore, the properties of triptolide and the recent approval of its water-soluble form (PG490-88) for entry into Phase I clinical trials suggested that this drug was a promising candidate to test for antitumor activity against prostate cells. Experiments presented here demonstrated that triptolide had dose-dependent effects on both normal and cancer-derived primary cultures of human prostatic epithelial cells. Low concentrations of triptolide inhibited cell proliferation and induced a senescence-like phenotype. Higher concentrations of triptolide induced apoptosis that was unexpectedly associated with nuclear accumulation of p53. Paradoxically, levels of the p53 target genes, p21(WAF1/CIP1) and hdm-2, were reduced, as was bcl-2. Our preclinical studies suggest that triptolide might be an effective preventive as well as therapeutic agent against prostate cancer and that triptolide may activate a functional p53 pathway in prostate cells.