RESUMO
Critical processes underlying cancers must be better understood to develop strategies for treatment and prevention. A chemotherapeutic strategy is proposed that is based upon re-establishment, with a drug, of nullified programmed cell death (apoptosis) in cancer cells, which to survive have mutated to block apoptosis. A chemotherapy that is specific against tumors implanted in mice demonstrated the feasibility of this principle. This therapy is specific because it affects a process unique to cancer cells. It also has the advantage of killing these cells, in contrast to reversibly blocking their proliferation. The anti-apoptotic transcription factor NF-kappaB provides a potential therapeutic target in estrogen receptor negative (ER-) breast cancers that over-express the epidermal growth factor family of receptors (EGFR). Further investigations of the pathways utilize dominant negative protein inhibitory peptide, and small inhibitory RNAs (siRNAs) to block the production of relevant enzymes.
Assuntos
Apoptose , NF-kappa B/antagonistas & inibidores , Neoplasias/patologia , Animais , Antineoplásicos/farmacologia , Humanos , Neoplasias/tratamento farmacológico , RNA Interferente Pequeno/fisiologiaRESUMO
Cyclin E1 (formerly called cyclin E) and the recently described cyclin E2 belong to the family of E-type cyclins that operate during the G(1)/S phase progression in mammalian cells. The two E-cyclins share a catalytic partner, cyclin-dependent kinase 2 (CDK2), and activate their associated kinase activities at similar times during cell cycle progression. Despite these similarities, it is unknown whether the two proteins perform distinct functions, or, alternatively, they control S-phase entry of different cell types in a tissue-specific fashion. To start addressing in vivo functions of E-cyclins, we determined the expression pattern of cyclins E1 and E2 during normal mouse development. We found that the two E-cyclins showed very similar patterns of expression; both were expressed within the proliferating compartment during embryo development. Analyses of cells and tissues lacking members of the retinoblastoma (pRB) family of proteins revealed that the expression of both cyclins is controlled in a pRB-dependent, but p107- and p130-independent fashion, likely through the pRB-dependent E2F transcription factors. We also found that cyclins E1 and E2 are expressed at high levels in mouse breast tumors driven by the Myc oncogene. Last, we found that cyclin E2 is overexpressed in approximately 24% of analyzed human mammary carcinomas. Collectively these findings suggest that the expression of cyclins E1 and E2 is governed by similar molecular circuitry.
Assuntos
Neoplasias da Mama/genética , Ciclina E/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Animais , Northern Blotting , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário e Fetal/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Camundongos , Proteína do Retinoblastoma/fisiologia , Células-Tronco/metabolismoRESUMO
The effect of a kinase inhibitor Go6796 on growth of epidermal growth factor (EGF)-stimulated estrogen receptor negative (ER-) breast cancer cells in vivo and role of nuclear factor kappa B (NF-kappaB) on tumorogenesis have been investigated. This was studied in an animal model by implanting ER- mouse mammary epithelial tumor cells (CSMLO) in syngeneic A-J mice. (i) Local administration of Go6976 an inhibitor of protein kinases C alpha and beta inhibited growth of tumors and caused extensive necrotic degeneration and regression of the tumors without causing any microscopically detectable damage to the vital organs liver and lung. (ii) Stable expression of dominant-negative mutants of the beta subunit (dnIkkbeta) of the inhibitory kappa B (IkappaB) kinase (dnIkk) that selectively blocked activation of NF-kappaB caused loss of tumorigenic potential of CSMLO cells. Stable expression of dnIkkbeta also blocked phorbol 12-myristate 13-acetate (PMA)-induced activation of NF-kappaB and overexpression of cyclin D1, concomitantly with the loss or reduced tumorigenic potential of these cells. Thus, results from in vivo and in vitro experiments strongly suggest the involvement of NF-kappaB in ER- mammary epithelial cell-mediated tumorigenesis. We propose that blocking NF-kappaB activation not only inhibits cell proliferation, but also antagonizes the antiapoptotic role of this transcription factor in ER- breast cancer cells. Thus, NF-kappaB is a potential target for therapy of EGFR family receptor-overexpressing ER- breast cancers.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , NF-kappa B/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Animais , Sequência de Bases , Neoplasias da Mama/genética , Carbazóis/farmacologia , Divisão Celular/efeitos dos fármacos , Ciclina D1/metabolismo , Primers do DNA/genética , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Quinase I-kappa B , Indóis/farmacologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos A , Mutação , NF-kappa B/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
BACKGROUND: The action of the steroid hormone estradiol (E2) is mediated via interaction with a specific receptor (ER) that initiates a series of events downstream, leading to the modulation of hormone-responsive genes and cell proliferation. Antihormones also bind, but do not confer the active configuration to ER, thereby, blocking the transmission of E2-ER-initiated signals for cell proliferation. Although these compounds qualify for successful therapy of ER-positive [ER (+)] breast cancer patients, only a fraction of patients responds to antihormone treatment. In this study, the functional status of ER is determined to identify alternative targets for therapy of antihormone-resistant ER (+) breast cancers. METHOD: The interaction of ER with a specific DNA sequence, designated as E2 response element (ERE), was targeted to assess the functional state of ER. ER-ERE complex formation was measured by electrophoretic mobility shift assay (EMSA) and by a newly developed technique, based on the preferential binding of DNA-protein complex to a nitrocellulose membrane (NMBA) that measures both total and functional fraction of ER. RESULTS: The NMBA assay identified functional variants of ER among ER (+) breast cancer cell lines and breast tumor biopsy specimens. ER of (21PT) cells did not bind E2 and these cells were tamoxifen (TAM) resistant. However 21PT cells were sensitive to a calmodulin (CaM) antagonist, W7, that blocked ERE-ER complex formation. CONCLUSIONS: ER variants of the 21PT type were detected among breast cancer biopsy specimens, emphasizing the significance of an alternative therapeutic target for TAM-resistant ER (+) human breast cancers with compounds such as W7.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Receptores de Estrogênio/metabolismo , Elementos de Resposta/efeitos dos fármacos , Adulto , Idoso , Bioquímica/métodos , Biópsia , Calmodulina/antagonistas & inibidores , Colódio/metabolismo , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Estradiol/metabolismo , Feminino , Hormônios/farmacologia , Humanos , Pessoa de Meia-Idade , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Receptores de Progesterona/metabolismo , Sulfonamidas/farmacologia , Tamoxifeno/farmacologia , Resultado do Tratamento , Células Tumorais CultivadasRESUMO
Chemotherapy of cancer is limited by its toxicity to normal cells. On the basis of discoveries in signal transduction and cell cycle regulation, novel mechanism-based therapeutics are being developed. Although these cell cycle modulators were designed to target cancer cells, some of them can also be applied for a different purpose, i.e., to protect normal cells against the lethality of chemotherapy. Loss of sensitivity of cancer cells to cell cycle inhibitors can be exploited for selective protection of normal cells that retain this response. Indeed, inhibition of redundant or overactivated pathways (e.g., growth factor-activated pathways) or stimulation of absent pathways in cancer cells (e.g., p53, Rb, and p16) may not arrest cycling of cancer cells. But growth arrest of normal cells will then permit selective killing of cancer cells by cycle-dependent chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Animais , Antineoplásicos/efeitos adversos , Divisão Celular/efeitos dos fármacos , HumanosRESUMO
Early detection is an effective means of reducing cancer mortality. Here, we describe a highly sensitive high-throughput screen that can identify panels of markers for the early detection of solid tumor cells disseminated in peripheral blood. The method is a two-step combination of differential display and high-sensitivity cDNA arrays. In a primary screen, differential display identified 170 candidate marker genes differentially expressed between breast tumor cells and normal breast epithelial cells. In a secondary screen, high-sensitivity arrays assessed expression levels of these genes in 48 blood samples, 22 from healthy volunteers and 26 from breast cancer patients. Cluster analysis identified a group of 12 genes that were elevated in the blood of cancer patients. Permutation analysis of individual genes defined five core genes (P < or = 0.05, permax test). As a group, the 12 genes generally distinguished accurately between healthy volunteers and patients with breast cancer. Mean expression levels of the 12 genes were elevated in 77% (10 of 13) untreated invasive cancer patients, whereas cluster analysis correctly classified volunteers and patients (P = 0.0022, Fisher's exact test). Quantitative real-time PCR confirmed array results and indicated that the sensitivity of the assay (1:2 x 10(8) transcripts) was sufficient to detect disseminated solid tumor cells in blood. Expression-based blood assays developed with the screening approach described here have the potential to detect and classify solid tumor cells originating from virtually any primary site in the body.
Assuntos
Neoplasias da Mama/sangue , Perfilação da Expressão Gênica , Células Neoplásicas Circulantes , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Análise por Conglomerados , DNA Complementar , Humanos , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Suberoylanilide hydroxamic acid (SAHA) is a prototype of the newly developed, second-generation, hybrid polar compounds. It is a novel histone deacetylase inhibitor with high potency for inducing cell differentiation of cultured murine erythroleukemia cells. Studies with SAHA have primarily been performed with hematopoietic tumor cells. Here we extent these studies with SAHA to human breast cancer cell lines in an attempt to find better therapeutic agents for breast cancer treatment. MATERIALS AND METHODS: Human breast cancer cell lines, MCF7, MDA-MB-231, and MDA-MB-435, as well as normal cells, including the normal breast epithelial cell line MCF-10A, and fibroblasts, were treated with SAHA. Cells assayed for cell survival by using trypan blue exclusion assay, colony formation assay, and cell cycle and apoptosis analysis. The effects of SAHA on cell cycle and apoptosis regulatory proteins were examined by Western blots analysis. The identification of additional target genes was carried out by differential display (DD) and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: SAHA inhibited clonogenic growth of MCF7, MDA-MB-231, and MDA-MB-435 breast cancer cells. These cells were more sensitive to SAHA-mediated cytotoxic effects than normal breast epithelial cells and fibroblasts. The cytotoxic effects of SAHA on breast cancer cells were manifested by G1 and G2/M cell cycle arrest and eventual apoptosis. The pan-caspase inhibitor, Z-VAD.fmk, blocked SAHA-induced cell death, DNA laddering, and cleavage of poly(ADP-ribose) polymerase, indicating the involvement of caspases in SAHA-mediated apoptosis. In addition, SAHA modulated cell cycle and apoptosis regulatory proteins. For example, cyclin-dependent kinase (CDK) inhibitors p21WAF1/Cip1 and p27Kip1 were induced, and retinoblastoma protein pRb was hypophosphorylated. Moreover, SAHA induced several genes associated with differentiation and/ or growth inhibition. These genes encode gelsolin, isopentenyl-diphosphate delta isomerase (IDI1), and 1,25-dihydroxyvitamin D-3 up-regulated protein 1 (VDUP1), the last two of which were identified by DD. Induction of these genes may contribute to SAHA-mediated pro-differentiating and antiproliferative effects. CONCLUSIONS: SAHA induced growth inhibition, cell cycle arrest, and eventual apoptosis in human breast cancer cells, possibly by modulating cell cycle and apoptosis regulatory proteins, such as CDK inhibitors p21 and p27, pRb, and other differentiation and/or growth inhibition-associated genes, including gelsolin, IDI1 and VDUP1. This, together with the low toxicity in normal cells, suggests that SAHA might have therapeutic potential for the treatment of human breast cancers.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Ácidos Hidroxâmicos/uso terapêutico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sequência de Bases , Inibidores de Caspase , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Primers do DNA , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacos , VorinostatRESUMO
Suberoylanilide hydroxamic acid (SAHA) is a novel histone deacetylase inhibitor with high potency in inducing differentiation of cultured murine erythroleukemia cells. We have recently demonstrated that SAHA induces cell cycle arrest and apoptosis in human breast cancer cells, accompanied by up-regulation of the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, via a p53-independent mechanism. In this study, we used p21 gene expression as a model system to elucidate the molecular mechanism(s) underlying SAHA-mediated gene activation. Treatment of human breast cancer cell line MCF7 cells with SAHA induced p21 mRNA as a consequence of an immediate-early gene activation. Moreover, SAHA activated the p21 promoter primarily through two Spl sites located at -82 and -69 relative to the transcription start site. Furthermore, Sp1 and Sp3 proteins were the major factors binding to the Spl site of the p21 promoter. However, SAHA did not alter their DNA binding activities, suggesting that SAHA mediates p21 promoter activity by a mechanism other than altering the DNA binding activities of Sp1 and Sp3. Further studies using the GAL4 luciferase assay system demonstrated that both GAL4-Sp1 and GAL4-Sp3 fusion proteins supported SAHA-mediated gene activation from a promoter driven by five GAL4 DNA binding sites, and that GAL4-Sp3 fusion protein was suppressive in the absence of SAHA treatment. Collectively, our results suggest that SAHA activates the p21 promoter through the Spl sites, and that both Spl and Sp3 proteins can mediate SAHA-induced gene activation.
Assuntos
Ciclinas/genética , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3 , Fatores de Transcrição/genética , Ativação Transcricional , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , VorinostatRESUMO
The level of estrogen receptor (ER) is a key determinant for the management of ER-positive [ER(+)] breast cancer patients. Growth of many human breast cancers is regulated by estrogen (E2) and progesterone (Pr). Generally, the ER in ER(+) breast cancer patients is targeted for therapy with antihormones. However 40% of ER(+) patients do not respond to antihormone therapy. Thus, the identification of antihormone resistant ER(+) breast cancers is essential for therapeutic predictions. Although 3H-E2 binding and immunodetection can identify ER, these procedures do not assess the functional state of the receptor molecule. In this study we describe a novel and rapid assay for the detection of ER and its functional state on the basis of the downstream interaction with its response element (ERE) based on the preferential binding of DNA-protein complex (ERE-ER) to a nitrocellulose membrane (NMBA). This method permits measurement of both the total and the functional fraction of ER. The ER status was examined in breast cancer cell lines and in breast cancer biopsy specimens by (i) 3H-E2 binding assay, (ii) immunodetection assays and (iii) by its interaction with 32P-ERE. The sensitive NMBA assay was validated with well-characterized ER(+) breast cancer cell lines and also identified functional variants of ER among breast tumor biopsy specimens.
Assuntos
Neoplasias da Mama/metabolismo , Receptores de Estrogênio/metabolismo , Biópsia , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Colódio/metabolismo , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Estrogênios/metabolismo , Feminino , Humanos , Imunoensaio/métodos , Ligantes , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Isoformas de Proteínas/metabolismo , Elementos de Resposta/genética , Células Tumorais CultivadasRESUMO
The epidermal growth factor (EGF) family of receptors (EGFR) is overproduced in estrogen receptor (ER) negative (-) breast cancer cells. An inverse correlation of the level of EGFR and ER is observed between ER- and ER positive (+) breast cancer cells. A comparative study with EGFR-overproducing ER- and low-level producing ER+ breast cancer cells suggests that EGF is a major growth-stimulating factor for ER- cells. An outline of the pathway for the EGF-induced enhanced proliferation of ER- human breast cancer cells is proposed. The transmission of mitogenic signal induced by EGF-EGFR interaction is mediated via activation of nuclear factor kappaB (NF-kappaB). The basal level of active NF-kappaB in ER- cells is elevated by EGF and inhibited by anti-EGFR antibody (EGFR-Ab), thus qualifying EGF as a NF-kappaB activation factor. NF-kappaB transactivates the cell-cycle regulatory protein, cyclin D1, which causes increased phosphorylation of retinoblastoma protein, more strongly in ER- cells. An inhibitor of phosphatidylinositol 3 kinase, Ly294-002, blocked this event, suggesting a role of the former in the activation of NF-kappaB by EGF. Go6976, a well-characterized NF-kappaB inhibitor, blocked EGF-induced NF-kappaB activation and up-regulation of cell-cycle regulatory proteins. This low molecular weight compound also caused apoptotic death, predominantly more in ER- cells. Thus Go6976 and similar NF-kappaB inhibitors are potentially novel low molecular weight therapeutic agents for treatment of ER- breast cancer patients.
Assuntos
Neoplasias da Mama/metabolismo , Fator de Crescimento Epidérmico/metabolismo , NF-kappa B/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Carbazóis/farmacologia , Ciclo Celular , Ciclina D1/metabolismo , Ciclina D1/fisiologia , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Feminino , Humanos , Indóis/farmacologia , Cinética , NF-kappa B/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
Human SIX1 (HSIX1) is a member of the Six class of homeodomain proteins implicated in muscle, eye, head, and brain development. To further understand the role of HSIX1 in the cell cycle and cancer, we developed an HSIX1-specific antibody to study protein expression at various stages of the cell cycle. Our previous work demonstrated that HSIX1 mRNA expression increases as cells exit S phase and that overexpression of HSIX1 can attenuate a DNA damage-induced G(2) cell cycle checkpoint. Overexpression of HSIX1 mRNA was observed in 44% of primary breast cancers and 90% of metastatic lesions. Now we demonstrate that HSIX1 is a nuclear phosphoprotein that becomes hyperphosphorylated at mitosis in both MCF7 cells and in Xenopus extracts. The pattern of phosphorylation observed in mitosis is similar to that seen by treating recombinant HSIX1 with casein kinase II (CK2) in vitro. Apigenin, a selective CK2 inhibitor, diminishes interphase and mitotic phosphorylation of HSIX1. Treatment of MCF7 cells with apigenin leads to a dose-dependent arrest at the G(2)/M boundary, implicating CK2, like HSIX1, in the G(2)/M transition. HSIX1 hyperphosphorylated in vitro by CK2 loses its ability to bind the MEF3 sites of the aldolase A promoter (pM), and decreased binding to pM is observed during mitosis. Because CK2 and HSIX1 have both been implicated in cancer and in cell cycle control, we propose that HSIX1, whose activity is regulated by CK2, is a relevant target of CK2 in G(2)/M checkpoint control and that both molecules participate in the same pathway whose dysregulation leads to cancer.
Assuntos
Ciclo Celular , Proteínas de Homeodomínio/metabolismo , Sequência de Aminoácidos , Animais , Caseína Quinase II , Humanos , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transdução de Sinais , XenopusRESUMO
A major objective of current cancer research is to develop a detailed molecular characterization of tumor cells and tissues that is linked to clinical information. Toward this end, we have identified approximately one-quarter of all genes that were aberrantly expressed in a breast cancer cell line using differential display. The cancer cells lost the expression of many genes involved in cell adhesion, communication, and maintenance of cell shape, while they gained the expression of many synthetic and metabolic enzymes important for cell proliferation. High-density, membrane-based hybridization arrays were used to study mRNA expression patterns of these genes in cultured cells and archived tumor tissue. Cluster analysis was then used to identify groups of genes, the expression patterns of which correlated with clinical information. Two clusters of genes, represented by p53 and maspin, had expression patterns that strongly associated with estrogen receptor status. A third cluster that included HSP-90 tended to be associated with clinical tumor stage, whereas a forth cluster that included keratin 14 tended to be associated with tumor size. Expression levels of these clinically relevant gene clusters allowed breast tumors to be grouped into distinct categories. Gene expression fingerprints that include these four gene clusters have the potential to improve prognostic accuracy and therapeutic outcomes for breast cancer patients.
Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Genes Supressores de Tumor , Genes p53/genética , Proteínas de Choque Térmico HSP90/genética , Humanos , Queratinas/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Estrogênio/fisiologia , Reprodutibilidade dos Testes , Serpinas/genética , Células Tumorais CultivadasRESUMO
A gene related to cell differentiation was identified by differential display as a candidate suppressor of metastases in colon cancer. This gene, with a full-length cDNA of 3 kb, is expressed in normal colon and primary colon cancer tissues and cell lines but not in their metastatic counterparts. A GenBank search found that it is identical to a recently cloned gene, differentiation-related gene-1 (Drg-1), isolated from differentiated HT-29 colon cancer cells. Stable transfection of the SW620 metastatic colon cancer cell line with Drg-1 cDNA induced morphological changes consistent with differentiation and up-regulated the expression of several colonic epithelial cell differentiation markers (alkaline phosphatase, carcinoembryonic antigen, and E-cadherin). Moreover, the expression of Drg-1 is controlled by several known cell differentiation reagents, such as ligands of peroxisome proliferator-activated receptor gamma (troglitazone and BRL46593) and of retinoid X receptor (LG268), and histone deacetylase inhibitors (trichostatin A, suberoylanilide hydroxamic acid, and tributyrin). A synergistic induction of Drg-1 expression was seen with the combination of tributyrin and a low dose of 5'-aza-2'-dexoycytidine (100 nM), an inhibitor of DNA methylation. Functional studies revealed that overexpression of Drg-1 in metastatic colon cancer cells reduced in vitro invasion through Matrigel and suppressed in vivo liver metastases in nude mice. We propose that Drg-1 suppresses colon cancer metastasis by inducing colon cancer cell differentiation and partially reversing the metastatic phenotype.
Assuntos
Proteínas de Ciclo Celular , Neoplasias do Colo/genética , Genes Supressores de Tumor , Proteínas/genética , Animais , Diferenciação Celular , Neoplasias do Colo/patologia , Metilação de DNA , Regulação para Baixo , Feminino , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , RNA Mensageiro/análise , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores do Ácido Retinoico/fisiologia , Receptores X de Retinoides , Fatores de Transcrição/fisiologia , Células Tumorais CultivadasRESUMO
We describe a method to enhance the sensitivity of eukaryotic cDNA detection in differential display (DD). Typically, DD protocols require between 200 and 500 ng RNA for each reverse transcription reaction. The addition of Escherichia coli RNA before reverse transcription of eukaryotic RNA increases the detection of DD patterns more than tenfold. The method broadens the applicability of DD and allows the identification of genes that are differentially expressed when the amount of eukaryotic RNA is limited.
Assuntos
DNA Complementar/análise , RNA Bacteriano/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Desoxirribonuclease I/metabolismo , Eletroforese em Gel de Ágar , Escherichia coli/genética , Expressão Gênica , Humanos , RNA Mensageiro/análise , Células Tumorais CultivadasRESUMO
BACKGROUND: Human multiple myeloma (MM) remains an incurable hematological malignancy. We have reported that beta-lapachone, a pure compound derived from a plant, can induce cell death in a variety of human carcinoma cells, including ovary, colon, lung, prostate, pancreas, and breast, suggesting a wide spectrum of anticancer activity. MATERIALS AND METHODS: We first studied antisurvival effects of beta-lapachone in human MM cells by colony formation assay. To determine whether the differential inhibition of colony formation occurs through antiproliferative activity, we performed MTT assays. The cytotoxicity of beta-lapachone on human peripheral blood mononuclear cells was also measured by MTT assay. To determine whether the cell death induced by beta-lapachone occurs through necrosis or apoptosis, we used the propidium iodide staining procedure to determine the sub-GI fraction, Annexin-V staining for externalization of phosphatidylserine, and fragmentation of cellular genomic DNA subjected to gel electrophoresis. To investigate the mechanism of anti-MM activity, we examined Bcl-2 expression, cytochrome C release, and poly (ADP ribose) polymerase cleavage by Western blot assay. RESULTS: We found that beta-lapachone (less than 4 microM) inhibits cell survival and proliferation by triggering cell death with characteristics of apoptosis in ARH-77, HS Sultan, and MM.1S cell lines, in freshly derived patient MM cells (MM.As), MM cell lines resistant to dexamethasone (MM.1R), doxorubicin (DOX.40), mitoxantrone (MR.20), and mephalan (LR5). Importantly, after treatment with beta-lapachone, we observed no apoptosis in peripheral blood mononuclear cells in either quiescent or proliferative states, freshly isolated from healthy donors. In beta-lapachone treated ARH-77, cytochrome C was released from mitochondria to cytosol, and poly (ADP ribose) polymerase was cleaved, signature events of apoptosis. Finally, the apoptosis induced by beta-lapachone in MM cells was not blocked by either interleukin-6 or Bcl-2, which confer multidrug resistance in MM. CONCLUSIONS: Our results suggest potential therapeutic application of beta-lapachone against MM, particularly to overcome drug resistance in relapsed patients.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Naftoquinonas/farmacologia , Western Blotting , Divisão Celular , Sobrevivência Celular , Grupo dos Citocromos c/metabolismo , Fragmentação do DNA , Relação Dose-Resposta a Droga , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Recidiva , Células Tumorais CultivadasRESUMO
Ablation of tumor colonies was seen in a wide spectrum of human carcinoma cells in culture after treatment with the combination of beta-lapachone and taxol, two low molecular mass compounds. They synergistically induced death of cultured ovarian, breast, prostate, melanoma, lung, colon, and pancreatic cancer cells. This synergism is schedule dependent; namely, taxol must be added either simultaneously or after beta-lapachone. This combination therapy has unusually potent antitumor activity against human ovarian and prostate tumor prexenografted in mice. There is little host toxicity. Cells can commit to apoptosis at cell-cycle checkpoints, a mechanism that eliminates defective cells to ensure the integrity of the genome. We hypothesize that when cells are treated simultaneously with drugs activating more than one different cell-cycle checkpoint, the production of conflicting regulatory signaling molecules induces apoptosis in cancer cells. beta-Lapachone causes cell-cycle delays in late G(1) and S phase, and taxol arrests cells at G(2)/M. Cells treated with both drugs were delayed at multiple checkpoints before committing to apoptosis. Our findings suggest an avenue for developing anticancer therapy by exploiting apoptosis-prone "collisions" at cell-cycle checkpoints.
Assuntos
Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Naftoquinonas/farmacologia , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Ovarianas/enzimologia , Células Tumorais CultivadasRESUMO
HIV infection is accompanied by an early immune dysfunction limiting host control of virus and likely contributing to difficulties in achieving a successful vaccine against HIV. We report here that the HIV Tat protein is strongly immunosuppressive, both immediately after immunization of mice with soluble protein (sTat), and in seroconverting humans, and propose that Tat-induced suppression cripples immune surveillance to HIV infection. We show that macrophages are sensitive to sTat stimulation at concentrations 1,000-fold lower (500 pM) than T cells, and this stimulation is accompanied by the immunosuppressive induction of Fas ligand on the macrophage. T cell proliferative defects induced by sTat in vitro can be completely (at lower concentrations of sTat) or partially (at higher concentrations) reversed by antagonists to Fas/Fas ligand interaction. We further report a method to preserve immunogenicity while inactivating Tat immunosuppression through oxidation, which advances the use of oxidized Tat as a component of an anti-HIV vaccine. These observations define additional methods to study the immunosuppressive functions of sTat that now may be rapidly applied to primary isolates from individuals with differing clinical courses. Our findings have immediate relevance for vaccine development, by describing and supporting a strategy that includes inactivated sTat in a multicomponent, anti-HIV vaccine.
Assuntos
Produtos do Gene tat/farmacologia , Tolerância Imunológica/fisiologia , Vacinas contra a AIDS/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Proteína Ligante Fas , Citometria de Fluxo , Produtos do Gene tat/imunologia , Produtos do Gene tat/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Humanos , Immunoblotting , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/farmacologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
This study describes a new potential role in human cancer for a gene, HLM, isolated by differential display, that bears homology to an oxysterol-binding protein. A significant association between increased expression of HLM with metastatic disease was found. HLM mRNA levels were increased in circulating tumor cells in patients' peripheral blood and in primary human epithelial cells expressing the human papillomavirus16 E6 and E7 proteins. HLM mRNA was not detected in most normal human tissues, including peripheral blood and lymph node. These findings indicate that HLM may function as a potential marker for tumor dissemination.