RESUMEN
The list of cancer markers of current interest has grown considerably, but none of the markers used in clinical work is a true tumor marker. These cancer biomarkers are based on the determination of tumor antigens. Here, we report a single method of autoantibody enzyme immunoassay (EIA) screens for a spectrum of serum tumor markers. A comparison of the autoantibody-based EIA to conventional antigen EIA kits, using receiver operating characteristic (ROC) plots, showed that the autoantibody EIA can significantly enhance the sensitivity and specificity of tumor markers. The detection of serum autoantibodies for a spectrum of serum tumor markers, as demonstrated here, suggests that most, if not all, serum cancer biomarkers produce autoantibodies. A unique autoantibody biomarker screening method, as presented here, might therefore facilitate achieving the accurate and early diagnosis of cancer.
Asunto(s)
Anticuerpos Antineoplásicos/sangre , Antígenos de Neoplasias/inmunología , Autoanticuerpos/sangre , Biomarcadores de Tumor/sangre , Proteínas Quinasas Dependientes de AMP Cíclico/inmunología , Neoplasias/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunoglobulina G/sangre , Neoplasias/inmunología , Subunidades de Proteína/inmunología , Curva ROC , Sensibilidad y EspecificidadRESUMEN
DNA (antisense and other oligonucleotides) drug design represents a direct genetic approach for cancer treatment. Such an approach takes advantage of mechanisms that activate genes known to confer a growth advantage to neoplastic cells. The ability to block the expression of these genes allows exploration of normal growth regulation. Progress in DNA drug technology has been rapid, and the traditional antisense inhibition of gene expression is now viewed on a genomic scale. This global view has led to a new vision in antisense technology, the elimination of nonspecific and undesirable side effects, and ultimately the generation of more effective and less toxic nucleic acid medicines. Several antisense oligonucleotides are in clinical trials, are well tolerated, and are potentially active therapeutically. DNA drugs are promising molecular medicines for treating human cancer in the near future.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , ADN/química , Diseño de Fármacos , Animales , Ensayos Clínicos como Asunto , Terapia Combinada , Terapia Genética , Humanos , Imitación Molecular , Oligonucleótidos/síntesis química , Oligonucleótidos/farmacología , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/farmacologíaRESUMEN
Among the technologies available for gene knockdown RNase H-dependent antisense oligonucleotides and RNAi are very popular. Both offer specificity and efficient knockdown of the genes; both are useful tools to study gene functions. Antisense and RNAi methods share many practical problems such as site selection, toxicity at high concentration, and the difficulty of transfection in certain cell types. We will focus in this review on the most important issues in the development of both methods and their possible use in gene-silencing therapy.
Asunto(s)
ADN sin Sentido/uso terapéutico , ARN Interferente Pequeño/uso terapéutico , Silenciador del Gen , HumanosRESUMEN
BACKGROUND: Inactivation of the human type Ialpha regulatory subunit (RIalpha) of cyclic AMP dependent protein kinase (PKA) (PRKAR1A) leads to altered kinase activity, primary pigmented nodular adrenocortical disease (PPNAD), and sporadic adrenal and other tumours. METHODS AND RESULTS: A transgenic mouse carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline responsive promoter (the Tg(Prkar1a*x2as)1Stra, Tg(tTAhCMV)3Uh or tTA/X2AS line) developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia and other features reminiscent of PPNAD, including late onset weight gain, visceral adiposity, and non-dexamethasone suppressible hypercorticosteronaemia, with histiocytic, epithelial hyperplasias, lymphomas, and other mesenchymal tumours. These lesions were associated with allelic losses of the mouse chromosome 11 Prkar1a locus, an increase in total type II PKA activity, and higher RIIbeta protein levels; the latter biochemical and protein changes were also documented in Carney complex tumours associated with PRKAR1A inactivating mutations and chromosome 17 PRKAR1A locus changes. CONCLUSION: We conclude that the tTA/X2AS mouse line with a downregulated Prkar1a gene replicates several of the findings in Carney complex patients and their affected tissues, supporting the role of RIalpha as a candidate tumour suppressor gene.
Asunto(s)
Neoplasias de las Glándulas Endocrinas/enzimología , Proteínas/fisiología , Enfermedades de la Corteza Suprarrenal/enzimología , Enfermedades de la Corteza Suprarrenal/genética , Neoplasias de las Glándulas Suprarrenales/enzimología , Neoplasias de las Glándulas Suprarrenales/genética , Alelos , Animales , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación hacia Abajo , Neoplasias de las Glándulas Endocrinas/genética , Eliminación de Gen , Genes Supresores de Tumor , Humanos , Pérdida de Heterocigocidad , Ratones , Ratones Transgénicos , Mutación , Síndromes Neoplásicos Hereditarios/enzimología , Síndromes Neoplásicos Hereditarios/genética , Fenotipo , Proteínas/genética , Neoplasias de la Tiroides/enzimología , Neoplasias de la Tiroides/genéticaRESUMEN
GEM 231 is a second-generation antisense oligonucleotide targeted against the RIalpha regulatory subunit of cAMP-dependent protein kinase type I (PKA-I). Excessive expression of PKA-I is associated with cell proliferation and transformation, and increased levels of secreted extracellular PKA (ECPKA) are found in the serum of cancer patients. Preclinical studies have demonstrated single-agent antitumor activity of GEM 231 in a variety of human cancer xenograft models, and additive or synergistic antitumor activity has been observed with taxane and/or camptothecin-based combinations. Based on prior safety (MTD) data demonstrating dose-dependent, reversible, and cumulative transaminitis, and high peak plasma concentration (Cmax)-dependent changes in activated partial thromboplastin time (aPTT) with GEM 231 2-h twice-weekly infusions, an alternative schedule of GEM 231 given as a single agent was evaluated in patients with advanced solid tumors. Fourteen patients (median age approximately 60 yrs) with advanced solid malignancies received a total of 78 weeks of therapy. GEM 231 was infused via a CADD pump at 80 mg/m2/day (d) for 3 d/wk (n = 1), then for 5 d/wk at 80 (n = 3), 120 (n = 8), and 180 mg/m2/d (n = 2). One cycle was defined as 4 weeks of therapy. Apparent dose dependency for the occurrence of transaminitis was readily reversible. At 180 mg/m2/d, 2 of 2 patients had cycle 1 dose-limiting toxicity (DLT) transaminitis. One patient treated at 120 mg/m2/d experienced grade 3 transaminase elevations after 8 weeks of therapy, but when serum transaminase values rapidly improved he resumed treatment at 80 mg/m2/d for 6 weeks until tumor progression was documented. Another patient at 120 mg/m2/d developed grade 3 esophagitis after 3 weeks, limiting further dosing. One patient (lung cancer) demonstrated stable disease for 9 weeks. Overall, plasma aPTT was minimally prolonged and changes were transient, peaked at the end of each infusion, and were not associated with spontaneous bleeding. A constitutive symptom (e.g., low-grade fatigue) was common, cumulative, and reversible following discontinuation of therapy. Serum ECPKA was measured by enzymatic assay and Western blotting from blood drawn at the beginning and end of each infusion. Serum ECPKA levels demonstrated a trend to decline with the treatment. In addition to single agent schedules, combination trials were undertaken to assess safety and possible interaction of GEM 231 with taxanes (paclitaxel, docetaxel), given once every 3 weeks (one cycle). While trials using the 2-h twice-weekly GEM 231 infusions are ongoing, preliminary results from both studies show that it is safe to combine paclitaxel or docetaxel with GEM 231. Overall, it is also feasible to administer GEM 231 in combination with taxane or nontaxane chemotherapy (e.g., camptothecins). Phase I combination studies are currently underway to further explore the clinical, pharmacokinetic, and biologic profile of GEM 231 with chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Neoplasias/tratamiento farmacológico , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos/farmacología , ARN Mensajero/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , HumanosRESUMEN
The regulatory (R) subunits of cAMP-dependent protein kinase (PKA) are implicated in the regulation of cell proliferation and differentiation. There are two isoforms of PKA that are distinguished by two types of R subunit, RI and RII. Evidence suggests that RI is associated with proliferation and RII is associated with cell differentiation. Previous work in this laboratory has demonstrated that depletion of the RIalpha subunit by treatment with an antisense oligonucleotide (ODN) induces differentiation in leukemia cells and growth arrest and apoptosis in epithelial cancer cells. Using the prostate cancer cell line PC3M as a model system, we have developed a cell line that overexpresses a retroviral vector construct containing the RIalpha antisense gene. This cell line has been characterized and the effectiveness of the construct determined. In the work presented here, we demonstrate by immunocytochemistry that treatment with RIalpha antisense ODN induces translocation of the Calpha subunit of PKA to the nucleus of PC3M prostate cancer cells. The translocation of Calpha triggered by exogenous antisense ODN treatment mirrors that observed in cells endogenously overexpressing the antisense gene. Triggering the nuclear translocation of the Calpha subunit of PKA in the cell may be an important mechanism of action of RIalpha antisense that regulates cell growth independent of adenylate cyclase and cellular cAMP levels. The nuclear localization of the Calpha subunit of PKA may be an essential step in revealing the mechanism whereby this critical kinase regulates cell growth.
Asunto(s)
Dominio Catalítico , Núcleo Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Oligonucleótidos Antisentido/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Colforsina/farmacología , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Humanos , Masculino , Oligonucleótidos Antisentido/genética , Subunidades de Proteína , Especificidad por Sustrato , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Antisense oligodeoxynucleotides can selectively block disease-causing genes, and cancer genes have been chosen as potential targets for antisense drugs to treat cancer. However, nonspecific side effects have clouded the true antisense mechanism of action and hampered clinical development of antisense therapeutics. Using DNA microarrays, we have conducted a systematic characterization of gene expression in cells exposed to antisense, either exogenously or endogenously. Here, we show that in a sequence-specific manner, antisense targeted to protein kinase A RIalpha alters expression of the clusters of coordinately expressed genes at a specific stage of cell growth, differentiation, and activation. The genes that define the proliferation-transformation signature are down-regulated, whereas those that define the differentiation-reverse transformation signature are up-regulated in antisense-treated cancer cells and tumors, but not in host livers. In this differentiation signature, the genes showing the highest induction include genes for the G proteins Rap1 and Cdc42. The expression signature induced by the exogenously supplied antisense oligodeoxynucleotide overlaps strikingly with that induced by endogenous antisense gene overexpression. Defining antisense DNAs on the basis of their effects on global gene expression can lead to identification of clinically relevant antisense therapeutics and can identify which molecular and cellular events might be important in complex biological processes, such as cell growth and differentiation.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , ADN sin Sentido/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Adenocarcinoma/patología , Adenocarcinoma/terapia , Animales , Diferenciación Celular , División Celular , ADN sin Sentido/uso terapéutico , ADN Complementario/genética , Diseño de Fármacos , Terapia Genética , Humanos , Masculino , Ratones , Ratones Desnudos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Oligodesoxirribonucleótidos Antisentido/uso terapéutico , Fenotipo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Subunidades de Proteína , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Tionucleótidos/química , Células Tumorales Cultivadas/trasplante , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
8-Cl-cyclic adenosine monophosphate (8-Cl-cAMP) has been known to induce growth inhibition and differentiation in a variety of cancer cells by differential modulation of protein kinase A isozymes. To understand the anticancer activity of 8-Cl-cAMP further, we investigated the effect of 8-Cl-cAMP on apoptosis in human cancer cells. Most of the tested human cancer cells exhibited apoptosis upon treatment with 8-Cl-cAMP, albeit with different sensitivity. Among them, SH-SY5Y neuroblastoma cells and HL60 leukemic cells showed the most extensive apoptosis. The effect of 8-Cl-cAMP was not reproduced by other cAMP analogues or cAMP-elevating agents, showing that the effect of 8-Cl-cAMP was not caused by simple activation of protein kinase A (PKA). However, competition experiments showed that the binding of 8-Cl-cAMP to the cAMP receptor was essential for the induction of apoptosis. After the treatment of 8-Cl-cAMP, cells initially accumulated at the S and G2/M phases of the cell cycle and then apoptosis began to occur among the population of cells at the S/G2/M cell cycle phases, indicating that the 8-Cl-cAMP-induced apoptosis is closely related to cell cycle control. In support of this assumption, 8-Cl-cAMP-induced apoptosis was blocked by concomitant treatment with mimosine, which blocks the cell cycle at early S phase. Interestingly, 8-Cl-cAMP did not induce apoptosis in primary cultured normal cells and non-transformed cell lines, showing that 8-Cl-cAMP-induced apoptosis is specific to transformed cells. Taken together, our results show that the induction of apoptosis is one of the mechanisms through which 8-Cl-cAMP exerts anticancer activity.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/toxicidad , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Ciclo Celular/fisiología , Células 3T3 , Animales , Neoplasias de la Mama , Células CHO , Ciclo Celular/efectos de los fármacos , Cricetinae , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Fase G2 , Células HL-60 , Células HeLa , Humanos , Células K562 , Ratones , Mitosis , Neuroblastoma , Neoplasias Ováricas , Receptores de AMP Cíclico/efectos de los fármacos , Receptores de AMP Cíclico/fisiología , Fase S , Células Tumorales CultivadasRESUMEN
The CRE (cyclic AMP response element)-transcription factor complex plays a critical role in response to hormonal signals for cell proliferation, differentiation, and apoptosis. We have reported previously that the CRE-transcription factor decoy oligonucleotide specifically slows tumor cell proliferation and inhibits CRE- and Ap-1-directed transcription in vivo (Park et al., 1999). We have investigated the effect of inhibiting CRE-directed transcription on ovarian cancer cell growth. Here, we report that CRE-decoy oligonucleotide treatment results in the inhibition of cell growth and a marked reduction in the expression of the regulatory and catalytic subunits of protein kinase A and the type I and type II protein kinase A holoenzymes. Growth inhibition was accompanied by changes in cell morphology, appearance of apoptotic nuclei, and DNA fragmentation. In addition, MMP-9 (matrix methalloproteinase-9) activity was markedly reduced in CRE-decoy treated cells. Indirect immunofluorescence revealed that CRE-decoy oligonucleotide treatment promoted export of the CRE-binding protein, CREB, from the nucleus to the cytoplasm, while importing the catalytic subunit of protein kinase A from the cytoplasm to the nucleus. The results indicate that the decoy oligonucleotide, by binding specifically to CRE-transcription factors, interferes with CRE-directed transcription in vivo. These results show a critical role for CRE-directed transcription in ovarian cancer cell growth. Thus, the CRE-decoy oligonucleotide may provide a powerful means to combat ovarian cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neoplasias Ováricas/patología , Factor de Transcripción AP-1/antagonistas & inhibidores , Apoptosis/genética , Western Blotting , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Isoenzimas/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Células Tumorales CultivadasRESUMEN
We have previously demonstrated that the activation of p53 signaling may contribute to tumor growth inhibition by the CRE-decoy oligonucleotide containing CRE sequence (5'-TGACGTCA-3') (Lee et al., Biochemistry 39, 4863-4868, 2000). However, growth inhibition by CRE-decoy treatment was also observed in tumor cells containing a mutant p53 (Park et al., J. Biol. Chem. 274, 1573-1580, 1999). To understand additional mechanisms of the decoy oligonucleotide, we investigated the effect on cyclin D1 expression and a cyclin D1/Cdk4/retinoblastoma protein (pRB) signaling pathway. Here we show that in MCF7 breast cancer cells the CRE-decoy competed with cyclin D1-CRE (5'-TAACGTCA-3') for binding transcription factors and reduced cyclin D1 gene expression (in reporter gene assay, Northern blotting and Western blotting) to modulate cyclin D1/Cdk4/pRB signaling and G1-S progression in a steady state and/or under estrogen stimulation. Decrease of cyclin D1 protein level by CRE-decoy treatment was also observed in p53-mutated cancer cells. Cyclin D1 expression was also diminished in MCF7 cells stably expressing dominant negative mutant CREB indicating that the nonspecific effect of oligonucleotide or its degradation products could be excluded. These data suggest that inhibition of cyclin D1 expression contributes to the growth inhibition induced by the decoy oligonucleotide in MCF7 cells through a cyclin D1/Cdk4/pRB signaling pathway. Downregulation of cyclin D1 expression also provides a mechanism of CRE-decoy-induced growth inhibition in tumor cells having p53 mutation.
Asunto(s)
Neoplasias de la Mama/metabolismo , Ciclina D1/genética , Quinasas Ciclina-Dependientes/metabolismo , Oligonucleótidos/farmacología , Proteínas Proto-Oncogénicas , Proteína de Retinoblastoma/metabolismo , Neoplasias de la Mama/patología , División Celular/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina , Estrógenos/farmacología , Femenino , Humanos , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Elementos de Respuesta , Transducción de Señal , Activación Transcripcional , Transfección , Células Tumorales CultivadasRESUMEN
Nucleic acid molecules with high affinities for a target transcription factor can be introduced into cells as decoy cis-elements to bind these factors and alter gene expression. This review discusses a synthetic single-stranded palindromic oligonucleotide, which self-hybridizes to form a duplex/hairpin and competes with cAMP response element (CRE) enhancers for binding transcription factors. This oligonucleotide inhibits CRE- and Ap-1-directed gene transcription and promotes growth inhibition in vitro and in vivo in a broad spectrum of cancer cells, without adversely affecting normal cell growth. Evidence presented here suggests that the CRE-decoy oligonucleotide can provide a powerful new means of combating cancers, viral diseases, and other pathological conditions by regulating the expression of cAMP-responsive genes.
Asunto(s)
División Celular/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Oligodesoxirribonucleótidos/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Elementos de Facilitación Genéticos , Genes fos , Humanos , Oligodesoxirribonucleótidos/uso terapéutico , Factor de Transcripción AP-1/metabolismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
In a recent report (Cho et al., Proc. Natl. Acad. Sci. USA 97, 835-840, 2000), we showed that cancer cells of various cell types secrete cAMP-dependent protein kinase (PKA) into the conditioned medium and that in the serum of cancer patients this extracellular PKA (ECPKA) is upregulated 10-fold as compared with normal serum. Here, we characterized the enzymatic properties of ECPKA that is present in the conditioned medium of PC3M prostate cancer cells and in the serum of cancer patients, and we compared ECPKA with PKA found in the cell extracts of PC3M cells. ECPKA present in the conditioned medium and human serum was not activated by cAMP addition, but intracellular PKA activity was totally dependent on the addition of cAMP. This indicates that the ECPKA is present in active, free C subunit form, whereas intracellular PKA is present in inactive holoenzyme form. ECPKA activity increased in a substrate concentration- and time-dependent manner, as did intracellular PKA. Both ECPKA and intracellular PKA activities were specifically inhibited by the PKA inhibitor protein, PKI. However, ECPKA activity was more temperature-sensitive than intracellular PKA; after two cycles of freezing/thawing, only 20% of initial ECPKA activity was detected compared with over 40% of intracellular PKA activity. Western blot analysis revealed the presence of a 40 kDa C(alpha) subunit of PKA in both conditioned medium and in the serum of cancer patients. These results suggest that ECPKA, out of the context of cAMP regulation, may function as a growth factor promoting cell growth and transformation; thus, it may serve as a tumor biomarker.
Asunto(s)
Biomarcadores de Tumor/sangre , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neoplasias/sangre , Medios de Cultivo Condicionados , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/sangre , Proteínas Quinasas Dependientes de AMP Cíclico/química , Humanos , Cinética , Masculino , Neoplasias/enzimología , Oligopéptidos/farmacología , Neoplasias de la Próstata , Subunidades de Proteína , Células Tumorales CultivadasRESUMEN
Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness.
Asunto(s)
Adhesión Celular/genética , ADN sin Sentido/genética , Receptores ErbB/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Transactivadores , Animales , Cadherinas/genética , División Celular/genética , Proteínas del Citoesqueleto/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Vectores Genéticos , Humanos , Ratones , Ratones Desnudos , Neoplasias Ováricas/fisiopatología , Receptor ErbB-3/análisis , Receptor ErbB-3/genética , Transcripción Genética/genética , Transfección , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , alfa Catenina , beta CateninaRESUMEN
The cyclic AMP-dependent protein kinase (PKA) exists in two isoforms, PKA-I (type I) and PKA-II (type II), that contain an identical catalytic (C) subunit but distinct regulatory (R) subunits, RI and RII, respectively. Increased expression of RIalpha/PKA-I has been shown in human cancer cell lines, in primary tumors, in cells after transformation, and in cells upon stimulation of growth. We have shown previously that a single-injection RI, antisense treatment results in a reduction in RIalpha and PKA-I expression and sustained inhibition of human colon carcinoma growth in athymic mice (M. Nesterova and Y. S. Cho-Chung, Nat. Med., 1: 528-533, 1995). Growth inhibition accompanied reduction in RIalpha/PKA-I expression and compensatory increases in RIIbeta protein and PKA-IIbeta, the RIIbeta-containing holoenzyme. Here, we report that these in vivo findings are consistent with observations made in cancer cells in culture. We demonstrate that the antisense depletion of RIalpha in cancer cells results in increased RIIbeta protein without increasing the rate of RIIbeta synthesis or RIIbeta mRNA levels. Pulse-chase experiments revealed a 3-6-fold increase in the half-life of RIIbeta protein in antisense-treated colon and prostate carcinoma cells with little or no change in the half-lives of RIalpha, RIIalpha, and Calpha proteins. Compensation by RIIbeta stabilization may represent a novel biochemical adaptation mechanism of the cell in response to sequence-specific loss of RIalpha expression, which leads to sustained down-regulation of PKA-I activity and inhibition of tumor growth.
Asunto(s)
Neoplasias del Colon/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Oligonucleótidos Antisentido/farmacología , Neoplasias de la Próstata/enzimología , Northern Blotting , Western Blotting , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Estabilidad de Enzimas , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Humanos , Masculino , Oligonucleótidos Antisentido/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fase S/efectos de los fármacos , Fase S/fisiología , Especificidad por Sustrato , Tionucleótidos/genética , Tionucleótidos/farmacología , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Carney complex (CNC) is a multiple neoplasia syndrome characterized by spotty skin pigmentation, cardiac and other myxomas, endocrine tumours and psammomatous melanotic schwannomas. CNC is inherited as an autosomal dominant trait and the genes responsible have been mapped to 2p16 and 17q22-24 (refs 6, 7). Because of its similarities to the McCune-Albright syndrome and other features, such as paradoxical responses to endocrine signals, genes implicated in cyclic nucleotide-dependent signalling have been considered candidates for causing CNC (ref. 10). In CNC families mapping to 17q, we detected loss of heterozygosity (LOH) in the vicinity of the gene (PRKAR1A) encoding protein kinase A regulatory subunit 1-alpha (RIalpha), including a polymorphic site within its 5' region. We subsequently identified three unrelated kindreds with an identical mutation in the coding region of PRKAR1A. Analysis of additional cases revealed the same mutation in a sporadic case of CNC, and different mutations in three other families, including one with isolated inherited cardiac myxomas. Analysis of PKA activity in CNC tumours demonstrated a decreased basal activity, but an increase in cAMP-stimulated activity compared with non-CNC tumours. We conclude that germline mutations in PRKAR1A, an apparent tumour-suppressor gene, are responsible for the CNC phenotype in a subset of patients with this disease.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , Neoplasias de las Glándulas Endocrinas/genética , Mutación , Mixoma/genética , Neoplasias/genética , Neurilemoma/genética , Pigmentación de la Piel/genética , Acromegalia/genética , Alelos , Western Blotting , Cromatografía Líquida de Alta Presión , Cromosomas Humanos Par 17 , Cromosomas Humanos Par 2 , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Análisis Mutacional de ADN , ADN Complementario/metabolismo , Exones , Etiquetas de Secuencia Expresada , Salud de la Familia , Femenino , Genotipo , Mutación de Línea Germinal , Homocigoto , Humanos , Intrones , Pérdida de Heterocigocidad , Masculino , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Ácidos Nucleicos Heterodúplex , Linaje , Fenotipo , Polimorfismo GenéticoRESUMEN
8-Cl-cAMP induces apoptotic cell death in human cancer cells. To look at this more closely, we examined the changes in the levels of Bcl-2 family proteins during 8-Cl-cAMP-induced apoptosis of SH-SY5Y human neuroblastoma cells. Following the treatment with 8-Cl-cAMP, Bcl-2 was transiently down-regulated and Bad was increased continuously up to day 5. In addition, overexpression of Bcl-2 efficiently blocked the 8-Cl-cAMP-induced apoptosis, suggesting Bcl-2 family proteins may be involved in the 8-Cl-cAMP-induced apoptosis. The contribution of the apoptotic cell death and the inhibition of cell proliferation in the 8-Cl-cAMP-induced growth inhibition was closely monitored in the Bcl-2-overexpressing cells. Though the apoptosis was reduced significantly, no significant difference was observed in the inhibition of cell proliferation up to day 2 of 8-Cl-cAMP treatment. These results suggest that 8-Cl-cAMP exerts anticancer activity by two distinct mechanisms, i.e. , through the inhibition of cell proliferation as well as the induction of apoptosis. Supporting this notion was the observations that (1) suppression of apoptosis by zVAD did not abrogate 8-Cl-cAMP-induced inhibition of cell proliferation, and (2) 8-Cl-cAMP did not show additive inhibition of cell proliferation in RIIbeta-overexpressing cells.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Inhibidores de Caspasas , Línea Celular , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Expresión Génica , Genes bcl-2 , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Células Tumorales CultivadasRESUMEN
The CRE, 5'-TGACGTCA-3', has been described as the consensus sequence for the cis-element that directs cAMP-regulated gene expression. Many transcription factors bind to this element and regulate the expression of a wide variety of cellular and viral genes. We have shown that CRE-transcription factor decoy oligonucleotide restrains the growth of cancer cells in vitro and in vivo [Park, Y. G., Nesterova, M., Agrawal, S., and Cho-Chung, Y. S. (1999) J. Biol. Chem. 274, 1573-1580]. The growth inhibition was accompanied by changes in cell morphology and apoptosis. To elucidate the molecular mechanism(s) of the growth inhibition by the CRE-decoy oligonucleotide, we investigated the p53 signaling pathway. Herein, we report that CRE-decoy oligonucleotide treatment results in an increase in the p53 protein level in MCF-7 human breast cancer cells that express wild-type p53. The p21WAF1/Cip1 protein levels were also increased in the CRE-decoy oligonucleotide treated cells accompanying a reduction in Cdk2- and cyclin E-dependent kinase activity and pRb phosphorylation. Pulse-chase experiments reveal that the p53 upregulation was due to increased stability of the protein. The decoy oligonucleotide treatment also enhanced the p53 promotor-directed transcription in vivo along with the increase in p53-CBP (CREB-binding protein) complex formation. Thus, the stabilization and activation of p53 may have contributed to the growth inhibition induced by CRE-transcription factor decoy oligonucleotide in MCF-7 breast cancer cells. This decoy oligonucleotide approach offers great promise as a tool for defining cellular regulatory processes and treating cancer and other diseases.
Asunto(s)
Neoplasias de la Mama/patología , Quinasas CDC2-CDC28 , AMP Cíclico/fisiología , Oligonucleótidos/farmacología , Elementos de Respuesta/genética , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/metabolismo , Proteína de Unión a CREB , División Celular/efectos de los fármacos , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Nucleares/metabolismo , Oligonucleótidos/genética , Oligonucleótidos/uso terapéutico , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Retinoblastoma/metabolismo , Termodinámica , Transactivadores/metabolismo , Activación Transcripcional/efectos de los fármacos , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Both cAMP and retinoids play a role in cell differentiation and the control of cell growth. A site-selective cAMP analog, 8-Cl-cAMP and retinoic acid synergistically inhibit growth and induce apoptosis in certain cancer cells. In advanced or recurrent malignant diseases, retinoic acid (RA) is not effective even at doses that are toxic to the host. The objective of our present study was to examine the mechanism(s) of synergistic effects of retinoic acid (9-cis, 13-cis or all-trans RA) and 8-Cl-cAMP on apoptosis in human ovarian cancer NIH: OVCAR-3 and OVCAR-8 cells. RA induced growth inhibition and apoptosis in OVCAR-3 and OVCAR-8 cells. 8-Cl-cAMP acted synergistically with RA in inducing and activating retinoic acid receptor beta (RARbeta) which correlates with growth inhibition and apoptosis in both cell types. In addition, induction of apoptosis by RA plus 8-Cl-cAMP requires caspase-3 activation followed by cleavage of anti-poly(ADP-ribose) polymerase. Furthermore, mutations in CRE-related motif within the RARbeta promoter resulted in loss of both transcriptional activation of RARbeta and synergy between RA and 8-Cl-cAMP. RARbeta expression appears to be associated with induction of apoptosis. Introduction of the RARbeta gene into OVCAR-3 cells resulted in gain of RA sensitivity. Loss of RARbeta expression, therefore, may contribute to the tumorigenicity of human ovarian cancer cells. Thus, combined treatment with RA and 8-Cl-cAMP may provide an effective means for inducing RARbeta expression leading to apoptosis in ovarian cancer cells.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Antineoplásicos/farmacología , Apoptosis , Neoplasias Ováricas/patología , Receptores de Ácido Retinoico/biosíntesis , Tretinoina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Caspasa 3 , Caspasas/fisiología , División Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , Células Tumorales CultivadasRESUMEN
To examine the role of protein kinase A (EC 2.7.1.37) isozymes in the retinoic acid-induced growth inhibition and neuronal differentiation, we investigated the changes of protein kinase A isozyme patterns in retinoic acid-treated SH-SY5Y human neuroblastoma cells. Retinoic acid induced growth inhibition and neuronal differentiation of SH-SY5Y cells in a dose- and time-dependent manner. Neuronal differentiation was evidenced by extensive neurite outgrowth, decrease of N-Myc oncoprotein, and increase of GAP-43 mRNA. Type II protein kinase A activity increased by 1.5-fold in differentiated SH-SY5Y cells by retinoic acid treatment. The increase of type II protein kinase A was due to the increase of RIIbeta and Calpha subunits. Since type II protein kinase A and RIIbeta have been known to play important role(s) in the growth inhibition and differentiation of cancer cells, we further investigated the role of the increased type II protein kinase A by overexpressing RIIbeta in SH-SY5Y cells. The growth of RIIbeta-overexpressing cells was slower than that of parental cells, being comparable to that of retinoic acid-treated cells. Retinoic acid treatment further increased the RIIbeta level and further inhibited the growth of RIIbeta-overexpressing cells, showing strong correlation between the level of RIIbeta and growth inhibition. However, RIIbeta-overexpressing cells did not show any sign of neuronal differentiation and responded to retinoic acid in the same way as parental cells. These data suggest that protein kinase A participates in the retinoic acid-induced growth inhibition through the up-regulation of RIIbeta/type II protein kinase A.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neuroblastoma , Tretinoina/farmacología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico , Proteína Quinasa Tipo II Dependiente de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , ARN Mensajero/metabolismo , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/enzimologíaRESUMEN
Overexpression of cAMP-dependent protein kinase (PKA) type I isozyme is associated with cell proliferation and neoplastic transformation. The presence of PKA on the external surface of LS-174T human colon carcinoma cells has been shown. Here, we show that cancer cells of various cell types excrete PKA into the conditioned medium. This extracellular PKA (ECPKA) is present in active, free catalytic subunit (C subunit) form, and its activity is specifically inhibited by PKA inhibitory protein, PKI. Overexpression of the Calpha or RIalpha subunit gene of PKA in an expression vector, which up-regulates intracellular PKA type I, markedly up-regulates ECPKA expression. In contrast, overexpression of the RIIbeta subunit, which eliminates PKA type I, up-regulates PKA type II, and reverts the transformed phenotype, down-regulates ECPKA. A mutation in the Calpha gene that prevents myristylation allows the intracellular PKA up-regulation but blocks the ECPKA increase, suggesting that the NH(2)-terminal myristyl group of Calpha is required for the ECPKA expression. In serum of cancer patients, the ECPKA expression is up-regulated 10-fold as compared with normal serum. These results indicate that the ECPKA expression is an ordered cellular response of a living cell to actively exclude excess intracellular PKA molecules from the cell. This phenomenon is up-regulated in tumor cells and has an inverse relationship with the hormone dependency of breast cancer. Thus, the extracellular PKA may serve as a potential diagnostic and prognostic marker for cancer.