RESUMEN
All-trans-retinoic acid (RA) treatment of acute promyelocytic leukemia (APL) cases expressing the t(15;17) product, PML/RARalpha, is a successful example of differentiation therapy. Uncovering RA target genes is of considerable interest in APL. This study comprehensively examines in APL cells transcriptional and post-transcriptional regulation of the novel candidate RA target gene, G0S2, the G0/G1 switch gene. Reverse transcription (RT)-polymerase chain reaction (PCR) and heteronuclear PCR assays performed +/- treatment with the protein synthesis inhibitor cycloheximide (CHX) revealed G0S2 induction within 3 h of RA-treatment. Treatment with the RNA synthesis inhibitor actinomycin D did not implicate G0S2 transcript stabilization in the RA-mediated increase of G0S2 mRNA expression. Promoter elements of G0S2 were cloned into a reporter plasmid and retinoic acid receptor (RAR) co-transfection assays confirmed transcriptional activation after RA-treatment. Consistent with G0S2 being a direct RA target gene, retinoic acid response element (RARE) half-sites were found in this promoter. Mutation of these sites blocked RA-transcriptional activation of G0S2. To extend analyses to the protein expression level, a polyclonal anti-G0S2 antibody was derived and detected murine and human G0S2 species. G0S2 protein was rapidly induced in cultured NB4-S1 human APL cells and in APL transgenic mice treated with RA. An RAR pan-antagonist confirmed dependence on RARs for this induction. That these findings are clinically relevant was shown by analyses of APL cells derived directly from patients. These leukemic cells induced both a prominent increase in the cellular differentiation marker nitrotetrazolium blue (NBT) staining and marked increase in G0S2 expression. Taken together, these findings indicate G0S2 is an RA target gene. The functional role of G0S2 in retinoid response of APL warrants further study.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Tretinoina/farmacología , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Expresión Génica/efectos de los fármacos , Humanos , Immunoblotting , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , ARN Mensajero/análisis , Receptores de Ácido Retinoico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Elemento de Respuesta a la Vitamina D/efectos de los fármacos , Elemento de Respuesta a la Vitamina D/fisiologíaRESUMEN
Acute promyelocytic leukemia (APL) is characterized by expression of promyelocytic leukemia (PML)/retinoic acid (RA) receptor alpha (RARalpha) protein and all-trans-RA-mediated clinical remissions. RA treatment can confer PML/RARalpha degradation, overcoming dominant-negative effects of this oncogenic protein. The present study uncovered independent retinoid degradation mechanisms, targeting different domains of PML/RARalpha. RA treatment is known to repress PML/RARalpha and augment ubiquitin-activating enzyme-E1-like (UBE1L) protein expression in NB4-S1 APL cells. We previously reported RA-induced UBE1L and the IFN-stimulated gene, 15-kDa protein ISG15ylation in APL cells. Whether the ubiquitin-like protein ISG15 directly conjugates with PML/RARalpha was not explored previously and is examined in this study. Transient transfection experiments with different PML/RARalpha domains revealed that RA treatment preferentially down-regulated the RARalpha domain, whereas UBE1L targeted the PML domain for repression. As expected, ubiquitin-specific protease 18 (UBP43/USP18), the ISG15 deconjugase, opposed UBE1L but not RA-dependent PML/RARalpha degradation. In contrast, the proteasomal inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal, inhibited both UBE1L- and RA-mediated PML/RARalpha degradation. Notably, UBE1L induced ISG15ylation of the PML domain of PML/RARalpha, causing its repression. These findings confirmed that RA triggers PML/RARalpha degradation through different domains and distinct mechanisms. Taken together, these findings advance prior work by establishing two pathways converge on the same oncogenic protein to cause its degradation and thereby promote antineoplastic effects. The molecular pharmacologic implications of these findings are discussed.
Asunto(s)
Citocinas/metabolismo , Regulación Leucémica de la Expresión Génica , Leucemia Promielocítica Aguda/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Enzimas Activadoras de Ubiquitina/farmacología , Ubiquitinas/metabolismo , Animales , Antineoplásicos/farmacología , Bronquios/citología , Bronquios/metabolismo , Células COS , Células Cultivadas , Chlorocebus aethiops , Endopeptidasas/metabolismo , Humanos , Immunoblotting , Inmunoprecipitación , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patología , Leupeptinas/farmacología , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transfección , Tretinoina/farmacología , Ubiquitina TiolesterasaRESUMEN
Cancer is characterized by uncontrolled cell division resulting from multiple mutagenic events. Cancer chemoprevention strategies aim to inhibit or reverse these events using natural or synthetic pharmacologic agents. Ideally, this restores normal growth control mechanisms. Diverse classes of compounds have been identified with chemopreventive activity. What unites many of them is an ability to inhibit the cell cycle by specifically modulating key components. This delays division long enough for cells to respond to mutagenic damage. In some cases, damage is repaired and in others cellular damage is sufficient to trigger apoptosis. It is now known that pathways responsible for targeting G1 cyclins for proteasomal degradation can be engaged pharmacologically. Emergence of induced cyclin degradation as a target for cancer therapy and chemoprevention in pre-clinical models is discussed in this article. Evidence for cyclin D1 as a molecular pharmacologic target and biological marker for clinical response is based on experience of proof of principle trials.
Asunto(s)
Ciclinas/metabolismo , Neoplasias/tratamiento farmacológico , Ciclo Celular , Ciclina D1/metabolismo , Ciclina G , Ciclina G1 , Sistemas de Liberación de Medicamentos/métodos , Humanos , Neoplasias/prevención & control , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Tobacco carcinogen treatment of immortalized human bronchial epithelial (HBE) cells has uncovered novel targets for cancer chemoprevention. Experiments were conducted with HBE cells and independent treatments with tobacco carcinogens along with the chemopreventive agent all-trans-retinoic acid (RA). That work highlighted D-type and E-type cyclins as novel molecular pharmacologic targets of several chemopreventive agents. G1 cyclins are often aberrantly expressed in bronchial preneoplasia and lung cancers. This implicated these species as targets for clinical cancer chemoprevention. Retinoid regulation mechanisms of D-type cyclins in lung cancer chemoprevention have been comprehensively explored. Retinoid chemoprevention has been mechanistically linked to proteasomal degradation of cyclin D1 and cyclin D3. Threonine 286 mutation stabilized cyclin D1, implicating phosphorylation in this retinoid chemoprevention. Studies with a phospho-specific anti-cyclin D1 antibody confirmed this hypothesis. Glycogen synthase kinase (GSK) inhibitors established a role for this kinase in the retinoid regulation of cyclin D1, but not cyclin D3. Involvement of D-type cyclins in this chemoprevention was shown using small interfering RNAs (siRNAs). Gene profiling experiments highlighted the E1-like ubiquitin-activating enzyme (UBE1L) in the retinoid regulation of cyclin D1. Proof of principle trials have translated these studies into the clinic and established that chemopreventive agents can target D-type cyclins. These findings have been built upon with a targeted combination regimen that cooperatively affects D-type cyclins. Taken together, these preclinical and clinical findings strongly implicate these cyclins as novel molecular pharmacological targets for cancer chemoprevention.
Asunto(s)
Transformación Celular Neoplásica/efectos de los fármacos , Quimioprevención/métodos , Ciclina D1/efectos de los fármacos , Ciclina D1/metabolismo , Neoplasias/prevención & control , Animales , Transformación Celular Neoplásica/metabolismo , Ensayos Clínicos como Asunto , HumanosRESUMEN
BACKGROUND: Cells from patients with t(15;17) acute promyelocytic leukemia (APL) express the fusion protein between the promyelocytic leukemia protein and retinoic acid receptor alpha (PML/RAR alpha). Patients with APL respond to differentiation therapy with all-trans-retinoic acid, which induces PML/RAR alpha degradation. When resistance to all-trans-retinoic acid develops, an effective treatment is arsenic trioxide (arsenite), which also induces this degradation. We investigated the mechanism of arsenite-induced PML/RAR alpha degradation. METHODS: NB4-S1 APL cells were treated with clinically relevant concentrations of arsenite. Lysosomes were visualized with a lysosome-specific dye. Lysosomal protein esterase was measured by immunoblot analysis. Lysosomal cathepsin L was detected by immunogold labeling and transmission electron microscopy, and its activity was measured in cytosolic cellular fractions. In vitro degradation assays of PML/RAR alpha in cell lysates were performed with and without protease inhibitors and assessed by immunoblot analysis. Only nonparametric two-sided statistical analyses were used. The nonparametric Wilcoxon test was used for group comparison, and the nonlinear regression technique was used for analysis of dose-response relationship as a function of arsenite concentration. RESULTS: Arsenite treatment destabilized lysosomes in APL cells. Lysosomal proteases, including cathepsin L, were released from lysosomes 5 minutes to 6 hours after arsenite treatment. PML/RAR alpha was degraded by lysate from arsenite-treated APL cells, and the degradation was inhibited by protease inhibitors. At both 6 and 24 hours, substantially fewer arsenite-treated APL cells, than untreated cells, contained cathepsin L clusters, a reflection of cathepsin L delocalization. Cells with cathepsin L clusters decreased as a function of arsenite concentration at rates of -2.03% (95% confidence interval [CI] = -4.01 to -.045; P = .045) and -2.39% (95% CI = -4.54 to -.024; P = .029) in 6- and 24-hour treatment groups, respectively, per 1.0 microM increase in arsenite concentration. Statistically significantly higher cytosolic cathepsin L activity was detected in lysates of arsenite-treated APL cells than in control lysates. For example, the mean increase in cathepsin activity at 6 hours and 1.0 microM arsenite was 26.3% (95% CI = 3.3% to 33%; P < .001), compared with untreated cells. CONCLUSIONS: In APL cells, arsenite may cause rapid destabilization of lysosomes.
Asunto(s)
Antineoplásicos/farmacología , Arsenitos/farmacología , Leucemia Promielocítica Aguda/tratamiento farmacológico , Lisosomas/efectos de los fármacos , Proteínas de Fusión Oncogénica/metabolismo , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Catepsina L , Catepsinas/metabolismo , Cisteína Endopeptidasas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Leucemia Promielocítica Aguda/metabolismo , Lisosomas/metabolismo , Microscopía Electrónica de Transmisión , Proteínas de Fusión Oncogénica/efectos de los fármacos , Péptido Hidrolasas/metabolismo , Proyectos de Investigación , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Decades of hypothesis-driven research have identified candidate targets for cancer therapy and chemoprevention. Recently, genomic, proteomic, and tissue-based microarray approaches have made possible another scientific approach. This is one that interrogates comprehensively the complex profile of mRNA or protein expression present in normal, preneoplastic, or malignant cells and tissues. This in turn can uncover critical targets for cancer pharmacology and also lead to a better understanding of the known or novel networks of gene expression that play a rate-limiting role in carcinogenesis. This chapter addresses the use of mRNA expression profiling to uncover candidate target genes active in cancer pharmacology by citing as an example how this has already proven useful to reveal that retinoids (natural and synthetic derivatives of vitamin A) signal through pathways, which promote tumor cell differentiation, induce growth suppression, trigger apoptosis or affect other growth regulatory pathways. Pathways involved in the regulation of protein stability will be highlighted as these play a critical role in mediating pharmacological effects of the retinoids in cancer therapy or chemoprevention.
Asunto(s)
Perfilación de la Expresión Génica , Neoplasias/genética , Retinoides/genética , Anticarcinógenos/farmacología , Antineoplásicos/farmacología , Apoptosis , Diferenciación Celular , Quimioprevención , Sistemas de Liberación de Medicamentos , Regulación Neoplásica de la Expresión Génica , Neoplasias/tratamiento farmacológico , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Unión al ARN/genética , Transducción de Señal/genética , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Células Tumorales CultivadasRESUMEN
Retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and chemoprevention. We reported previously that all-trans-retinoic acid (RA) treatment prevented carcinogen-induced transformation of immortalized human bronchial epithelial (HBE) cells. To identify cancer chemopreventive mechanisms, immortalized (BEAS-2B), carcinogen-transformed (BEAS-2B(NNK)), and RA-chemoprevented (BEAS-2B(NNK/RA)) HBE cells were used to conduct microarray analyses independently. Species increased in chemoprevented as compared with immortalized HBE cells (group I) and those augmented in chemoprevented as compared with transformed HBE cells (group II) included known RA-target genes as well as previously unrecognized RA-target genes in HBE cells. Unexpectedly, both groups were also enriched for interferon-stimulated genes. One interferon-stimulated gene of particular interest was UBE1L, the ubiquitin-activating enzyme E1-like protein. UBE1L expression was also induced after prolonged RA-treatment of immortalized HBE cells. UBE1L mRNA was shown previously as repressed in certain lung cancer cell lines, directly implicating UBE1L in lung carcinogenesis. Notably, UBE1L immunoblot expression was reduced in a subset of malignant as compared with adjacent normal lung tissues that were examined. Immunohistochemical analyses were performed using a new assay developed to detect this species using rabbit polyclonal anti-UBE1L antibodies independently raised against the amino- or carboxyl-termini of UBE1L. Studies done on paraffin-embedded and fixed tissues revealed abundant UBE1L, but low levels of cyclin D1 expression in the normal human bronchial epithelium, indicating an inverse relationship existed between these species. To study this further, cotransfection into HBE cells of wild-type or mutant UBE1L species was accomplished. In a dose-dependent manner, wild-type but not mutant UBE1L species repressed cyclin D1 expression. This implicated UBE1L in a retinoid chemoprevention mechanism involving cyclin D1 repression described previously. Taken together, these findings directly implicate UBE1L as a candidate-pharmacologic target for lung cancer chemoprevention. These findings also provide a mechanistic basis for the tumor suppressive effects of UBE1L through cyclin D1 repression.
Asunto(s)
Anticarcinógenos/uso terapéutico , Neoplasias Pulmonares/prevención & control , Tretinoina/uso terapéutico , Enzimas Activadoras de Ubiquitina/genética , Línea Celular Tumoral , Genes Supresores de Tumor , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Enzimas Activadoras de Ubiquitina/análisis , Enzimas Activadoras de Ubiquitina/fisiologíaRESUMEN
All-trans-retinoic acid (RA) treatment induces remissions in acute promyelocytic leukemia (APL) cases expressing the t(15;17) product, promyelocytic leukemia (PML)/RA receptor alpha (RARalpha). Microarray analyses previously revealed induction of UBE1L (ubiquitin-activating enzyme E1-like) after RA treatment of NB4 APL cells. We report here that this occurs within 3 h in RA-sensitive but not RA-resistant APL cells, implicating UBE1L as a direct retinoid target. A 1.3-kb fragment of the UBE1L promoter was capable of mediating transcriptional response to RA in a retinoid receptor-selective manner. PML/RARalpha, a repressor of RA target genes, abolished this UBE1L promoter activity. A hallmark of retinoid response in APL is the proteasome-dependent PML/RARalpha degradation. UBE1L transfection triggered PML/RARalpha degradation, but transfection of a truncated UBE1L or E1 did not cause this degradation. A tight link was shown between UBE1L induction and PML/RARalpha degradation. Notably, retroviral expression of UBE1L rapidly induced apoptosis in NB4 APL cells, but not in cells lacking PML/RARalpha expression. UBE1L has been implicated directly in retinoid effects in APL and may be targeted for repression by PML/RARalpha. UBE1L is proposed as a direct pharmacological target that overcomes oncogenic effects of PML/RARalpha by triggering its degradation and signaling apoptosis in APL cells.