RESUMEN
We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3' terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys382-p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G1-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors.
Asunto(s)
Daño del ADN , Resistencia a Antineoplásicos , MicroARNs/metabolismo , ARN Neoplásico/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Femenino , Fase G1 , Células HeLa , Humanos , MicroARNs/genética , ARN Neoplásico/genética , Puntos de Control de la Fase S del Ciclo Celular , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Proteínas de Transporte Vesicular/genéticaRESUMEN
Differential display PCR analysis (DD-PCR) was used to identify novel genes that respond to IGF-I treatment in human MCF-7 breast cancer cells. Fifty-three cDNAs showed alterations in their mRNA levels in IGF-I treated cells. One of these genes showed a significant increase in the mRNA level in IGF-I treated cells in comparison to non-treated cells. We named this gene HIRF1 (human IGF-I regulated factor 1). Nucleotide blast analysis revealed that this gene has a 100% sequence identity with the sequence for BTG1 (B-cell translocation gene) binding factor 1 (human CCR4-associated factor 1 gene, hCAF1). By alignment of cloned HIRF1 cDNA and genomic DNA 8p21.3-p22 sequence, we were able to determine the exon-intron structure of the cloned HIRF1 gene on chromosome 8. Northern blot and real-time PCR analysis showed that BTG1 and c-fos reached their maximal expression fairly early within 10 min to 1 h, and decreased to basal levels after 3 h of IGF-I treatment. HIRF1/hCAF1 expression reached maximal stimulation after 3 h of IGF-I treatment and then gradually decreased to basal level. HIRF1 and BTG1 mRNA was inhibited by inhibitors of the cell signaling pathways, PI3/Akt kinase and MAPK kinases (ERK1/2 and p38). In summary, cloned HIRF1/hCAF1 is coregulated with BTG1 in response to IGF-I. The regulation of these genes as early response genes may have an important role in differentiation, growth and proliferation of breast cancer cells.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas de Neoplasias/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Humanos , Imidazoles/farmacología , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Datos de Secuencia Molecular , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piridinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor IGF Tipo 1/genética , Homología de Secuencia de Aminoácido , Transcripción Genética/efectos de los fármacosRESUMEN
PURPOSE: The purpose of this study was to determine whether curcumin would trigger cell death in the head and neck squamous cell carcinoma (HNSCC) cell lines CCL 23, CAL 27, and UM-SCC1 in a dose-dependent fashion. EXPERIMENTAL DESIGN: HNSCC cells were treated with curcumin and assayed for in vitro growth suppression using 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide and fluorescence-activated cell sorting analyses. Expression of p16, cyclin D1, phospho-Ikappabeta, and nuclear factor-kappabeta (NF-kappabeta) were measured by Western blotting, gel shift, and immunofluorescence. RESULTS: Addition of curcumin resulted in a dose-dependent growth inhibition of all three cell lines. Curcumin treatment resulted in reduced nuclear expression of NF-kappabeta. This effect on NF-kappabeta was further reflected in the decreased expression of phospho-Ikappabeta-alpha. Whereas the expression of cyclin D1, an NF-kappabeta-activated protein, was also reduced, there was no difference in the expression of p16 at the initial times after curcumin treatment. In vivo growth studies were done using nude mice xenograft tumors. Curcumin was applied as a noninvasive topical paste to the tumors and inhibition of tumor growth was observed in xenografts from the CAL27 cell line. CONCLUSIONS: Curcumin treatment resulted in suppression of HNSCC growth both in vitro and in vivo. Our data support further investigation into the potential use for curcumin as an adjuvant or chemopreventive agent in head and neck cancer.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Curcumina/farmacología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Animales , Anexina A5/química , Antineoplásicos/administración & dosificación , Western Blotting , Línea Celular Tumoral , Núcleo Celular/metabolismo , Separación Celular , Supervivencia Celular , Curcumina/administración & dosificación , Ciclina D1/biosíntesis , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Humanos , Proteínas I-kappa B/metabolismo , Técnicas In Vitro , Ratones , Ratones Desnudos , Microscopía Fluorescente , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Trasplante de Neoplasias , Fosforilación , Sales de Tetrazolio/farmacología , Tiazoles/farmacologíaRESUMEN
OBJECTIVES: To determine expression of cell cycle and apoptotic genes, biochemical analysis of CCL23 and antisense cyclin D1-transfected CCL23 (CCL23AS) cells in the presence of cisplatin was performed. In addition, biochemical analysis of CAL27 cells before and after treatment with cisplatin was performed to determine expression of cell cycle genes. DESIGN: CCL23, CCL23AS, and CAL27 cell lines were treated with cisplatin. Western blot analysis, fluorescence-activated cell sorting, and apoptosis assays were performed. SETTING: In vitro study of head and neck cancer cell lines CCL23, CCL23AS, and CAL27. INTERVENTION: CCL23, CCL23AS, and CAL27 cells were treated with cisplatin. MAIN OUTCOME MEASURES: Expression of p16, p21, p53, Bcl-xL, Bcl-xS, p27, DP1, MDM2, Bcl-2, c-Jun, and Jun-D were assessed using Western blot analysis. RESULTS: There was increased expression of p16, p21, p53, BCLxL, and BCLxS genes with cisplatin treatment in the CCL23 and CCL23AS cells. Expression of p27, DP1, MDM2, BCL2, c-iun, and jun-D remained unaltered after treatment. There was decreased phosphorylation of Rb protein with complete absence of hyperphosphorylated Rb in the maximally sensitized antisense cyclin D1-transfected (CCL23AS) cells. Fluorescence-activated cell sorter analysis revealed a decreased G2 phase of the cell cycle and an increased proportion of apoptotic cells in the CCL23AS cell line compared with parental CCL23 cells. Cell killing also occurred in the presence of caspase-3 inhibitor. While CCL23 cells contain wild-type p53, the CAL27 cells have a point mutation in codon 193 (A-->T transversion) of exon 6. However, CAL27 cells still exhibited increased expression of p21 after treatment with cisplatin. CONCLUSIONS: These results, in combination with increased expression of the p53 downstream effecter p21, indicate that the cisplatin-induced cell cycle arrest operates through the p16/p53-dependent pathway, and a caspase-independent pathway may be involved. Combination treatment of head and neck squamous cell carcinoma via cell cycle inhibition and cisplatin holds promise as a potential therapy in the clinical setting.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Escamosas/patología , Cisplatino/farmacología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis , Neoplasias de Cabeza y Cuello/patología , Proteína p53 Supresora de Tumor/análisis , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasas/análisis , Muerte Celular , Citometría de Flujo , Antígeno Nuclear de Célula en Proliferación/análisis , Proteínas Proto-Oncogénicas c-jun/análisis , Proteínas Proto-Oncogénicas c-mdm2/análisis , Proteína de Retinoblastoma/metabolismo , Células Tumorales Cultivadas , Proteína bcl-X/análisisRESUMEN
Previous molecular genetic studies on HeLa cell (a cervical cancer cell line) derived non-tumorigenic and tumorigenic hybrids have localized a tumor suppressor gene to the long arm of chromosome 11. Analysis of cervical cancer cell lines using chromosome 11 specific probes showed deletion and translocation of 11q13 sequences in five out of eight cell lines. Fluorescence in situ hybridization (FISH), using 11q13 specific probes, has shown interstitial deletion of 11q13 sequences in the HeLa cells. In order to determine whether 11q13 deletions occur in primary cervical tumors, we analysed 36 tumors using 20 different microsatellite and RFLP markers. Semi automated fluorescein based allelotyping was performed to identify loss of heterozygosity (LOH) in tumors. The results showed allelic loss in 17 (47%) tumors. Three different regions of loss, one near MEN1, the second near D11S913, and the third near INT2 locus were observed. The smallest region of deletion overlap at the D11S913 locus was localized to a 300 Kb distance between D11S4908 and D11S5023. Fluorescence in situ hybridization (FISH), using 11q13 specific cosmid and BAC (bacterial artificial chromosome) probes, confirmed allelic deletion in the tumors. PCR analysis further identified homozygous deletion of 11q13 sequences in a primary tumor, in HeLa cells and in two HeLa cell derived tumorigenic hybrid cell lines. The homozygous deletion in the cell lines was mapped to a 5.7 kb sequence of 11q13. We hypothesize therefore that a putative cervical cancer tumor suppressor gene exists within the 300 kb of chromosome 11q13.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 11/genética , ADN de Neoplasias/análisis , Genes Supresores de Tumor , Proteínas Proto-Oncogénicas , Neoplasias del Cuello Uterino/genética , Centrómero/genética , Mapeo Cromosómico , Cromosomas Artificiales Bacterianos/genética , Cromosomas Artificiales Bacterianos/metabolismo , Cartilla de ADN/química , Endometrio/patología , Femenino , Células HeLa , Humanos , Células Híbridas , Procesamiento de Imagen Asistido por Computador , Hibridación Fluorescente in Situ , Cariotipificación , Pérdida de Heterocigocidad , Metafase , Repeticiones de Microsatélite , Proteínas de Neoplasias/genética , Polimorfismo Conformacional Retorcido-Simple , Neoplasias del Cuello Uterino/patologíaRESUMEN
Nonrandom deletion of chromosome 11q13 sequences is a significant event in a number of human tumors. We have recently identified a 300-kb minimal area of deletion in primary cervical tumors that overlaps with deletions observed in endocrine and nasopharyngeal tumors. We have also observed a 5.7-kb homozygous deletion within this interval in HeLa cells (a cervical cancer cell line), HeLa cell-derived tumorigenic hybrids, and a primary cervical tumor, suggesting the presence of a tumor suppressor gene in this region. In the present investigation, we have constructed a 700-kb contig map encompassing the 300-kb deletion using the human genome sequence database and confirmed the map using various STS markers from the region. Our map also shows the overlap of a previously published rare, heritable fragile site, FRA11A, with the cervical cancer deletion locus. The mapped region contains highly repetitive GC-poor sequences. We have identified and characterized eight different polymorphic microsatellite markers from the sequences within and surrounding the deletion. Further, expression studies performed with 18 different ESTs localized adjacent to the homozygous deletion showed the presence of a transcript for only one of the ESTs, AA282789. This EST mapping within the homozygous deletion is also expressed in HeLa cells, thereby excluding the EST as the putative tumor suppressor gene. Additionally, analysis of four candidate genes (SF3B2, BRMS1, RIN1, and RAB1B) from the region showed expression of the expected size message in both the nontumorigenic and the tumorigenic HeLa cell hybrids, thereby excluding them as the putative tumor suppressor gene(s). However, Northern blot analysis with a fifth candidate gene, PACS1 (phosphofurin acidic cluster sorting protein), mapped to the deletion/FRA11A overlap region showed the expression of an 8-kb transcript in HeLa and five other tumor cell lines in addition to the expected 4.5-kb transcript. Since the gene shows abundant expression in normal tissues and an altered transcript is observed in tumor cell lines, we hypothesize that this gene could represent sequences of the putative tumor suppressor gene. Finally, we have observed a perfect 48-bp CAG/CCG repeat 99 kb proximal to D11S913, the marker linked to the neurodegenerative disorder spinocerebellar ataxia 5. The physical and transcription maps and the microsatellite markers of the 700-kb region of chromosome 11q13 should be helpful in the cloning of the cervical cancer tumor suppressor gene.
Asunto(s)
Cromosomas Humanos Par 11/genética , Genes Supresores de Tumor , Mapeo Físico de Cromosoma , Neoplasias del Cuello Uterino/genética , Femenino , Células HeLa , HumanosRESUMEN
Nasopharyngeal carcinoma (NPC) is a malignancy that is particularly prevalent among populations from Southern China and Southeast Asian countries. Evidence for a genetic contribution to the disease has been documented, although the genetic basis for NPC development is not yet fully understood. Previous functional evidence of tumor-suppressive activity on chromosome band 11q13 in NPC was obtained using a microcell-mediated chromosome-transfer approach with HONE1 NPC cells. In the present study, this region was subjected to a detailed investigation of microcell hybrids and their tumor segregants using microsatellite analysis to narrow down the region of tumor-suppressive activity. Fluorescence in situ hybridization was also performed with BAC and cosmid probes to confirm the microsatellite data. The critical region responsible for tumor suppression was narrowed down to a 1.8-Mb interval, which does not tolerate an additional normal allele by chromosome transfer. One or two alleles from either endogenous or exogenous chromosomes at 11q13 were consistently eliminated during tumor growth. Results of this study suggest that a candidate tumor-suppressor gene, not the MEN1 gene, maps between D11S4907 and GSTP1 in NPC.
Asunto(s)
Mapeo Cromosómico/métodos , Cromosomas Humanos Par 11/genética , Genes Supresores de Tumor , Neoplasias Nasofaríngeas/genética , Marcadores Genéticos/genética , Humanos , Hibridación Fluorescente in Situ , Repeticiones de Microsatélite , Neoplasia Endocrina Múltiple Tipo 1/genética , Células Tumorales CultivadasRESUMEN
Studies on nontumorigenic and tumorigenic human cell hybrids derived from the fusion of HeLa (a cervical cancer cell line) with GM00077 (a normal skin fibroblast cell line) have demonstrated "functional" tumor-suppressor activity on chromosome 11. It has been shown that several of the neoplastically transformed radiation-induced hybrid cells called GIMs (gamma ray induced mutants), isolated from the nontumorigenic CGL1 cells, have lost one copy of the fibroblast chromosome 11. We hypothesized, therefore, that the remaining copy of the gene might be mutated in the cytogenetically intact copy of fibroblast chromosome 11. Because a cervical cancer tumor suppressor locus has been localized to chromosome band 11q13, we performed deletion-mapping analysis of eight different GIMs using a total of 32 different polymorphic and microsatellite markers on the long arm (q arm) of chromosome 11. Four irradiated, nontumorigenic hybrid cell lines, called CONs, were also analyzed. Allelic deletion was ascertained by the loss of a fibroblast allele in the hybrid cell lines. The analysis confirmed the loss of a fibroblast chromosome 11 in five of the GIMs. Further, homozygous deletion (complete loss) of chromosome band 11q13 band sequences, including that of D11S913, was observed in two of the GIMs. Detailed mapping with genomic sequences localized the homozygous deletion to a 5.7-kb interval between EST AW167735 and EST F05086. Southern blot hybridization using genomic DNA probes from the D11S913 locus confirmed the existence of homozygous deletion in the two GIM cell lines. Additionally, PCR analysis showed a reduction in signal intensity for a marker mapped 31 kb centromeric of D11S913 in four other GIMs. Finally, Northern blot hybridization with the genomic probes revealed the presence of a novel >15-kb transcript in six of the GIMs. These transcripts were not observed in the nontumorigenic hybrid cell lines. Because the chromosome 11q13 band deletions in the tumorigenic hybrid cell lines overlapped with the minimal deletion in cervical cancer, the data suggest that the same gene may be involved in the development of cervical cancer and in radiation-induced carcinogenesis. We propose that a gene localized in proximity to the homozygous deletion is the candidate tumor-suppressor gene.