RESUMEN
BACKGROUND/AIM: Chronic inflammation generates large quantities of reactive oxygen and nitrogen species that damage DNA. DNA repair is important for cellular viability and genome integrity. MATERIALS AND METHODS: Expression levels of the DNA repair proteins OGG1, XPA, MLH1, PARP1, and XRCC6, which function in base excision repair, nucleotide excision repair, mismatch repair, single-strand break repair and double-strand break repair, respectively, were assessed using immunohistochemistry in ulcerative colitis and sporadic colorectal cancer biopsies. Levels of oxidative/ nitrosative stress biomarkers were also assessed. RESULTS: Ulcerative colitis and colorectal cancer lesions expressed significantly higher levels of all DNA repair proteins and oxidative/ nitrosative stress biomarkers compared to normal colonic mucosa. Ulcerative colitis had the highest XPA and XRCC6 expression. CONCLUSION: Oxidative/nitrosative stress is prevalent in the colon of both diseases. Nucleotide excision repair and non-homologous end-joining double-strand break repair may be compromised in colorectal cancer, but not in ulcerative colitis.
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Colitis Ulcerosa/genética , Neoplasias Colorrectales/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , ADN/genética , Estrés Nitrosativo/genética , Estrés Oxidativo/genética , Biomarcadores de Tumor/genética , Colitis Ulcerosa/patología , Colon/patología , Neoplasias Colorrectales/patología , Daño del ADN/genética , Humanos , Mucosa Intestinal/patología , Oxidación-ReducciónRESUMEN
BACKGROUND: Keratoacanthoma (KA) has a unique life cycle of rapid growth and spontaneous regression that shows similarities to the hair follicle cycle, which involves an active Wnt signaling during physiological regeneration. We analyzed the expression of the Wnt signaling proteins ß-catenin, Lef1, Sox9, and Cyclin D1 in young and old human KAs to investigate a possible role for Wnt signaling in KAs. AIM: To investigate the role of the Wnt/ß-catenin signaling pathway in human KAs. METHODS AND RESULTS: Formalin-fixed, paraffin-embedded tissue samples of 67 KAs were analyzed for protein expression using immunohistochemistry. The majority of KAs were positive for Sox9 and Cyclin D1 but not for nuclear-localized ß-catenin or Lef-1. No significant differences in protein expressions were seen between young and old KAs. However, we found a significant association between Ki67 and Cyclin D1 proteins (P= .008). CONCLUSIONS: The Wnt signaling pathway does not appear to play a significant role in the biogenesis of human KA. Sox9 overexpression may be indicative of inhibition of Wnt signaling. Sox-9 and Cyclin D1 are proliferation markers that are most likely transactivated by alternate signaling pathways.
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Queratoacantoma/etiología , Vía de Señalización Wnt/fisiología , Ciclina D1/análisis , Humanos , Queratoacantoma/metabolismo , Queratoacantoma/patología , Antígeno Ki-67/análisis , Factor de Unión 1 al Potenciador Linfoide/análisis , Factor de Transcripción SOX9/análisis , beta Catenina/análisisRESUMEN
Background: The colon and rectum are continuously exposed to oxidative stress that generates reactive oxygen species, which are a major cause of DNA double-strand breaks (DSB). Furthermore, chronic inflammatory diseases such as ulcerative colitis (UC) are characterized by an excess of reactive nitrogen species that can also lead to DNA double-strand breakage and genomic instability. We investigated the expression of the nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) protein in UC and sporadic colorectal cancer (CRC) due to its involvement in both DNA double-strand break repair and inflammatory signaling. Methods: NUCKS1 expression and expression of the DNA double-strand break marker gamma-H2AX (γH2AX) were assessed in formalin-fixed, paraffin-embedded UC and CRC patient biopsies using peroxidase immunohistochemistry. Expression levels for both proteins were evaluated together with previously published expression-level data for hTERT and TP53 proteins in the same material. Results: Nondysplastic UC lesions had 10-fold lower γH2AX expression and approximately 4-fold higher NUCKS1 expression compared with sporadic CRC, indicating minimal DNA DSB damage and heightened DNA DSB repair in these lesions, respectively. NUCKS1 expression in UC tended to decrease with increasing grades of dysplasia, whereas γH2AX, hTERT, and TP53 expression tended to increase with increasing grades of dysplasia. The highest γH2AX expression was seen in sporadic CRC, indicating considerable DNA DSB damage, whereas the highest NUCKS1 expression and hTERT expression were seen in nondysplastic UC. Conclusions: Overall, our data suggest that NUCKS1 may be involved in DNA DSB repair and/or inflammatory signaling in UC, but a more thorough investigation of both pathways in UC is warranted.
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Colitis Ulcerosa/metabolismo , Neoplasias Colorrectales/metabolismo , Roturas del ADN de Doble Cadena , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Anciano , Colitis Ulcerosa/genética , Colitis Ulcerosa/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Femenino , Marcadores Genéticos , Inestabilidad Genómica , Histonas/genética , Humanos , Inmunohistoquímica , Masculino , Proteínas Nucleares/genética , Fosfoproteínas/genética , Telomerasa/genética , Telomerasa/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Data are limited on oncogene mutation frequencies in polyps from principally asymptomatic participants of population-based colorectal cancer screening studies. In this study, DNA from 204 polyps, 5 mm or larger, were collected from 176 participants of the NORCCAP screening study and analyzed for mutations in KRAS, BRAF, and PIK3CA including the rarely studied KRAS exons 3 and 4 mutations. KRAS mutations were identified in 23.0% of the lesions and were significantly associated with tubulovillous adenomas and large size. A significantly higher frequency of KRAS mutations in females was associated with mutations in codon 12. The KRAS exon 3 and 4 mutations constituted 23.4% of the KRAS positive lesions, which is a larger proportion compared to previous observations in colorectal cancer. BRAF mutations were identified in 11.3% and were associated with serrated polyps. None of the individuals were diagnosed with de novo or recurrent colorectal cancer during the follow-up time (median 11.2 years). Revealing differences in mutation-spectra according to gender and stages in tumorigenesis might be important for optimal use of oncogenes as therapeutic targets and biomarkers.
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BACKGROUND: Keratoacanthoma (KA) is a common keratinocytic skin neoplasm that typically develops rapidly and undergoes complete spontaneous regression. As the pro-apoptotic p53 protein may be involved in the lifecycle of KA, we studied the p53 status throughout the main stages of KA that include proliferation, maturation and regression in a large series of lesions. METHODS: One-hundred and twenty-four KAs were characterized with respect to age of the lesions both clinically and histopathologically, in addition to phenotypic characteristics such as cellular atypia, infiltration, inflammation and fibrosis. Tp53 mutations were detected by capillary electrophoresis, and p53 protein levels were assessed by immunohistochemistry. RESULTS: Tp53 mutations were detected in 49 cases (39.5%) and were associated with high p53 protein levels (p = 0.007) and histopathologic age of the lesions (p = 0.044). Significant association was also seen between high p53 protein levels and atypia (p = 0.036), whereas the association with infiltration showed borderline significance (p = 0.057). High p53 protein levels were significantly associated with gene mutations in transplanted, but not in non-transplanted patients. CONCLUSION: We show a high frequency of Tp53 mutations in KAs that is associated with increased p53 levels. The results indicate a role for the p53 protein in KA development.
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Queratoacantoma/patología , Neoplasias Cutáneas/patología , Proteína p53 Supresora de Tumor/biosíntesis , Adulto , Anciano , Análisis Mutacional de ADN , Electroforesis Capilar , Femenino , Humanos , Inmunohistoquímica , Queratoacantoma/genética , Queratoacantoma/metabolismo , Masculino , Persona de Mediana Edad , Mutación , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Proteína p53 Supresora de Tumor/análisis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: The glycerophospholipid lysophosphatidic acid (LPA), which is present in most tissues and in high concentrations in saliva, may exert profound effects on oral cancer cells. We have investigated mitogenic signalling induced by LPA in the two oral carcinoma cell lines, D2 and E10, focusing on the role of EGFR transactivation and downstream pathways. METHODS: Two oral squamous carcinoma cell lines, D2 and E10, were analysed for effects of LPA on signalling pathways and induction of DNA synthesis. Pathway activation was investigated by examining phosphorylation of signalling proteins and by the use of specific pathway inhibitors. RESULTS: The D2 cells had higher levels of activated signalling proteins and higher DNA synthesis activity in the basal condition than E10 cells. EGF did not induce proliferation in D2 cells, whereas LPA induced proliferation in both cell lines, by mechanisms depending on EGFR transactivation. Release of EGFR ligands was involved in basal and LPA-induced proliferation in both D2 and E10 cells. The proliferation in D2 cells was dependent on the PI3K/Akt pathway, but not the MEK/ERK pathway. In E10 cells, the PI3K/Akt, MEK/ERK and p38 pathways were all involved in the proliferation. CONCLUSION: Transactivation of EGFR is required for LPA-induced DNA synthesis in D2 and E10 cells. Our results also show that although proliferation of oral carcinoma cells is regulated by several pathways, and differentially in E10 and D2 cells, the PI3K pathway has a crucial role in both cell lines.
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Carcinoma de Células Escamosas/metabolismo , ADN de Neoplasias/biosíntesis , Neoplasias de Cabeza y Cuello/metabolismo , Lisofosfolípidos/farmacología , Neoplasias de la Boca/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Replicación del ADN/fisiología , ADN de Neoplasias/genética , Activación Enzimática , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Redes y Vías Metabólicas/efectos de los fármacos , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Fosforilación , Carcinoma de Células Escamosas de Cabeza y Cuello , Activación Transcripcional/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Aneuploidy is a common feature in the colonic mucosa of patients suffering from the inflammatory bowel disease ulcerative colitis (UC) and often precedes the development of dysplasia and cancer. Aneuploidy is assumed to be caused by missegregation of chromosomes during mitosis, often due to a faulty spindle assembly checkpoint. p53 is a tumour suppressor protein known to regulate the spindle assembly checkpoint and is frequently mutated in aneuploid cells. Aurora A is a presumed oncoprotein, also involved in regulation of the spindle assembly checkpoint. In the present study, we examined the mutational frequency of TP53 and the protein levels of p53 in a set of 20 progressor and 10 non-progressor colectomies from patients suffering from longstanding UC. In addition, we re-examined previously published immunohistochemical data on Aurora A expression using the same material. Levels of Aurora A were re-examined with regard to DNA ploidy status and dysplasia within the progressors, as well as in relation to p53 accumulation and TP53 mutational status. We detected p53 accumulation only within the progressor colectomies, where it could be followed back 14 years prior to the colectomies, in pre-colectomy biopsies. TP53 mutations were detected in both progressors and non-progressors. Expression levels of Aurora A were similar in the progressors and non-progressors. Within the group of progressors however, low levels of Aurora A were associated with areas of DNA aneuploidy, as well as with increasing degrees of dysplasia. Our results indicate that alterations in p53 may be an early biomarker of a progressor colon, and that p53 is accumulated early in UC-related carcinogenesis. Furthermore, a decreased Aurora A expression is associated with the development of DNA aneuploidy, as well as with dysplasia in UC progressors.
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Aurora Quinasa A/genética , Colitis Ulcerosa/genética , Colitis Ulcerosa/patología , Expresión Génica , Mutación , Proteína p53 Supresora de Tumor/genética , Adolescente , Adulto , Aurora Quinasa A/metabolismo , Carcinogénesis/genética , Niño , Colectomía , Colitis Ulcerosa/cirugía , Análisis Mutacional de ADN , Progresión de la Enfermedad , Exones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ploidias , Proteína p53 Supresora de Tumor/metabolismo , Adulto JovenRESUMEN
Longstanding ulcerative colitis (UC) is a disease of chronic inflammation of the colon. It is associated with the development of colorectal cancer through a multistep process including increasing degrees of dysplasia and DNA-ploidy changes. However, not all UC patients will develop these characteristics even during lifelong disease, and patients may therefore be divided into progressors who develop dysplasia or cancer, and non-progressors who do not exhibit such changes. In the present study, the amount of hTERT, the catalytic subunit of the enzyme telomerase, was estimated by using peroxidase immunohistochemistry (IHC) in a set of progressor and non-progressor UC colectomies. The protein levels in the colonic mucosa of the progressors and nonprogressors were compared, and further comparisons between different categories of dysplastic development and to DNA-ploidy status within the progressors were made. Levels of hTERT were elevated in the colonic mucosa of the progressors and non-progressors when compared to non-UC control samples, but no difference was observed between the hTERT levels in the mucosa of progressors and non-progressors. The levels of hTERT associated with levels of Ki67 to a significant degree within the non-progressors. hTERT expression in lesions with DNA-aneuploidy were decreased as compared to diploid lesions, when stratified for different classes of colonic morphology. Our results indicate an association between hTERT protein expression and aneuploidy in UC-progressor colons, and also a possible protective mechanism in the association between hTERT and Ki67, against development of malignant features within the mucosa of a UC-colon.
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Colitis Ulcerosa/metabolismo , Colon/metabolismo , Mucosa Intestinal/metabolismo , Telomerasa/metabolismo , Adolescente , Adulto , Aneuploidia , Niño , Neoplasias del Colon/metabolismo , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Telomerasa/genética , Adulto JovenRESUMEN
BACKGROUND: Ulcerative colitis (UC) is a chronic, inflammatory bowel disease which may lead to dysplasia and adenocarcinoma in patients when long-lasting. Short telomeres have been reported in mucosal cells of UC patients. Telomeres are repetitive base sequences capping the ends of linear chromosomes, and protect them from erosion and subsequent wrongful recombination and end-to-end joining during cell division. Short telomeres are associated with the development of chromosomal instability and aneuploidy, the latter being risk factors for development of dysplasia and cancer. Specifically, the abrupt shortening of one or more telomeres to a critical length, rather than bulk shortening of telomeres, seems to be associated with chromosomal instability. METHODS: We investigated possible associations between dysplasia, aneuploidy and telomere status in a total of eight lesions from each of ten progressors and four nonprogressors suffering from longstanding UC. We have analyzed mean telomere length by qPCR, as well as the amount of ultra-short telomeres by the Universal STELA method. RESULTS: An increased amount of ultra-short telomeres, as well as general shortening of mean telomere length are significantly associated with dysplasia in longstanding UC. Furthermore, levels of ultra-short telomeres are also significantly increased in progressors (colons harbouring cancer/dysplasia and/or aneuploidy) compared to nonprogressors (without cancer/dysplasia/aneuploidy), whereas general shortening of telomeres did not show such associations. CONCLUSIONS: Our data suggest that ultra-short telomeres may be more tightly linked to colorectal carcinogenesis through development of dysplasia in UC than general telomere shortening. Telomere status was not seen to associate with DNA aneuploidy.
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Adenocarcinoma/genética , Aneuploidia , Transformación Celular Neoplásica/genética , Colitis Ulcerosa/genética , Neoplasias del Colon/genética , ADN/análisis , Acortamiento del Telómero , Adenocarcinoma/química , Transformación Celular Neoplásica/patología , Inestabilidad Cromosómica , Colitis Ulcerosa/patología , Neoplasias del Colon/química , Diploidia , Progresión de la Enfermedad , Femenino , Humanos , Mucosa Intestinal/química , MasculinoRESUMEN
BACKGROUND: We previously reported that a pool of low molecular weight peptides can be extracted by alkali treatment of DNA preparations obtained from prokaryotic and eukaryotic cells after intensive deproteinization. This class of peptides, isolated from wheat bud chromatin, induces growth inhibition, DNA damage, G2 checkpoint activation and apoptosis in HeLa cells. In this work we studied their mechanism of action by investigating their ability to interfere with DNA synthesis. METHODS: BrdUrd comet assays were used to detect DNA replication defects during S phase. DNA synthesis, cell proliferation, cell cycle progression and DNA damage response pathway activation were assessed using 3H-thymidine incorporation, DNA flow cytometry and Western blotting, respectively. RESULTS: BrdUrd labelling close to DNA strand discontinuities (comet tails) detects the number of active replicons. This number was significantly higher in treated cells (compared to controls) from entry until mid S phase, but markedly lower in late S phase, indicating the occurrence of defective DNA synthesis. In mid S phase the treated cells showed less 3H-thymidine incorporation with respect to the controls, which supports an early arrest of DNA synthesis. DNA damage response activation was also shown in both p53-defective HeLa cells and p53-proficient U2OS cells by the detection of the phosphorylated form of H2AX after peptide treatment. These events were accompanied in both cell lines by an increase in p21 levels and, in U2OS cells, of phospho-p53 (Ser15) levels. At 24 h of recovery after peptide treatment the cell cycle phase distribution was similar to that seen in controls and CDK1 kinase accumulation was not detected. CONCLUSION: The data reported here show that the antiproliferative effect exhibited by these chromatin peptides results from their ability to induce genomic stress during DNA synthesis. This effect seems to be S-phase specific since surviving cells are able to progress through their normal cell cycle when the peptide fraction is removed from the culture medium. It is likely that the subsequent apoptosis is a consequence of the failed attempt of the tumour cells to repair the DNA damage induced by the peptides.
RESUMEN
We studied patterns of DNA damage signaling and cell cycle response to clinically-relevant (bolus) and high doses of 5-fluorouracil (5-FU) in three colorectal cancer cell lines with differing MMR and TP53 status in an attempt to better understand how 5-FU exerts its cytotoxicity. The ATM/CHEK2/ CHEK1 signaling pathway was not activated in response to bolus 5-FU in the MMR-deficient cell lines HCT116 (TP53-proficient or TP53-depleted) and HCT15 (TP53-deficient), consistent with negligible/reparable DNA damage and no cell death. The pattern of DNA damage checkpoint activation in bolus 5-FU-treated HT29 (TP53-deficient/MMR-proficient) cultures suggested SSB formation (CHEK1 activation) followed by DSB formation (CHEK2 activation and increased phospho-H2AX levels), but no cell death suggested that DNA repair capacity was not overwhelmed. High-dose 5-FU treatment led to activation of ATM/CHEK2/TP53 (not CHEK1) in TP53-proficient and TP53-depleted HCT116 (later CHEK2 activation relative to TP53-proficient) cultures; HCT15 cultures had ATM activation only. These data and increased phospho-H2AX levels indicated DSB formation; apoptosis was induced in both cell lines indicating irreparable DNA damage. TP53-depleted HCT116 cultures also had DSBs after high-dose 5-FU treatment but experienced a (transient) G1/S cell cycle arrest that protected them from apoptosis. TP53 phosphorylation at Ser20/33/37 was seen in TP53-proficient HCT116 cultures regardless of 5-FU concentration at ≥4 h following treatment, indicating TP53 stabilization/transcriptional activation. Overall, activation of ATM, CHEK1 and/or CHEK2 and phospho-H2AX levels reflected the nature of 5-FU-induced DNA damage and indi-cated when DNA damage was significant (5-FU-dose-dependent). DNA repair and cell cycle responses to 5-FU-induced DNA damage were distinctly affected by MMR and TP53 (role in BER/NER) functionalities, but MMR deficiency especially seemed to confer less overall sensitivity to 5-FU.
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Antimetabolitos Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Daño del ADN , Reparación de la Incompatibilidad de ADN , Fluorouracilo/farmacología , Ciclo Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Genes p53 , Células HCT116 , Células HT29 , Humanos , Masculino , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
Cell cycle checkpoints ensure that eukaryotic cells do not enter mitosis after ionizing irradiation (IR). The G(2)-arrest after IR is the result of activation of multiple signalling pathways, the contributions of which vary with time after irradiation. We have studied the time evolution of the IR-induced G(2)-arrest in human B-lymphocyte cancer cell lines, as well as the molecular mechanisms responsible for the arrest. Cells that were in G(2) phase at the time of irradiation experienced a transient arrest that blocked entry into mitosis at 0-2 hours after IR (0.5 or 4 Gy). Activation of ATM and CHEK2 occurred at the same time as this early arrest and was, like the arrest, abrogated by the ATM-inhibitor KU-55933. A late, permanent and ATM-independent arrest (≥6 hours after IR) of cells that were in G(2)/S/G(1) at the time of irradiation (4 Gy) was inactivated by caffeine. This late G(2)-arrest could not be explained by down-regulation of genes with functions in G(2)/mitosis (e.g. PLK1, CCNB1/2), since the down-regulation was transient and not accompanied by reduced protein levels. However, the persistent phosphorylation of CHEK1 after 4 Gy suggested a role for CHEK1 in the late arrest, consistent with the abrogation of the arrest in CHEK1-depleted cells. TP53 was not necessary for the late G(2)-arrest, but mediated an intermediate arrest (2-10 hours after IR) independently of ATM and CHEK1. In conclusion, the IR-induced arrest in G(2) is mediated by ATM immediately after irradiation, with TP53 for independent and transient back-up, while CHEK1 is necessary for the late arrest.
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Fase G2/efectos de la radiación , Radiación Ionizante , Proteínas de la Ataxia Telangiectasia Mutada , Cafeína/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2 , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Humanos , Mitosis , Morfolinas/farmacología , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Pironas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: Spindle checkpoint proteins such as Mad2 and BubR1 are important for chromosome segregation during mitosis. The aim of the present study was to examine their possible impact on prognosis in tonsillar carcinomas and their relation to clinical variables, the prevalence of human papillomavirus (HPV), p53 status, and Ki-67 positivity. METHODS: We examined the expression of Mad2 and BubR1 by immunohistochemistry on tissue microarrays from 105 patients with tonsillar carcinomas. RESULTS: BubR1 and Mad2 were both expressed in tonsillar carcinomas. Expression of BubR1 was a significant prognostic factor in univariate survival analysis. In multivariate analyses, BubR1 was a significant prognostic factor together with stage, age, and HPV status p < .01), whereas Mad2 did not show any significant correlations. CONCLUSION: We have shown that BubR1 expression is a novel and strong prognostic factor in tonsillar carcinomas, giving additional information to the TNM stage and other known prognostic factors.
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Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidad , Proteínas Serina-Treonina Quinasas/metabolismo , Neoplasias Tonsilares/metabolismo , Neoplasias Tonsilares/mortalidad , Factores de Edad , Proteínas de Unión al Calcio/metabolismo , Carcinoma de Células Escamosas/patología , Proteínas de Ciclo Celular/metabolismo , Femenino , Humanos , Inmunohistoquímica , Proteínas Mad2 , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Infecciones por Papillomavirus/epidemiología , Pronóstico , Proteínas Represoras/metabolismo , Neoplasias Tonsilares/patologíaRESUMEN
CONCLUSIONS: This study confirms a high prevalence of human papillomavirus (HPV)-positive tonsillar tumours (52%). The survival of the HPV-positive group was significantly better in males. OBJECTIVES: We assessed the prevalence of HPV in 137 patients with tonsillar carcinomas, measured the p53- and Ki-67-positive tumour cell fractions and correlated the results with clinical variables. PATIENTS AND METHODS: Tumour DNA from patients with squamous cell carcinoma of the tonsillar region was amplified by PCR and sequenced for detection of HPV subtypes. RESULTS: HPV was found in 71/137 (52%) of the tumours; HPV-16 was the most frequent subtype (87%). HPV positivity did not correlate with gender, stage, T- and N categories, Ki-67 expression or p53 positivity. The HPV-positive group had a significantly better survival (p < 0.01) compared with the HPV-negative group in males. In a multivariate analysis HPV status gave prognostic information in addition to the earlier established factors, i.e. age, gender and stage (p < 0.05).
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Carcinoma de Células Escamosas/epidemiología , Carcinoma de Células Escamosas/virología , Papillomavirus Humano 16/aislamiento & purificación , Papiloma/epidemiología , Papiloma/virología , Neoplasias Tonsilares/epidemiología , Neoplasias Tonsilares/virología , Anciano , Biopsia , Carcinoma de Células Escamosas/genética , Femenino , Genes p53/genética , Humanos , Inmunohistoquímica , Incidencia , Antígeno Ki-67/genética , Masculino , Persona de Mediana Edad , Noruega/epidemiología , Tonsila Palatina/patología , Tonsila Palatina/virología , Papiloma/genética , Prevalencia , Pronóstico , Neoplasias Tonsilares/genéticaRESUMEN
BACKGROUND: Defective expression of genes involved in mitotic chromosome segregation (e.g. AURKA, BUB1B), DNA damage response (e.g. TP53, BRCA1), and telomere function (e.g. TERT) may play a role in the development of tumor aneuploidy. MATERIALS AND METHODS: The levels of TP53, BRCA1 and TERT were assessed in 55 sporadic colorectal tumors and 37 normal mucosas using tissue microarrays and immunohistochemical detection, and their associations with DNA aneuploidy, levels of mitotic spindle proteins AURKA, AURKB, MAD2L1 and BUB1B and clinicopathological parameters were investigated. RESULTS: DNA aneuploidy was associated only with TP53 alterations. BRCA1 expression in tumors was significantly correlated with individual mitotic spindle protein expressions, and TERT and MAD2L1 expressions were moderately correlated in the tumor group, suggesting a putative role for TERT in MAD2L1 regulation. CONCLUSION: Loss of TP53 function appears to be involved in the development of aneuploidy, but not in the deregulation of mitotic spindle protein function.
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Aneuploidia , Proteína BRCA1/biosíntesis , Neoplasias Colorrectales/genética , Telomerasa/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Aurora Quinasa A , Aurora Quinasa B , Aurora Quinasas , Proteína BRCA1/genética , Biopsia , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Humanos , Inmunohistoquímica , Proteínas Mad2 , Masculino , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Telomerasa/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: Deproteinized DNA from eukaryotic and prokaryotic cells still contains a low-molecular weight peptidic fraction which can be dissociated by alkalinization of the medium. This fraction inhibits RNA transcription and tumor cell growth. Removal from DNA of normal cells causes amplification of DNA template activity. This effect is lower or absent in several cancer cell lines. Likewise, the amount of active peptides in cancer cell DNA extracts is lower than in DNA preparation of the corresponding normal cells. Such evidence, and their ubiquitous presence, suggests that they are a regulatory, conserved factor involved in the control of normal cell growth and gene expression. RESULTS: We report that peptides extracted from wheat bud chromatin induce growth inhibition, G2 arrest and caspase-dependent apoptosis in HeLa cells. The growth rate is decreased in cells treated during the S phase only and it is accompanied by DNA damage and DNA synthesis inhibition. In G2 cells, this treatment induces inactivation of the CDK1-cyclin B1 complex and an increase of active chk1 kinase expression. CONCLUSION: The data indicate that the chromatin peptidic pool inhibits HeLa cell growth by causing defective DNA replication which, in turn, arrests cell cycle progression to mitosis via G2 checkpoint pathway activation.
Asunto(s)
Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proteínas de Unión al ADN/farmacología , Fase G2/efectos de los fármacos , Péptidos/farmacología , Proteínas de Plantas/farmacología , Triticum/química , Proteína Quinasa CDC2/metabolismo , Procesos de Crecimiento Celular/efectos de los fármacos , Cromatina/metabolismo , Ciclina B/metabolismo , Ciclina B1 , Daño del ADN/efectos de los fármacos , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Fase S/efectos de los fármacosRESUMEN
The cellular response to chemoradiotherapy was investigated in cells of the HCT116 (wild-type TP53) and HT29 (mutated TP53) human colorectal cancer cell lines to better understand how the chemotherapeutic agent 5-fluorouracil (5-FU) acts as a radiosensitizer in vitro and how it contributes to the well-documented greater efficacy of chemoradiotherapy compared to radiotherapy (or chemotherapy) alone. A bolus 5-FU treatment protocol that simulated actual clinical clearance kinetics was used with a radiation dose given within 90 min after drug addition. The involvements of key signaling pathways (DNA damage response, cell cycle progression, cell proliferation, cell death) in cell responses were investigated concurrently, allowing for direct correlations of numerous treatment response phenotypes. Early DNA damage response, substantial cell death, loss of clonogenicity, and senescence characterized both radiotherapy- and chemoradiotherapy-treated cultures but not chemotherapy-treated cultures. The largest G(2)/M arrests and strongest correlation of senescence with non-clonogenicity were seen in radiotherapy- and chemoradiotherapy-treated HCT116 cell cultures, suggesting that functional TP53 could play a role in maintaining/inducing these cellular phenotypes. Overall, chemoradiotherapy proved to be the most effective treatment modality since it resulted in the strongest growth inhibitions, largest G(2)/M arrests, largest fractions of senescent cells, and complete loss of clonogenicity in both cell lines.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/radioterapia , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Neoplasias Colorrectales/patología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Daño del ADN , Células HCT116 , Células HT29 , Histonas/metabolismo , Humanos , Mitosis/efectos de los fármacos , Mitosis/efectos de la radiación , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Interleukin (IL)-33 is a novel member of the IL-1 family of cytokines that promotes Th2 responses in lymphocytes as well as the activation of both mast cells and eosinophils via the ST2 receptor. Additionally, IL-33 has been proposed to act as a chromatin-associated transcriptional regulator in both endothelial cells of high endothelial venules and chronically inflamed vessels. Here we show that nuclear IL-33 is expressed in blood vessels of healthy tissues but down-regulated at the earliest onset of angiogenesis during wound healing; in addition, it is almost undetectable in human tumor vessels. Accordingly, IL-33 is induced when cultured endothelial cells reach confluence and stop proliferating but is lost when these cells begin to migrate. However, IL-33 expression was not induced by inhibiting cell cycle progression in subconfluent cultures and was not prevented by antibody-mediated inhibition of VE-cadherin. Conversely, IL-33 knockdown did not induce detectable changes in either expression levels or the cellular distribution of either VE-cadherin or CD31. However, activation of endothelial cell cultures with either tumor necrosis factor-alpha or vascular endothelial growth factor and subcutaneous injection of these cytokines led to a down-regulation of vascular IL-33, a response consistent with both its rapid down-regulation in wound healing and loss in tumor endothelium. In conclusion, we speculate that the proposed transcriptional repressor function of IL-33 may be involved in the control of endothelial cell activation.
Asunto(s)
Núcleo Celular/metabolismo , Endotelio/metabolismo , Inflamación/metabolismo , Interleucinas/metabolismo , Neovascularización Patológica/metabolismo , Animales , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/metabolismo , Recuento de Células , Movimiento Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Células Cultivadas , Citocinas/farmacología , Regulación hacia Abajo/efectos de los fármacos , Endotelio/efectos de los fármacos , Endotelio/patología , Femenino , Salud , Humanos , Interleucina-33 , Neoplasias/metabolismo , Neoplasias/patología , Ratas , Factor de Necrosis Tumoral alfa/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
BACKGROUND: Use of irinotecan (CPT-11) as second-line therapy for metastatic colorectal cancer has shown some promise in cases where 5-fluorouracil (5-FU) has failed. Cross-resistance to both drugs may however be a potential clinical problem. The cellular response to CPT-11 was investigated in two human colon cancer cell lines that demonstrate a differential response to 5-FU. MATERIALS AND METHODS: Cell cycle progression, clonogenic survival, DNA damage checkpoint activation, apoptosis induction and senescence development were assessed during 48 hours of treatment and 72 hours of recovery. RESULTS: Both cell lines had similar cellular response patterns to CPT-11. Growth inhibition, loss of clonogenicity, ataxia telangiectasia mutated (ATM) activation, H2AX phosphorylation, TP53 stabilization, CDKN1A induction, G2/M arrests, endoreduplication, negligible cell death and appearance of a senescence-associated beta-galactosidase phenotype were observed. CONCLUSION: Cross-resistance to 5-FU and CPT-11 was not demonstrated. The appearance of a senescence phenotype in response to CPT-11 treatment may have potential clinical relevance for treatment regimens.
Asunto(s)
Camptotecina/análogos & derivados , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/genética , Daño del ADN/efectos de los fármacos , Resistencia a Antineoplásicos , Fluorouracilo/farmacología , Apoptosis/efectos de los fármacos , Camptotecina/farmacología , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Células HCT116 , Humanos , IrinotecánRESUMEN
Loss of TP53 function may contribute to 5-fluorouracil (5-FU) resistance in colorectal cancer since TP53-deficient cells may be unable to undergo apoptosis in response to 5-FU-induced DNA damage. 5-FU treatment of TP53-deficient cells would provide useful information on the apoptotic response to drug-induced DNA damage in the absence of TP53 and its transcriptional targets. We investigated apoptosis induction and cell cycle alterations in response to short-term treatment with two different 5-FU concentrations following siRNA-mediated knockdown of TP53 in the TP53-proficient HCT116 colon cancer cell line. We focused on high-dose 5-FU treatment to investigate the apoptotic phenotype in 5-FU-treated cultures since this dose resulted in apoptosis induction at 24 h of treatment, whereas clinically-relevant bolus 5-FU treatment of HCT116 cultures did not. Gene expression alterations were also assessed in 5-FU-treated HCT116 cultures using whole genome expression arrays. Compared to 5-FU-treated TP53-proficient HCT116 cultures, 5-FU-treated TP53-depleted HCT116 cultures showed lack of CDKN1A induction, decreased apoptotic levels, decreased FAS and TNFRSF10B transcript levels and cleaved PARP protein levels, G1/S transition arrests, decreased CCND1 protein levels, and smaller intra-S phase arrests. Alterations in gene expression in 5-FU-treated TP53-depleted HCT116 cultures confirmed previously-reported TP53 target genes and suggested potentially novel TP53 target genes (e.g. APOBEC3C, BIRC3, JMJD2B, LAMP3, MYO1E, PRRG1, SULF2, TACSTD2, TncRNA, ZFYVE20) that may play a role in mediating the 5-FU-induced DNA damage response in TP53-proficient cells. Abrogation of TP53 function in 5-FU-treated HCT116 cultures results in reduced apoptosis, TP53- and CDKN1A-independent G1/S phase arrests that may be protective against apoptosis, smaller intra-S phase arrests, and transcript level decreases of both reported TP53 target genes as well as potentially novel TP53 target genes.