RESUMEN

Cancer cells are exposed to major compressive and shearing forces during invasion and metastasis, leading to extensive plasma membrane damage. To survive this mechanical stress, they need to repair membrane injury efficiently. Targeting the membrane repair machinery is thus potentially a new way to prevent invasion and metastasis. We show here that annexin-A2 (ANXA2) is required for membrane repair in invasive breast and pancreatic cancer cells. Mechanistically, we show by fluorescence and electron microscopy that cells fail to reseal shear-stress damaged membrane when ANXA2 is silenced or the protein is inhibited with neutralizing antibody. Silencing of ANXA2 has no effect on proliferation in vitro, and may even accelerate migration in wound healing assays, but reduces tumor cell dissemination in both mice and zebrafish. We expect that inhibiting membrane repair will be particularly effective in aggressive, poor prognosis tumors because they rely on the membrane repair machinery to survive membrane damage during tumor invasion and metastasis. This could be achieved either with anti-ANXA2 antibodies, which have been shown to inhibit metastasis of breast and pancreatic cancer cells, or with small molecule drugs.

Asunto(s)

Proteínas de la Membrana , Neoplasias Pancreáticas , Animales , Ratones , Línea Celular Tumoral , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias Pancreáticas/patología , Pez Cebra

RESUMEN

Oncolytic viruses are regulated by the tumor phenotype to replicate and lyse cancer cells selectively. To identify optimal strategies for breast cancer we compared five adenoviruses with distinct regulatory mechanisms: Ad-dl922-947 (targets G1-S checkpoint); Ad-Onyx-015 and Ad-Onyx-017 (target p53/mRNA export); Ad-vKH1 (targets Wnt pathway), and AdEHE2F (targets estrogen receptor/G1-S checkpoint/hypoxic signaling). The quantity of virus required to kill 50% of breast cancer cells after 6 days (EC(50), plaque-forming units per cell) was measured. The most potent virus was Ad-dl922-947 (EC(50), 0.01-5.4 in SkBr3, MDA-231, MDA-468, MCF7, and ZR75.1 cells), followed by wild-type (Ad-WT; EC(50), 0.3-5.5) and AdEHE2F (EC(50), 1.4-3.9). Ad-vKH1 (EC(50), 7.2-72.1), Ad-Onyx-017 (EC(50), 8.4-167), and Ad-Onyx-015 (EC(50), 17.7-377) showed less activity. Most viruses showed limited cytotoxicity in normal human cells, including breast epithelium MCF10A (EC(50), >722) and fibroblasts (EC(50), >192) and only moderate cytotoxicity in normal microvascular endothelial cells (HMVECs; EC(50), 42.8-149), except Ad-dl922-947, which was active in HMVECs (EC(50), 1.6). After injection into MDA-231 xenografts, Ad-WT, AdEHE2F, and Ad-dl922-947 showed replication, assessed by hexon staining and quantitative polymerase chain reaction measurement of viral DNA, and significantly inhibited tumor growth, leading to extended survival. After intravenous injection Ad-dl922-947 showed DNA replication (233% of the injected dose was measured in liver after 3 days) whereas AdEHE2F did not. Overall, AdEHE2F showed the best combination of low toxicity in normal cells and high activity in breast cancer in vitro and in vivo, suggesting that molecular targeting using estrogen response elements, hypoxia response elements, and a dysregulated G1-S checkpoint is a promising strategy for virotherapy of breast cancer.

Asunto(s)

Adenovirus Humanos/genética , Neoplasias de la Mama/terapia , Viroterapia Oncolítica/métodos , Virus Oncolíticos/genética , Adenovirus Humanos/fisiología , Animales , Secuencia de Bases , Neoplasias de la Mama/patología , Neoplasias de la Mama/virología , Ciclo Celular , Muerte Celular , Línea Celular , Línea Celular Tumoral , Cartilla de ADN/genética , Femenino , Humanos , Ratones , Ratones SCID , Trasplante de Neoplasias , Virus Oncolíticos/fisiología , Trasplante Heterólogo , Replicación Viral

RESUMEN

Oncolytic adenoviruses have shown some promise in cancer gene therapy. However, their efficacy in clinical trials is often limited, and additional therapeutic interventions have been proposed to increase their efficacies. In this context, molecular imaging of viral spread in tumours could provide unique information to rationalize the timing of these combinations. Here, we use the human sodium iodide symporter (hNIS) as a reporter gene in wild-type and replication-selective adenoviruses. By design, hNIS cDNA is positioned in the E3 region in a wild-type adenovirus type 5 (AdIP1) and in an adenovirus in which a promoter from the human telomerase gene (RNA component) drives E1 expression (AdAM6). Viruses show functional hNIS expression and replication in vitro and kinetics of spread of the different viruses in tumour xenografts are visualized in vivo using a small animal nano-SPECT/CT camera. The time required to reach maximal spread is 48 h for AdIP1 and 72 h for AdAM6 suggesting that genetic engineering of adenoviruses can affect their kinetics of spread in tumours. Considering that this methodology is potentially clinically applicable, we conclude that hNIS-mediated imaging of viral spread in tumours may be an important tool for combined anticancer therapies involving replicating adenoviruses

Asunto(s)

Neoplasias del Colon/diagnóstico por imagen , Neoplasias del Colon/terapia , Genes Reporteros , Terapia Genética/métodos , Viroterapia Oncolítica/métodos , Simportadores/genética , Tomografía Computarizada de Emisión de Fotón Único , Adenoviridae/genética , Infecciones por Adenoviridae/diagnóstico por imagen , Animales , Neoplasias del Colon/virología , Expresión Génica , Humanos , Inyecciones Intralesiones , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Transducción Genética/métodos , Trasplante Heterólogo , Replicación Viral

RESUMEN

The Wnt signaling pathway is activated by mutations in the adenomatous polyposis coli (APC) or beta-catenin genes in most colon cancers, leading to the transactivation of promoters containing binding sites for the Tcf/LEF family of transcription factors. We have previously shown that it is possible to confer colon cancer specificity on autonomous parvoviruses by inserting Tcf sites into the viral P4 promoter. The mutant Tcf promoters were responsive to activation of the Wnt pathway but the viruses replicated poorly. We show here that reduction of the number of Tcf sites from four to two leads to an increase in the efficiency of replication and toxicity of the viruses in Co115 colon cancer cells, with only a small reduction in selectivity for cells with an active Wnt signaling pathway. Despite this improvement, virus production by most colon cancer cells remained low. Analysis of parental phH1 virus infection of SW480 colon cancer cells showed that the nonstructural and capsid proteins were expressed, but single stranded DNA and progeny virus were not produced. This defect reflects the dependence of autonomous parvoviruses on host functions for many steps in their replication cycle and represents a major limitation to the use of selectively replicating parvoviruses for colon cancer therapy.

Asunto(s)

Neoplasias del Colon/virología , Marcación de Gen , Parvovirus/fisiología , Regiones Promotoras Genéticas , Factores de Transcripción TCF/genética , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/virología , Sitios de Unión , Humanos , Parvovirus/patogenicidad , Células Tumorales Cultivadas/virología , Replicación Viral

RESUMEN

Clinical studies with oncolytic adenoviruses have shown that existing viruses are safe but lack efficacy. To selectively increase the toxicity of oncolytic adenoviruses targeting colon tumours, we have inserted the yeast cytosine deaminase gene (yCD) after the fibre gene in the major late transcript. yCD was expressed using either an internal ribosome entry site (IRES) or by alternative splicing of a new exon analogous to the Ad41 long fibre exon. The IRES-CD virus gave higher yCD expression on Western blots. Both approaches result in yCD expression restricted to the period after viral DNA replication. Viral burst size was reduced by less than approximately 10-fold by 5-fluorocytosine (5-FC), showing that expression of yCD as a late gene is compatible with virus replication. Cytopathic effect assays in colon cancer cell lines showed that both yCD viruses have approximately 10-fold increased toxicity in the presence of the prodrug 5-FC, which is converted to 5-fluorouracil (5-FU) by yCD. Toxicity was higher following addition of 5-FC immediately after infection. The largest gain in toxicity was seen in HT29 colon cancer cells, which are the least permissive colon cancer cells for the parental virus, indicating that the new 5-FC/yCD viruses may have broader applications for colon cancer therapy than their predecessors.

Asunto(s)

Neoplasias del Colon/terapia , Flucitosina/uso terapéutico , Terapia Genética/métodos , Profármacos/uso terapéutico , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Pez Cebra , Adenoviridae/genética , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Citosina Desaminasa/genética , Expresión Génica , Ingeniería Genética , Vectores Genéticos/administración & dosificación , Células HT29 , Humanos , Transcripción Genética , Replicación Viral , Proteínas Wnt

RESUMEN

The wnt signaling pathway is constitutively activated in colon tumors by mutations in the adenomatous polyposis coli and beta-catenin genes. We have modified the minute virus of mice (MVM) P4 promoter to make it responsive to wnt signaling by inserting binding sites for the heterodimeric beta-catenin/Tcf transcription factor. In luciferase assays we can see up to 20-fold selectivity of Tcf mutant P4 promoters for cells with activated wnt signaling. Hybrid MVM/H-1 viruses containing Tcf mutant promoters were tested for NS1 expression, viral DNA replication, virus replication, and cytopathic effect on colon, lung, kidney, and cervical cancer cell lines. Activation of the wnt pathway by expression of Delta N-beta-catenin increased NS1 expression and viral burst size in 293T and H1299 lung cancer cells, showing that the Tcf mutant P4 promoter can respond to wnt signals in the context of the virus. Compared to the parental virus, the burst size of the Tcf mutant viruses was reduced at least 1,000-fold in H1299, 293T, NB324K, and HeLa cells, which have inactive wnt signaling pathways. The burst size and cytopathic effect of the Tcf viruses was near wild-type levels in SW480 and Isreco1 colon cancer cell lines, which have high Tcf activity. The high specificity of these viruses should permit the development of H-1 virus-based vectors which combine high safety and greater efficacy in cancer therapy.

Asunto(s)

Neoplasias del Colon , Marcación de Gen/métodos , Vectores Genéticos , Parvovirus/fisiología , Replicación Viral , Proteínas de Pez Cebra , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/virología , Animales , Sitios de Unión , Neoplasias del Colon/genética , Neoplasias del Colon/virología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Humanos , Ratones , Virus Diminuto del Ratón/genética , Virus Diminuto del Ratón/fisiología , Mutación , Parvovirus/genética , Parvovirus/patogenicidad , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Proteínas Wnt , beta Catenina

RESUMEN

Mutation of the adenomatous polyposis coli and beta-catenin genes in colon cancer leads to constitutive activation of transcription from promoters containing binding sites for Tcf/LEF transcription factors. We have constructed adenoviruses with Tcf binding sites in the early promoters, in order to target viral replication to colon tumours. Tcf regulation of the E1A promoter confers a 100-fold selectivity for cells with activated wnt signalling in viral burst and cytopathic effect assays. p300 is a coactivator for beta-catenin, and E1A inhibits Tcf-dependent transcription through sequestration of p300, but mutation of the p300 binding site in E1A leads to a 10-fold reduction in cytopathic effect of all of the Tcf-regulated viruses. When Tcf sites are inserted in the E1A, E1B, E2 and E4 promoters the viruses show up to 100 000-fold selectivity for cells with activated wnt signalling.

Asunto(s)

Adenoviridae/genética , Neoplasias del Colon/genética , Regiones Promotoras Genéticas/genética , Transducción de Señal/genética , Transactivadores , Factores de Transcripción/genética , Poliposis Adenomatosa del Colon/genética , Adenoviridae/fisiología , Proteínas E1A de Adenovirus/genética , Proteínas del Citoesqueleto/genética , Marcación de Gen/métodos , Humanos , Células Tumorales Cultivadas , Replicación Viral , beta Catenina

RESUMEN

Assessment of the predictive value of p53 requires the testing of large numbers of samples from patients enrolled in prospective phase III clinical trials. The goal of this study was to determine whether p53 status can be determined by p53 yeast functional assay using the limiting amounts of material that can typically be obtained in prospective phase III trials (particularly when chemotherapy is given before surgery). All patients presenting with a clinically palpable tumour which could be considered large enough to perform a trucut biopsy (> or =2 cm breast tumour) were eligible for this study. Two trucut biopsies and one incisional biopsy were performed on the surgical specimens (mastectomy or tumourectomy). Samples were snap frozen and cryostat sections were taken for histology and p53 testing. Thirty patients were included. Three samples out of 90 failed to give any p53 PCR products, probably because these samples contained almost entirely fibrous tissue. Of the 87 samples that could be tested, the incisional and trucut biopsies results were fully concordant in every case. p53 could be defined in 97% of patients by double trucut biopsy. Eight out of 30 tumours tested were mutant for p53 (27%). p53 status can be reliably determined by yeast assay from single frozen sections of trucut biopsies. Histological examination before p53 testing is essential to exclude cases where the p53 result may reflect only the status of the normal cells in the biopsy.

Asunto(s)

Biomarcadores de Tumor/análisis , Neoplasias de la Mama/genética , ADN de Neoplasias/genética , Genes p53/genética , Proteína p53 Supresora de Tumor/biosíntesis , Levaduras/genética , Adulto , Bioensayo/métodos , Biopsia , Neoplasias de la Mama/patología , Criopreservación , Análisis Mutacional de ADN , Cartilla de ADN , Estudios de Factibilidad , Femenino , Mutación del Sistema de Lectura , Humanos , Mastectomía , Mutación Missense , Reacción en Cadena de la Polimerasa , Valor Predictivo de las Pruebas , Manejo de Especímenes

RESUMEN

p53 can adopt two forms in vitro, a latent form that binds naked DNA poorly and an active form that binds DNA well. Conversion of the latent form to the active form is thought to occur by an allosteric mechanism induced by phosphorylation and acetylation. Despite the large differences in affinity produced by regulatory modifications in vitro, mutation of putative regulatory sites has not produced correspondingly large effects on transcription of p53 target genes in vivo. To determine whether genotoxic stress regulates DNA binding by p53 in vivo, we have performed quantitative chromatin immunoprecipitation (ChIP) assays on tumor and normal cell lines containing wild-type p53. ChIP recovers several hundredfold more p21 and MDM2 promoter DNA from p53 wild-type than p53-null cells, indicating that the assay is specific for p53. Genotoxic stress induces much smaller increases in chromatin precipitation, which are matched by changes in the p53 protein level. Thus, in the experimental systems tested, allosteric regulation of DNA binding is not a major level of regulation of p53 activity. The p53 target genes tested can be divided into a group showing high promoter occupancy in vivo (p21, MDM2, and PUMA) and a group giving substantially weaker or background p53 binding (bax, AIP1, and PIG3). Neither group shows selective recruitment of p53 to the promoter in cells undergoing apoptosis, indicating that the decision to undergo apoptosis or cell cycle arrest depends on other changes in the cell.

Asunto(s)

Cromatina/química , Cromatina/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Sitio Alostérico , Apoptosis , Secuencia de Bases , ADN/metabolismo , Relación Dosis-Respuesta en la Radiación , Fibroblastos/metabolismo , Humanos , Datos de Secuencia Molecular , Fosforilación , Pruebas de Precipitina , Unión Proteica , ARN Mensajero/metabolismo , Transcripción Genética , Células Tumorales Cultivadas

RESUMEN

The human p53 protein acts mainly as a stress inducible transcription factor transactivating several genes involved in cell cycle arrest (e.g. p21) or apoptosis (e.g. Bax, PIG3). Roughly half of all human tumours contains p53 missense mutations. Virtually all tumour-derived p53 mutants are unable to activate Bax transcription but some retain the ability to activate p21 transcription. Identification of these mutants may have valuable clinical implications. We have determined the transactivation ability of 77 p53 mutants using reporter yeast strains containing a p53-regulated ADE2 gene whose promoter is regulated by p53 responsive elements derived from the regulatory region of the p21, Bax and PIG3 genes. We also assessed the influence of temperature on transactivation. Our results indicate that a significant proportion of mutants [16/77 (21%); 10/64 (16%) considering only tumour-derived mutants] are transcriptionally active, especially with the p21 promoter. Discriminant mutants preferentially affect less conserved (P<0.04, Fisher's exact test), more rarely mutated (P<0.006, Fisher's exact test) amino acids. Temperature sensitivity is frequently observed, but is more common among discriminant than non-discriminant mutants (P<0.003, Fisher's exact test). Finally, we extended the analysis to a group of mutants isolated in BRCA-associated tumours that surprisingly were indistinguishable from wild type in standard transcription, growth suppression and apoptosis assays in human cells, but showed gain of function in transformation assays. The incidence of transcriptionally active mutations among this group was significantly higher than in the panel of mutants studied previously (P<0.001, Fisher's exact test). Since it is not possible to predict the behaviour of a mutant from first principles, we propose that the yeast assay be used to compile a functional p53 database and fill the gap between the biophysical, pharmacological and clinical fields.

Asunto(s)

Ciclinas/genética , Mutación Missense , Regiones Promotoras Genéticas , Proteínas/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/genética , Activación Transcripcional , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Apoptosis , Sitios de Unión , Evolución Biológica , Carboxiliasas/genética , Ciclo Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Saccharomyces cerevisiae/genética , Temperatura , Transcripción Genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/química , Proteína X Asociada a bcl-2

RESUMEN

Several reports have noted epidemiological differences in the prevalence or prognostic significance of p53 mutants with arginine (R) or proline (P) at the codon 72 polymorphism (R72/P72) in certain cancer types, but the biological significance of these variants is unclear. The ability of p53 mutants to interact with and inactivate the p53 homolog p73 was recently reported to depend on the conformational state of the p53 protein and the residue at codon 72. Since the conformation of p53 mutants may influence their ability to transdominantly inhibit wild-type p53, we tested whether there was a correlation between the amino acid at codon 72 and the transdominance of p53 alleles found in tumors. The transdominance test was performed using a simple yeast transcription assay, and the amino acid at codon 72 was determined by sequencing. A total of 100 p53 mutants were tested. Compared with the germline frequency (R:P = 427:297), an extreme bias in favor of the R72 allele was observed with recessive mutants (R:P = 50:7, P < 0.0002), whereas no selection for the R72 allele was seen with transdominant mutants (R:P = 23:20). p53 and p73 are known to transactivate overlapping sets of target genes. We interpret the R72 bias with recessive mutants as evidence that decreased activation of p53 target genes provides a selective growth advantage to tumor cells during the stage of tumorigenesis in which a wild-type and mutant p53 allele coexist. We suggest that transdominant p53 mutants achieve this by inactivation of the remaining wild-type p53 allele, whereas recessive p53 mutants achieve it through inactivation of p73.

Asunto(s)

Alelos , Arginina/genética , Genes Dominantes , Mutación , Neoplasias/genética , Polimorfismo Genético , Proteína p53 Supresora de Tumor/genética , Humanos , Proteína p53 Supresora de Tumor/química

RESUMEN

OBJECTIVE: The p53 status is increasingly regarded as a marker predictive of response to particular cancer therapies, but for this approach it is self-evident that the p53 status must be determined correctly. METHODS: We have tested ovarian cancers with single-strand conformation polymorphism analysis (SSCP), immunohistochemical staining with DO-1 anti-p53 antibody (IHC), and yeast p53 functional assay (FASAY). RESULTS: These techniques commonly used to detect p53 mutations showed important differences in their sensitivity. Of 53 tumors tested with three indirect techniques, 27 (50%), 33 (62%) and 41 (77%) were positive by SSCP, IHC, and FASAY, respectively. In a subset of 32 tumors strongly suspected of containing mutations, 25 (78%), 26 (81%), 29 (91%) and 30 (94%) were positive by SSCP, immunostaining, DNA sequencing and yeast assay, respectively. CONCLUSIONS: Under comparable routine conditions, the FASAY reached the highest sensitivity. Since no single technique detected all mutations, we recommend the use of at least two different techniques in situations where the p53 status will affect patient management.

Asunto(s)

Análisis Mutacional de ADN/métodos , Genes p53/genética , Mutación , Neoplasias Ováricas/genética , Alelos , Femenino , Humanos , Inmunohistoquímica , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Análisis de Secuencia de ADN , Levaduras/genética

RESUMEN

Despite important advances in understanding the molecular basis of cancer, few treatments have been devised which rationally target known causal oncogenic defects. Selectively replicating viruses have a major advantage over nonreplicating viruses to target these defects because the therapeutic effect of the injected virus is augmented by virus produced within the tumor. To permit rational targeting of colon tumors, we have developed replicating adenoviruses that express the viral E1B and E2 genes from promoters controlled by the Tcf4 transcription factor. Tcf4 is constitutively activated by mutations in the adenomatous polyposis coli and beta-catenin genes in virtually all colon tumors and is constitutively repressed by Groucho and CtBP in normal tissue. The Tcf-E2 and Tcf-E1B promoters are active in many, but not all, cell lines with activation of the wnt pathway. Viruses with Tcf regulation of E2 expression replicate normally in SW480 colon cancer cells but show a 50- to 100-fold decrease in replication in H1299 lung cancer cells and WI38 normal fibroblasts. Activation of wnt signaling by transduction of a stable beta-catenin mutant into normal fibroblasts renders the cells permissive for virus replication. Insertion of Tcf4 sites in the E1B promoter has only small effects on replication in vitro but significantly reduces the inflammatory response in a rodent lung model in vivo. Replicating adenoviruses with Tcf regulation of both E1B and E2 transcription are potentially useful for the treatment of liver metastases from colorectal tumors, but additional changes will be required to produce a virus that can be used to treat all colon tumors.

Asunto(s)

Adenovirus Humanos/genética , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Activación Transcripcional , Replicación Viral , Proteínas de Pez Cebra , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Proteínas E2 de Adenovirus/genética , Proteínas E2 de Adenovirus/metabolismo , Adenovirus Humanos/fisiología , Animales , Secuencia de Bases , Línea Celular , Datos de Secuencia Molecular , Mutación , Neoplasias/genética , Neoplasias/virología , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , Ratas , Sigmodontinae , Factores de Transcripción TCF , Proteína 2 Similar al Factor de Transcripción 7 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Proteínas Wnt

RESUMEN

We examined the frequency of p53 mutations in 38 oropharyngeal squamous cell carcinomas (SCC), using both a yeast functional assay and a conventional immunohistochemical staining method (IHC) to detect p53 mutations. We also explored the clinical importance of p53 mutations in oropharyngeal SCC. An accumulation of p53 protein was detected in 17 of the 38 (45%) tumors by IHC, whereas the yeast-based assay detected 6 additional p53 mutations, for a total of 23 tumors (61%) with p53 mutations. The cDNA sequencing analysis revealed that the 6 mutations undetected by IHC consisted of 3 frameshift, 1 nonsense and 2 missense mutations. Thus, the yeast functional assay was more sensitive than conventional IHC for detecting p53 mutations. Subsequently, the relationship between p53 mutations and the clinico-pathological parameters in oropharyngeal SCC was evaluated using the results of the functional assay. Mutation of p53 was not associated with the patient age, sex, tumor stage or degree of tumor cell differentiation. Interestingly, heavy drinking had a significant positive correlation with the p53 mutation, but heavy smoking did not, suggesting that prolonged exposure to alcohol is more related to p53 mutation in oropharyngeal SCC than to tobacco consumption. Radiation sensitivity was examined by comparing tumor size on magnetic resonance images before and after completion of therapy with 45 Gy radiation, in the 18 cases of T2 oropharyngeal SCC that were initially treated by radiotherapy. The results showed that tumors with wild-type p53 decreased in size significantly compared to those with mutant p53. In 33 patients treated with curative intent, the overall survival after the completion of therapy was better in patients with a wild-type p53 tumor than in patients with a mutant p53 tumor. We conclude that p53 mutation is associated with radiation resistance and a decreased probability of survival in oropharyngeal SCC.

Asunto(s)

Carcinoma de Células Escamosas/genética , Mutación , Neoplasias Orofaríngeas/genética , Proteína p53 Supresora de Tumor/genética , Adulto , Anciano , Anciano de 80 o más Años , Bioensayo , Análisis Mutacional de ADN/métodos , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN , ARN Neoplásico/análisis , Levaduras/genética

RESUMEN

p53 status was tested in 180 patients with primary breast cancer using a yeast functional assay. Mutations were identified in 32% of cases. Only half were point missense mutations; the remainder were nonsense, insertion, deletion and splice site mutations. Twenty-two percent of mutations were located outside exons 5-8. For a median follow-up of 88 months, survival analysis showed that p53 mutation conferred a worse prognosis in the whole population and the node-positive subgroup but not in node-negative patients. p53 status, tumour size >2 cm, axillary lymph node metastasis and high histological grade were major adverse risk factors in univariate analysis. Multivariate analysis of 153 patients for whom full data were available showed that p53 status contributed prognostic information when tumour size and lymph node status were taken into account but not when histological grade was included. p53 status thus contributes only limited new prognostic information in breast cancer when established prognostic factors are taken into account. Int. J. Cancer (Pred. Oncol.) 84:587-593, 1999.

Asunto(s)

Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Análisis Mutacional de ADN/métodos , Genes p53/genética , Mutación , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/mortalidad , Femenino , Mutación del Sistema de Lectura , Eliminación de Gen , Genes Fúngicos , Humanos , Persona de Mediana Edad , Mutación Missense , Pronóstico , Empalme del ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Levaduras/genética

Asunto(s)

Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Proteínas del Citoesqueleto/genética , Proteínas de Unión al ADN , Mutación Missense , Proteínas Proto-Oncogénicas/genética , Proteínas Adaptadoras Transductoras de Señales , Proteína de la Poliposis Adenomatosa del Colon , Proteínas Portadoras , Femenino , Predisposición Genética a la Enfermedad , Humanos , Judíos , Masculino , Repeticiones de Microsatélite , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS , Proteínas de Neoplasias/genética , Proteínas Nucleares , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Linaje , Factores de Riesgo

RESUMEN

We have developed a new approach to photodynamic therapy based on adenoviral transduction of the rate-limiting enzyme in heme synthesis. Conventional phototherapy uses porphyrin-based chemical photosensitisers, including delta-aminolaevulinic acid (ALA) which is converted to protoporphyrin IX (PpIX) by the enzymes of the heme biosynthetic pathway. The lack of a specific mechanism for targeting chemical photosensitisers and PpIX to tumour cells means that therapeutic irradiation can damage normal tissue and exposure to sunlight following treatment can cause severe burns. The rate limiting enzyme in PpIX synthesis is ALA-synthase (ALA-S). We have developed a new yeast vector system for manipulation of the adeno- virus genome and used it to construct a virus expressing a mutant form of ALA-S lacking the iron response elements which regulate ALA-S translation and the heme regulatory motifs which regulate import of ALA-S into mitochondria. The virus induces a large increase in PpIX expression and confers photosensitivity on cultured cells. Unlike conventional photodynamic therapy, a viral approach makes it possible to restrict photosensitivity by biological rather than purely physical or chemical means. As with HSV thymidine kinase, ALA-S expression is a general mechanism for sensitisation to a therapeutic agent which can easily be adapted to whatever means of gene delivery is most effective.

Asunto(s)

5-Aminolevulinato Sintetasa/genética , Adenoviridae/genética , Vectores Genéticos/administración & dosificación , Fotoquimioterapia , Fármacos Fotosensibilizantes , Neoplasias Cutáneas/terapia , Secuencia de Aminoácidos , Apoptosis , Secuencia de Bases , Línea Celular , Citometría de Flujo , Terapia Genética/métodos , Humanos , Etiquetado Corte-Fin in Situ , Microscopía Fluorescente , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neoplasias Cutáneas/tratamiento farmacológico , Células Tumorales Cultivadas

RESUMEN

p53 mutants in tumours have a reduced affinity for DNA and a reduced ability to induce apoptosis. We describe a mutant with the opposite phenotype, an increased affinity for some p53-binding sites and an increased ability to induce apoptosis. The apoptotic function requires transcription activation by p53. The mutant has an altered sequence specificity and selectively fails to activate MDM2 transcription. Loss of MDM2 feedback results in overexpression of the mutant, but the mutant kills better than wild-type p53 even in MDM2-null cells. Thus the apoptotic phenotype is due to a combination of decreased MDM2 feedback control and increased or unbalanced expression of other apoptosis-inducing p53 target genes. To identify these genes, DNA chips were screened using RNA from cells expressing the apoptosis-inducing mutant, 121F, and a sequence-specificity mutant with the reciprocal phenotype, 277R. Two potential new mediators of p53-dependent apoptosis were identified, Rad and PIR121, which are induced better by 121F than wild-type p53 and not induced by 277R. The 121F mutant kills untransformed MDM2-null but not wild-type mouse embryo fibroblasts and kills tumour cells irrespective of p53 status. It may thus expand the range of tumours which can be treated by p53 gene therapy.

Asunto(s)

Apoptosis , Proteínas Nucleares , Proteína p53 Supresora de Tumor/metabolismo , Animales , Secuencia de Bases , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , ADN Complementario , Regulación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis , Fenotipo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética

RESUMEN

Nitric oxide (NO) and its derivatives can directly cause DNA damage and mutation in vitro and may play a role in the multistage carcinogenic process. It has been reported that NO induces mutation in the p53 tumor suppressor gene; we therefore analyzed the relationship between NO synthase (NOS) activity and p53 gene status in early-stage lung adenocarcinoma. Surgical samples were classified into two categories: 14 lung adenocarcinomas with high NOS activity (>25 pmol/min/g tissue, category A), and 16 with low NOS activity (<25 pmol/min/g tissue, category B). A yeast functional assay for p53 mutations disclosed a red colony that corresponded to a mutation in the p53 gene in 8 cases (57.1%) in category A and 3 cases (18.8%) in category B, the frequency being significantly higher in the former (P<0.05). A p53 DNA sequence analysis revealed that 5 of the 8 p53 mutation-positive samples in category A had a G:C-to-T:A transversion, which is reported to be a major target of NO. The mechanism of carcinogenesis of adenocarcinoma is not fully understood, but these results suggest that an excess of endogenously formed NO may induce a p53 gene mutation containing mainly G:C-to-T:A transversion in the early stage of lung adenocarcinoma. Our results suggest that NO has potential mutagenic and carcinogenic activity, and may play important roles in human lung adenocarcinoma.

Asunto(s)

Adenocarcinoma/genética , Genes p53 , Neoplasias Pulmonares/genética , Óxido Nítrico Sintasa/metabolismo , Adenocarcinoma/enzimología , Anciano , ADN/química , Femenino , Humanos , Neoplasias Pulmonares/enzimología , Masculino , Persona de Mediana Edad , Mutación , Óxido Nítrico/fisiología

RESUMEN

Monitoring the transduction efficiency is of paramount importance in gene therapy. To monitor adenovirus-mediated wild-type p53 gene transfer, we have used a quantitative assay which tests the ability of human p53 to activate transcription in yeast. Selective amplification of cellular and viral p53 transcripts followed by quantitative assessment of mutant p53 content with the assay permits measurement of the wild-type p53 transduction efficiency into SF-188, U251MG and HUG31 glioblastoma cells. One reverse transcription primer tracks the wild-type/mutant ratio of endogenous p53 mRNA (P2), and the other the wild-type/mutant ratio of both endogenous and exogenous p53 mRNA (P1). Following infection of cell lines homozygous for mutant p53, the apparent transduction efficiency calculated (tau 0 = [P1-P2]/[1 + P2]) correlated with the level of p21 expression. Transduction efficiency in heterozygous wild-type/mutant HUG31 cells increased linearly with multiplicity of infection (MOI) for tau 0 values between 0.5 and 5.9, and admixture of normal cell-derived RNA produced only a modest reduction in tau 0 value, in keeping with theoretical predictions. These results suggest that the yeast p53 functional assay may be a useful tool for monitoring p53 gene therapy.

Asunto(s)

Adenoviridae/genética , Genes p53 , Vectores Genéticos , Transducción Genética , Levaduras/genética , Northern Blotting , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Ciclinas/metabolismo , Inhibidores Enzimáticos/metabolismo , Expresión Génica , Humanos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo