RESUMEN

p53 loss of heterozygosity (p53LOH) is frequently observed in Li-Fraumeni syndrome (LFS) patients who carry a mutant (Mut) p53 germ-line mutation. Here, we focused on elucidating the link between p53LOH and tumor development in stem cells (SCs). Although adult mesenchymal stem cells (MSCs) robustly underwent p53LOH, p53LOH in induced embryonic pluripotent stem cells (iPSCs) was significantly attenuated. Only SCs that underwent p53LOH induced malignant tumors in mice. These results may explain why LFS patients develop normally, yet acquire tumors in adulthood. Surprisingly, an analysis of single-cell sub-clones of iPSCs, MSCs and ex vivo bone marrow (BM) progenitors revealed that p53LOH is a bi-directional process, which may result in either the loss of wild-type (WT) or Mut p53 allele. Interestingly, most BM progenitors underwent Mutp53LOH. Our results suggest that the bi-directional p53LOH process may function as a cell-fate checkpoint. The loss of Mutp53 may be regarded as a DNA repair event leading to genome stability. Indeed, gene expression analysis of the p53LOH process revealed upregulation of a specific chromatin remodeler and a burst of DNA repair genes. However, in the case of loss of WTp53, cells are endowed with uncontrolled growth that promotes cancer.

Asunto(s)

Alelos , Pérdida de Heterocigocidad , Células Madre/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

We reported previously on the development of a Bacillus anthracis vaccine strain expressing high levels of recombinant protective antigen (rPA) [Cohen et al., Infec Immun 2000;68(8):4549-58]. To further explore the potential of the B. anthracis platform, we generated several attenuated strains expressing lethal toxin components PA and LF, which are biologically inactive, yet retain their antigenic properties. A single injection of 5 x 10(7) spores of one of these strains, carrying PA mutation at a site involved in effector translocation (residues 313-314) was shown to resemble wild type PA in inducing production of high levels of anti-PA neutralizing antibodies and producing effective protective immunity for 12 months. Long-term protection and persistence of functional antibody titers was observed after the gradual elimination of spores from guinea pig tissues 3 months after injection and in the measurable absence of bacteria in tissues. The mutant toxin components could, thus be an effective alternatives to their native counterparts when presented to the immune system in context of a live B. anthracis strain. These live vaccine prototypes may serve as a platform for future multi-component vaccines.

Asunto(s)

Vacunas contra el Carbunco/administración & dosificación , Carbunco/prevención & control , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/administración & dosificación , Bacillus anthracis/inmunología , Toxinas Bacterianas/administración & dosificación , Esporas Bacterianas/inmunología , Animales , Carbunco/inmunología , Carbunco/microbiología , Vacunas contra el Carbunco/inmunología , Anticuerpos Antibacterianos/análisis , Antígenos Bacterianos/inmunología , Bacillus anthracis/fisiología , Toxinas Bacterianas/inmunología , Cobayas , Inmunización , Proteínas Recombinantes/inmunología , Esporas Bacterianas/ultraestructura , Vacunas Sintéticas/inmunología

RESUMEN

An attenuated nontoxinogenic nonencapsulated Bacillus anthracis spore vaccine expressing high levels of recombinant mutant protective antigen (PA), which upon subcutaneous immunization provided protection against a lethal B. anthracis challenge, was found to have the potential to serve also as an oral vaccine. Guinea pigs immunized per os with the recombinant spore vaccine were primed to B. anthracis vegetative antigens as well as to PA, yet only a fraction of the animals (30% to 50%) mounted a humoral response to all of these antigens. Protective immunity provided by per os immunization correlated with a threshold level of PA neutralizing antibody titers and was long-lasting. Protection conferred by per os immunization was attained when the vaccine was administered in the sporogenic form, which, unlike the vegetative cells, survived passage through the gastrointestinal tract. A comparison of immunization of unirradiated spores with immunization of gamma-irradiated spores demonstrated that germination and de novo synthesis of PA were prerequisites for mounting an immune protective response. Oral immunization of guinea pigs with attenuated B. anthracis spores resulted in a characteristic anti-PA immunoglobulin isotype profile (immunoglobulin [G1 IgG1] versus IgG2), as well as induction of specific anti-PA secretory IgA, indicating development of mucosal immunity.

Asunto(s)

Vacunas contra el Carbunco/inmunología , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Vacunas Sintéticas/inmunología , Administración Oral , Animales , Anticuerpos Antibacterianos/sangre , Femenino , Cobayas , Inmunización , Proteínas Recombinantes/inmunología , Esporas Bacterianas/inmunología , Vacunas Atenuadas/inmunología

RESUMEN

We have recently reported Bacillus anthracis attenuated live vaccine strains efficiently expressing recombinant protective antigen (rPA) and have shown a direct correlation between the level of rPA secreted by these cells and efficacy (S. Cohen, I. Mendelson, Z. Altboum, D. Kobiler, E. Elhanany, T. Bino, M. Leitner, I. Inbar, H. Rosenberg, Y. Gozes, R. Barak, M. Fisher, C. Kronman, B. Velan, and A. Shafferman, Infect. Immun. 68:4549-4558, 2000). To isolate more potent Bacillus promoters for a further increase in the production of rPA, we developed a promoter trap system based on various gfp reporter genes adapted for use in both Bacillus subtilis and B. anthracis backgrounds. Accordingly, a B. anthracis library of 6,000 clones harboring plasmids with chromosomal B. anthracis DNA fragments inserted upstream from gfpuv was constructed. Based on fluorescence intensity, 57 clones carrying potentially strong promoters were identified, some of which were DNA sequenced. The most potent B. anthracis promoter identified (Pntr; 271 bp) was 500 times more potent than the native pagA promoter and 70 times more potent than the alpha-amylase promoter (Pamy). This very potent promoter was tested along with the other promoters (which are three, six, and eight times more potent than Pamy) for the ability to drive expression of rPA in either B. subtilis or B. anthracis. The number of cell-associated pre-PA molecules in B. anthracis was found to correlate well with the strength of the promoter. However, there appeared to be an upper limit to the amount of mature PA secreted into the medium, which did not exceed that driven by Pamy. Furthermore, the rPA constructs fused to the very potent promoters proved to be deleterious to the bacterial hosts and consequently led to genetic instability of the PA expression plasmid. Immunization with attenuated B. anthracis expressing rPA under the control of promoters more potent than Pamy was less efficient in eliciting anti-PA antibodies than that attained with Pamy. The results are consistent with the notion that overexpression of PA leads to severe secretion stress and have practical implications for the design of second-generation rPA-based vaccines.

Asunto(s)

Carbunco/prevención & control , Antígenos Bacterianos/metabolismo , Bacillus anthracis/genética , Vacunas Bacterianas , Regiones Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Bacillus subtilis/genética , Bacillus subtilis/inmunología , Vacunas Bacterianas/genética , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/metabolismo , Diseño de Fármacos , Femenino , Genes Reporteros , Proteínas Fluorescentes Verdes , Cobayas , Humanos , Inmunización , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Vacunas Atenuadas

RESUMEN

Fibroblast growth factors (FGF) activate their receptors through the formation of trimolecular complexes, composed of a ligand, a receptor, and a heparan sulfate oligosaccharide, all of which are members of particularly large families capable of multiple interactions in a combinatorial fashion. Understanding this large network of interactions not only presents a great challenge, but is practically beyond the capacity of most classical techniques routinely used to study ligand receptor interactions. We have used the yeast two hybrid system to study protein-protein interactions in the FGF family. Both ligand and receptor ectodomains are properly folded and functional in the yeast. Basic FGF (bFGF) expressed in the yeast dimerizes spontaneously. This self-assembly occurs at low affinity, which can be greatly enhanced by the introduction of heparin, supporting a defined role for heparin in bFGF dimerization. Screening a rat embryo cDNA library with bFGF in the yeast two hybrid system identified a short variant of FGF receptor 1, found most frequently in embryonal and tumor cells and which possesses affinity toward bFGF that is significantly greater than that of the more abundant, full-length receptor. We find the yeast two hybrid system, a most suitable alternative method for the analysis of growth factor-receptor interactions as well as for screening for novel interacting proteins and modulators of FGF and its receptors.

Asunto(s)

Receptores de Factores de Crecimiento de Fibroblastos/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Técnicas del Sistema de Dos Híbridos , Animales , Clonación Molecular , ADN Complementario , Proteínas de Unión al ADN , Dimerización , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Heparina/farmacología , Ratas , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

The exposure of cells to DNA-damaging agents leads to the accumulation of wild-type p53 protein. Furthermore, overexpression of the wild-type p53, mediated by transfection of p53-coding cDNA, induced cells to undergo apoptosis or cell differentiation. In this study we found that the gamma-irradiation that caused the accumulation of wild-type p53 in 70Z/3 pre-B cells induced, in addition to apoptosis, cell differentiation. This was manifested by the expression of the kappa light chain immunoglobulin gene that coincided with the accumulation of cells at the G2 phase. Overexpression of mutant p53 in 70Z/3 cells interferes with both differentiation and accumulation of cells at the G2 phase, as well as with apoptosis, which were induced by gamma-irradiation. Furthermore, the increment in the wild-type p53 protein level following gamma-irradiation was disrupted in the mutant p53 overproducer-derived cell lines. This suggests that mutant p53 may exert a dominant negative effect in all of these activities. Data presented here show that while p53-induced apoptosis is associated with the G1 checkpoint, p53-mediated differentiation, which may be an additional pathway to escape the fixation of genetic errors, may be associated with the G2 growth arrest phase.

Asunto(s)

Expresión Génica/fisiología , Genes de Inmunoglobulinas/efectos de la radiación , Genes p53/efectos de la radiación , Cadenas kappa de Inmunoglobulina/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Apoptosis/efectos de la radiación , Linfocitos B/inmunología , Linfocitos B/fisiología , Linfocitos B/efectos de la radiación , Secuencia de Bases , Western Blotting , Ciclo Celular/fisiología , Ciclo Celular/efectos de la radiación , Línea Celular , Cartilla de ADN , Relación Dosis-Respuesta en la Radiación , Citometría de Flujo , Fase G2/fisiología , Fase G2/efectos de la radiación , Rayos gamma , Expresión Génica/efectos de la radiación , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteína p53 Supresora de Tumor/análisis

RESUMEN

Inactivation of the p53 tumor suppressor gene plays a major role in malignant transformation. The central question in this issue is concerned with the understanding of the function of p53 in normal cells and its deregulation in cancer cells. Several in vitro and in vivo experimental models have indicated that induction of cells to undergo differentiation involve up-regulation in the expression of the p53. In the case of B cell differentiation, p53 was found to be involved in several steps of the differentiation pathway. The conclusion that p53 plays a role in normal development and differentiation in vivo is substantiated by the observation that p53 is expressed during embryonic development and is detected at low levels in a number of organs of adult mice. Accentuated levels of p53 in testes of adult mice, suggests that p53 plays a role in the meiotic process of spermatogenesis. B cell differentiation and spermatogenesis are biological pathways which normally involve DNA reshuffling and rearrangements. In accordance with the notion that p53 is associated with DNA repair it is tempting to speculate that at least in these physiological pathways p53 functions as a master gene that controls genome integrity.

Asunto(s)

Diferenciación Celular , Proteína p53 Supresora de Tumor/fisiología , Animales , Linfocitos B/fisiología , Reparación del ADN , Humanos , Masculino , Espermatogénesis

Asunto(s)

Proteínas Nucleares , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , ADN/genética , ADN/metabolismo , Genes de Inmunoglobulinas , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Proto-Oncogenes , Ratas , Secuencias Repetitivas de Ácidos Nucleicos , Retroviridae/genética , Proteína p53 Supresora de Tumor/genética

RESUMEN

An analysis of cell lines representing different stages of the B-cell differentiation pathway indicated that about 50% of the cell lines examined expressed exclusively wild type p53 protein. These lines therefore offer a convenient system to study the involvement of p53 in cell differentiation. When 70Z/3, a pre-B cell line which expresses wild type p53, was treated with the differentiation inducer lipopolysaccharide (LPS), it was seen that increased levels of p53 mRNA preceded specific changes in kappa (kappa) immunoglobulin expression. This increased expression of kappa specific mRNA, which was evaluated by specific PCR analysis, was blocked following transfection with mutant p53 coding plasmids. Treatment of 13A60, another cell line which endogenously expresses wild type p53, with LPS caused a secretion of IgA antibodies, also accompanied by increased p53 mRNA expression. The conclusion was that induction of B-cell differentiation involves the transcription of the p53 gene. This was further substantiated by experiments showing that differentiation of stable clones derived from the 70Z/3 cell line, harboring a p53-promoter-CAT plasmid, induced increased CAT activity. Furthermore, wild type p53 transactivated the promoter control sequences of the kappa light chain gene. Taken together, these results suggest that p53 is involved in B-cell differentiation, a pathway which involves DNA rearrangements that may be accompanied by generation of faulty DNA. The fact that wild type p53 was shown to function as a transcriptional factor, coupled with the notion that it is associated with DNA repair systems, may designate p53 as a control protein in the B-cell differentiation pathway.

Asunto(s)

Linfocitos B/patología , Transformación Celular Neoplásica/patología , Proteína p53 Supresora de Tumor/fisiología , Animales , Linfocitos B/química , Linfocitos B/fisiología , Secuencia de Bases , Diferenciación Celular , Línea Celular Transformada , Transformación Celular Neoplásica/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Cloranfenicol O-Acetiltransferasa/fisiología , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Expresión Génica/genética , Inmunoglobulina A/análisis , Inmunoglobulina A/metabolismo , Cadenas kappa de Inmunoglobulina/análisis , Cadenas kappa de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/fisiología , Lipopolisacáridos/farmacología , Ratones , Datos de Secuencia Molecular , Mutación , Plasmacitoma/patología , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/análisis , ARN Mensajero/genética , Factores de Transcripción/fisiología , Transcripción Genética/genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba