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G Lozano

Publications and source records attributed to G Lozano.

At least 73 records · Page 4Linked to original sources
PubMed1994

NF-kappa B activation of p53. A potential mechanism for suppressing cell growth in response to stress.

The tumor suppressor p53 is a potent transcriptional activator that has been shown to regulate its own expression. In earlier studies, deletion analysis and site-specific mutagenesis identified the p53-responsive element that fits the p53 consensus sequence. In addition, the p53-responsive element was predicted to be a binding site for NF-kappa B. In this study, we showed that NF-kappa B present in HeLa nuclear extracts could bind the same DNA element in a sequence-specific manner. Co-transfection experiments showed that the p65 subunit of NF-kappa B, but not the p50 subunit, could activate the p53 promoter. In HeLa cells, tumor necrosis factor alpha (TNF-alpha) induced NF-kappa B activity. The p53 promoter was also induced by TNF-alpha under the same conditions. Both p65 transactivation and TNF-alpha induction of the p53 promoter depended on an intact NF-kappa B site. Detailed mutational analysis of the p53 and NF-kappa B responsive elements allowed differentiation of these two responses. Thus, we show that NF-kappa B activates p53 and that this activation is inducible by TNF-alpha. Since NF-kappa B induction occurs as a response to stress and p53 arrests cells in G1/S, where repair may be initiated, activation of p53 by NF-kappa B could be a mechanism by which cells can recover from stress.

Base Sequence↗
PubMed1994

PML, a growth suppressor disrupted in acute promyelocytic leukemia.

The nonrandom chromosomal translocation t(15;17)(q22;q21) in acute promyelocytic leukemia (APL) juxtaposes the genes for retinoic acid receptor alpha (RAR alpha) and the putative zinc finger transcription factor PML. The breakpoint site encodes fusion protein PML-RAR alpha, which is able to form a heterodimer with PML. It was hypothesized that PML-RAR alpha is a dominant negative inhibitor of PML. Inactivation of PML function in APL may play a critical role in APL pathogenesis. Our results demonstrated that PML, but not PML-RAR alpha, is a growth suppressor. This is supported by the following findings: (i) PML suppressed anchorage-independent growth of APL-derived NB4 cells on soft agar and tumorigenicity in nude mice, (ii) PML suppressed the oncogenic transformation of rat embryo fibroblasts by cooperative oncogenes, and (iii) PML suppressed transformation of NIH 3T3 cells by the activated neu oncogene. Cotransfection of PML with PML-RAR alpha resulted in a significant reduction in PML's transformation suppressor function in vivo, indicating that the fusion protein can be a dominant negative inhibitor of PML function in APL cells. This observation was further supported by the finding that cotransfection of PML and PML-RAR alpha resulted in altered normal cellular localization of PML. Our results also demonstrated that PML, but not PML-RAR alpha, is a promoter-specific transcription suppressor. Therefore, we hypothesized that disruption of the PML gene, a growth or transformation suppressor, by the t(15;17) translocation in APL is one of the critical events in leukemogenesis.

Animals↗
PubMed1994

Structural and functional analysis of p53: the acidic activation domain has transforming capability.

The p53 gene encodes a transcriptional activator that is able to suppress transformation. The protein can be divided into three functional domains: the acidic activation domain at the amino terminus; the oligomerization and nonspecific DNA binding regions in the carboxyl terminus; and the conformation domain, responsible for specific DNA binding, in the middle. To further examine the structural/functional relationship of p53, we undertook a functional study of deletion mutants of the protein. We assayed these mutants for their abilities to activate transcription, transform rat embryo fibroblasts, and oligomerize. Analysis of the results indicates that: (a) besides specific DNA binding, an intact conformation domain is necessary for the transactivation and oligomerization functions of p53; and (b) p53 mutants that contain the amino and carboxyl termini do not oligomerize with wild-type p53, yet they transform cells. In fact, the amino terminus alone transforms rat embryo fibroblasts. Transformation by these mutants is probably effected by the amino terminus binding and sequestration of factors essential for wild-type p53 function.

Acids↗
PubMed1993

The tumor suppressor p53 regulates its own transcription.

The ability of p53 to suppress transformation correlates with its ability to activate transcription. To identify targets of p53 transactivation, we examined the p53 promoter itself. Northern (RNA) analysis and transient transfection experiments showed that p53 transcriptionally regulated itself. A functionally inactive mutant p53 could not regulate the p53 promoter. Deletion analysis of the p53 promoter delineated sequences between +22 and +67 as being critical for regulation. Electrophoretic mobility shift analysis and methylation interference pinpointed the p53 DNA responsive element. When oligomerized in front of a heterologous minimal promoter, this element was regulated by wild-type p53 and not by mutant p53. Point mutations in the DNA element that eliminated protein-DNA interactions also resulted in a nonresponsive p53 promoter. The DNA element in the p53 promoter responsive to p53 regulation is similar to the p53 consensus sequence. However, we have been unable to detect a direct interaction of p53 with its promoter.

3T3 Cells↗
PubMed1993

SV40 T antigen abrogates p53-mediated transcriptional activity.

Recent evidence suggests that the tumor-suppressor protein p53 functions as a transcriptional regulator to control cell proliferation. An interaction with p53 is required for SV40 T antigen to transform primary cells; however, the effect of T antigen binding on p53 function is not known. In order to determine if an interaction with T antigen results in loss of p53-mediated transcriptional activity, we have used vectors expressing either a p53-GAL4 fusion protein or a wild-type p53 protein in transient co-transfection assays with T-antigen expression vectors. We have demonstrated that coexpression of T antigen significantly reduces both p53-GAL4-mediated transcription from a GAL4-dependent CAT reporter and p53-mediated transcription from a consensus p53 binding site in vivo. Moreover, T antigen was able to reduce binding of p53-GAL4 to its GAL4 binding sequence in gel shift experiments in vitro. These observed activities of T antigen were all dependent upon a functional p53-binding domain. In addition, coexpression of human papillomavirus type 18 E6 protein, able to bind to p53, was able to significantly reduce p53-mediated transcription. These results suggest that an interaction of certain viral oncoproteins with p53 results in loss of transcriptional activity of p53, a function that is important for maintaining normal cell growth.

3T3 Cells↗
PubMed1993

A functionally inactive p53 Li-Fraumeni syndrome mutant.

Germline mutations in the tumor-suppressor p53 have been recently identified in Li-Fraumeni syndrome patients. We analysed the function of one of these mutations, an arg-to-trp substitution at amino acid 245 in the murine p53 gene. This p53LFS mutant could not, unlike wild-type p53, suppress foci formation of rat embryo-fibroblasts. Like other p53 mutants it cooperated with activated ras to transform rat embryo fibroblasts. Overexpression of p53LFS thus resulted in a phenotype similar to other mutant p53s. The p53LFS protein was also transcriptionally inactive in contrast to previous studies using a p53LFS/GAL4 fusion protein. To better understand the functional domain disrupted in p53LFS, we developed a dimerization assay and showed that p53LFS still dimerized. In addition, p53LFS retained its ability to bind SV40 large T antigen and not hsc70, both characteristics of wild-type p53. Using immunofluorescence, we localized p53LFS to the nucleus. From these results we conclude that p53LFS represents an unusual p53 mutant in that it retains many characteristics of wild-type p53, however activities critical for growth suppression are lost.

Cell Nucleus↗
PubMed1992

Tissue-specific expression of the mouse alpha 2(I) collagen promoter. Studies in transgenic mice and in tissue culture cells.

We sought to determine the cis-acting elements responsible for the pattern of tissue specific expression of the mouse alpha 2(I) collagen gene. Using an RNase protection assay we first verified that expression of the alpha 2(I) collagen gene is mainly confined to tendons, bone, and skin in mice. Both transgenic mice and DNA transfection of tissue culture cells were used as experimental approaches. Transgenic mice lines were generated harboring chloramphenicol acetyltransferase (CAT) chimeric genes that contained either (a) 2000 base pairs (bp) of 5'-flanking sequences of the mouse alpha 2(I) collagen gene plus additional sequences between +418 and +1524 of the first intron of this gene or (b) the same promoter sequences without intron sequences or (c) the 350-bp proximal promoter sequences. Transgenic mice containing both types of 2000-bp promoters showed a pattern of CAT expression that was tissue specific. The presence of sequences of the first intron in the transgene did not increase the level of promoter activity. Transgenic mice harboring the 350-bp alpha 2(I) collagen promoter also showed a pattern that was tissue-specific except that high level expression also occurred in the brain. This suggests that negative regulation is an important component of tissue-specific expression. In order to analyze the first 350 bases in detail, we performed transient expression experiments, using promoter fragments attached to the luciferase reporter gene. Fibroblasts, which show a high level expression of the endogenous alpha 2(I) collagen gene, and B cells, in which the gene is silent, were transfected with a series of deletions and substitution mutations within the proximal 350-bp promoter. These experiments were unable to define unique cell-specific cis-acting elements. However, when the sequence between -315 and -284 was tandemly repeated upstream of a minimal alpha 2(I) collagen promoter (-41 to +54), the activity of this construction was considerably higher in fibroblasts than in B cells when compared with the minimal promoter itself. In gel retardation assays, the levels of complexes that bind to this sequence were higher in fibroblast nuclear extracts than in myeloma nuclear extracts. Our results are consistent with the hypothesis that the -315 to -284 DNA sequence participates in the cell-specific control of the alpha 2(I) collagen gene in fibroblasts.

Animals↗
PubMed1992

A convenient cloning vector containing the GAL4 DNA-binding domain.

A DNA fragment encoding the yeast GAL4 DNA-binding domain (amino acids 3-147) was cloned into a convenient vector. This vector contains unique restriction sites at both the 5' and 3' ends and allows the generation of fusion proteins containing the GAL4 DNA-binding domain. These fusion proteins can be tested for their ability to activate transcription.

Base Sequence↗
PubMed1992

Inhibition of the mutant p53 gene in transformation assays.

Mutation of the p53 gene is a key element in the development of several human cancers. Intron 4, a noncoding region of the p53 gene, is required for optimal expression of that gene. We have previously shown that nuclear protein binds intron 4 and have defined the protein-binding site. In this paper we address the question, "Does the mutant p53 gene's ability to transform cells to the malignant phenotype depend on protein binding to intron 4?" Using an in vitro assay in which the mutant p53 gene and Ha-ras oncogene cooperate in transformation of cells to the malignant phenotype, we determined the ability of mutant mouse p53 gene constructs, with and without two base pair substitutions at the intron 4 protein-binding site, to participate in malignant transformation. On Day 1, 5 x 10(5) rat embryo fibroblasts were transfected by the calcium phosphate procedure with 10 micrograms of both a mutant p53 gene construct and Ha-ras oncogene. Malignant transformation was evidenced by the formation of discrete foci of heaped-up cells. After 14 days of incubation at 37 degrees C in DMEM and 10% fetal calf serum (8% CO2), the cells were stained with cresyl violet and the foci counted. In three separate experiments, the presence of two base pair substitutions at the intron 4 protein-binding site caused a significant decrease in the number of foci formed (P less than 0.05).

Animals↗
PubMed1991

Reduced amounts of cartilage collagen fibrils and growth plate anomalies in transgenic mice harboring a glycine-to-cysteine mutation in the mouse type II procollagen alpha 1-chain gene.

We have generated transgenic mice harboring a glycine-to-cysteine mutation in residue 85 of the triple helical domain of mouse type II collagen. The offspring of different founders displayed a phenotype of severe chondrodysplasia characterized by short limbs and trunk, cranio-facial deformities, and cleft palate. The affected pups died of acute respiratory distress caused by an inability to inflate lungs at birth. Staining of the skeleton showed a severe retardation of growth for practically all bones. Light microscopic examination indicated a decrease in cartilage matrix density, a severe disorganization of growth plate architecture, and the presence of streaks of fibrillar material in the cartilage matrix. Electron microscopic analysis showed a pronounced decrease in the number of typical thin cartilage collagen fibrils, distension of the rough endoplasmic reticulum of chondrocytes, and the presence of abnormally large banded collagen fibril bundles. The level of expression of the mutant type II procollagen alpha 1 chain transgene in cartilage tissues was approximately equal to that of the endogenous gene in two of the strains. We propose that the principal consequence of the mutation is a considerable reduction in density of the typical thin cartilage collagen fibrils and that this phenomenon causes the severe disorganization of the growth plate. We also postulate that the abnormal thick collagen fibrils are probably related to a defect in crosslinking between the collagen molecules. The cartilage anomalies displayed by these transgenic mice are remarkably similar to those of certain human chondrodysplasias.

Animals↗
PubMed1991

An intron binding protein is required for transformation ability of p53.

Regulatory elements in intron sequences have been identified for several eukaryotic genes. The fourth intron of p53 is known to increase expression of p53 in a position dependent manner. We asked whether p53 intron 4 sequences interacted with DNA binding proteins to exact their effect. Three overlapping DNA fragments spanning the 5' end of p53 intron 4 were determined to specifically interact with protein in nuclear extracts from several cell lines by band shift analysis. Methylation interference experiments were used to identify purine residues involved in this protein-DNA interaction. Two G nucleotides were identified at intron 4 positions 33 and 44 and these were replaced by T and C, respectively. These two single base pair substitutions in the intron resulted in 1) lack of protein binding and 2) decreased expression of p53 as measured by a transformation assay. Thus the binding of protein to p53 intron 4 was shown to have functional significance. These experiments demonstrated a specific protein binding region in the 5' end of intron 4 critical for p53 expression and distinct from those elements already known to be involved in splicing.

Animals↗
PubMed1991

Tissue-specific expression of p53 in transgenic mice is regulated by intron sequences.

Transgenic mice were produced harboring the p53 murine cDNA clone regulated by the SV40 enhancer-promoter region 5' to the cDNA and the small t antigen splice sites, and poly(A) addition signals 3' to the cDNA. This construction was not expressed in these mice. The presence of several murine p53 introns in the cDNA, however, permitted expression of the transgene mRNA in several tissues of transgenic mice. The insertion of intron 4 led to the preferential expression of the transgene mRNA in spleen cells, where the endogenous p53 gene is also expressed at high levels. While intron 4 promoted high levels of p53 mRNA expression in a tissue-preferred manner in transgenic mice, there was no evidence that intron 4 could act as an enhancer of transcription in cell culture or in transgenic animals. The presence of some p53 introns appears to be critical for the regulation of this gene in vivo.

Animals↗
PubMed1991

Analysis of p53 mutants for transcriptional activity.

The wild-type p53 protein functions to suppress transformation, but numerous mutant p53 proteins are transformation competent. To examine the role of p53 as a transcription factor, we made fusion proteins containing human or mouse p53 sequences fused to the DNA binding domain of a known transcription factor, GAL4. Human and mouse wild-type p53/GAL4 specifically transactivated expression of a chloramphenicol acetyltransferase reporter in HeLa, CHO, and NIH 3T3 cells. Several mutant p53 proteins, including a mouse p53 mutant which is temperature sensitive for suppression, were also analyzed. A p53/GAL4 fusion protein with this mutation was also transcriptionally active only at the permissive temperature. Another mutant p53/GAL4 fusion protein analyzed mimics the mutation inherited in Li-Fraumeni patients. This fusion protein was as active as wild-type p53/GAL4 in our assay. Two human p53 mutants that arose from alterations of the p53 gene in colorectal carcinomas were 30- to 40-fold less effective at activating transcription than wild-type p53/GAL4 fusion proteins. Thus, functional wild-type p53/GAL4 fusion proteins activate transcription, while several transformation competent mutants do so poorly or not at all. Only one mutant p53/GAL4 fusion protein remained transcriptionally active.

Animals↗
PubMed1990

Transcriptional activation by wild-type but not transforming mutants of the p53 anti-oncogene.

The protein encoded by the wild-type p53 proto-oncogene has been shown to suppress transformation, whereas certain mutations that alter p53 become transformation competent. Fusion proteins between p53 and the GAL4 DNA binding domain were made to anchor p53 to a DNA target sequence and to allow measurement of transcriptional activation of a reporter plasmid. The wild-type p53 stimulated transcription in this assay, but two transforming mutations in p53 were unable to act as transcriptional activators. Therefore, p53 can activate transcription, and transformation-activating mutations result in a loss of function of the p53 protein. The inability of the p53 mutant proteins to activate transcription may enable them to be transformation competent.

Base Sequence↗