Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have studied the effects of human wild-type and mutant p53s on the long terminal repeat (LTR) promoter of human immunodeficiency virus type 1 (HIV). HeLa cells were cotransfected with a wild-type or mutant p53 expression plasmid and a plasmid containing a chloramphenicol acetyltransferase reporter gene under HIV LTR promoter control. As expected, expression of wild-type p53 inhibited promoter function. Expression of a p53 mutated at any one of the four amino acid positions 175, 248, 273, and 281 correlated with a significant increase of the HIV promoter activity. The HIV LTR was also significantly activated in Saos-2 cells that do not express endogenous p53. This finding suggests a gain-of-transactivation function by mutation of the p53 gene. Cotransfection of wild-type and mutant p53-281G expression plasmids indicated that either the wild type or the mutant was dominant in inhibiting or enhancing promoter activity, respectively, when transfected in excess of the other. Transfection experiments showed transactivation even when the Sp1, NF-kappa B, and TATA sites in the LTR were individually mutated. Synthetic minimal promoter constructs containing two Sp1 sites or two NF-kappa B sites or an ATF site are also significantly activated by the mutant p53-281G. Thus, the mutant protein may activate transcription through interaction with either a general transcription factor or a common factor that bridges the basal transcription machinery and the transcription factors Sp1, NF-kappa B, and ATF.
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PMID:Activation of the human immunodeficiency virus type 1 long terminal repeat by transforming mutants of human p53. 825 19

Chromosome 17p has been shown to be an early and frequent target for loss of heterozygosity through mitotic recombination in astrocytomas. These losses are frequently accompanied by point mutations in the p53 gene of the remaining allele, resulting in loss of wild type p53 function. However, a fraction of astrocytomas retain constitutional heterozygosity and do not have p53 mutations; some of these lose wild type p53 activity through binding to the protein product of amplified mdm2 genes. To test whether loss of wild type p53 biological function is a necessary step in astrocytoma progression we analyzed p53 expression and biological function in 13 glioma cell lines. All the cell lines expressed a 2.8-kilobase p53 transcript and showed various amounts of p53 protein by immunoprecipitation, except for cell line LN-Z308 which had only a small truncated p53 mRNA and no protein expression. To test whether the p53 expressed in these cell lines was functionally wild type or mutant we transfected them with a plasmid construct harboring a chloramphenicol acetyltransferase (CAT) reporter gene under the control of transcriptional elements that are induced by wild type but not mutant p53. Four lines were shown to retain wild type p53 function. Sequencing of the p53 gene in two of these cell lines confirmed the wild type genotype. These results show that inactivation of the p53 gene is not an obligatory step in glioblastoma genesis. This suggests either that two pathways (p53 inactivation dependent or independent) may lead to a tumor group classified histologically as glioblastoma or that in some cases p53 mutations are bypassed due to the presence of mutations in downstream effector genes.
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PMID:Analysis of the p53 gene and its expression in human glioblastoma cells. 830 26

Transgenic mice which carry hybrid p53 promoter-chloramphenicol acetyltransferase (CAT) transgenes were found to express CAT enzymatic activity predominantly in the testes. Endogenous levels of p53 mRNA and protein were lower than in the nontransgenic control mice. The various p53 promoter-CAT transgenic mice exhibited in their testes multinucleated giant cells, a degenerative syndrome resulting presumably from the inability of the tetraploid primary spermatocytes to complete meiotic division. The giant-cell degenerative syndrome was also observed in some genetic strains of homozygous p53 null mice. In view of the hypothesis that p53 plays a role in DNA repair mechanisms, it is tempting to speculate that the physiological function of p53 that is specifically expressed in the meiotic pachytene phase of spermatogenesis is to allow adequate time for the DNA reshuffling and repair events which occur at this phase to be properly completed. Primary spermatocytes which have reduced p53 levels are probably impaired with respect to DNA repair, thus leading to the development of genetically defective giant cells that do not mature.
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PMID:Mice with reduced levels of p53 protein exhibit the testicular giant-cell degenerative syndrome. 841 56

Wild-type p53 was shown to function as a transcription factor. The N-terminal region of the protein contains the transcription activation domain, while the C terminus is responsible for DNA binding. Localization of the DNA-binding domain of the p53 protein to the highly conserved carboxy-terminal region suggests that the interaction of p53 with DNA is important for its function. We have developed a strategy for studying the DNA sequence specificity of p53-DNA binding that is based on random sequence selection. We report here on the isolation of murine genomic DNA clones that are specifically bound by the wild-type p53 protein but are not bound by mutant p53 protein forms. The isolated p53 target gene contains the unique DNA-binding sequence GACACTGGTCACACTTGGCTGCTTAGGAAT. This fragment exhibits promoter activity as measured by its capacity to activate transcription of the chloramphenicol acetyltransferase reporter gene. Our results suggest that p53 directly binds DNA and functions as a typical transcription factor.
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PMID:Isolation and characterization of DNA sequences that are specifically bound by wild-type p53 protein. 844 83

The overexpression of P-glycoprotein is thought to be responsible for resistance to chemotherapy in some non-responsive cancers. The mechanism by which P-glycoprotein is overexpressed in human tumors is poorly understood. However, several lines of evidence suggest that the major regulatory mechanism of P-glycoprotein overexpression in human tumors is at the transcriptional level. During tumor progression one of the most commonly observed alterations is mutation of the p53 tumor-suppressor gene. It has been shown that the p53 protein plays a role in transcriptional regulation. To gain insight into the effect p53 protein may have on P-glycoprotein promoter activity, we transiently co-transfected plasmids containing the hamster pgp1 or human mdr1 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene with plasmids encoding either wild-type or mutant p53 protein into Chinese hamster ovary (CHO) cells. In this report, we show that wild-type p53 protein represses P-glycoprotein promoter activity, while mutant forms of p53 protein enhance P-glycoprotein promoter activity. Furthermore, we present data which indicate that the transcriptional regulatory effects of p53 are mediated through interactions with pgp1/mdr1 core promoter sequences. These findings have implications for our understanding of the molecular mechanism(s) by which p53 protein functions as a transcriptional regulator of gene expression. In addition, our results suggest a mechanism by which P-glycoprotein may be overexpressed in human cancers that also express mutant forms of p53 protein.
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PMID:The core promoter region of the P-glycoprotein gene is sufficient to confer differential responsiveness to wild-type and mutant p53. 850 78

P53 status may be a determinant of chemosensitivity of tumor cells; however, its involvement in cellular resistance to cisplatin remains uncertain. To investigate the relationships between p53 and the development of resistance to cisplatin, the p53 gene status was studied in ovarian carcinoma cell systems which included two cisplatin-resistant variants (IGROV-1/Pt 0.5 and IGROV-1/Pt 1) selected in vitro after prolonged drug exposure of the cisplatin-sensitive parental IGROV-1 cell line. IGROV-1/Pt 0.5 and IGROV-1/Pt 1 cell lines exhibited a degree of resistance of approximately 6 and 14, respectively, following 96-h exposure to the drug and were cross-resistant to other DNA-damaging agents (ionizing radiation and melphalan). Resistance to cisplatin paralleled a reduced cell susceptibility to cisplatin-induced apoptosis. DNA single-strand conformation polymorphism analysis of exons 5-9 demonstrated the presence of two mutants alleles at exon 8 in the two resistant cell lines, in contrast to the parental IGROV-1 cell line which exhibited the wild-type p53 gene. Direct DNA sequencing revealed that the mutations consist of two nucleotide changes in the DNA-binding domain at codons 270 (T/A) and 282 (C/T). The consecutive levels of p53 protein were lower in IGROV-1 than in IGROV-1/Pt cells. Following exposure to ionizing radiation or cisplatin, accumulation of the p53 protein was markedly enhanced only in the sensitive cells. Concomitantly, the expression of WAF-1 protein was strongly induced in the parental IGROV-1 cells, whereas WAF-1 protein remained undetectable in the IGROV-1/Pt 1 subline after DNA-damaging treatment. Consistent with this finding is the observation that ionizing radiation caused a different pattern of cell cycle perturbation in sensitive and resistant cells. Northern blot analysis demonstrated a marked reduction in bax mRNA levels in IGROV-1/Pt 1 cisplatin-resistant cells. Cotransfection assays with wild-type or mutant p53 expression plasmids and a reporter gene plasmid that utilized the bax gene promoter to drive transcription of chloramphenicol acetyltransferase were consistent with the role of p53 in regulation of bax expression in these cells. Taken together, these observations support a role for mutations of the p53 gene in the development of cisplatin resistance in ovarian cancer as a consequence of loss of the ability of p53 to transactivate bax, an apoptosis-inducing gene.
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PMID:Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems. 856 71

p53 is a nuclear phosphoprotein whose function is classified as tumor suppression. Studies have shown that p53 functions by binding to p53 DNA recognition sequences and regulates transcription of growth-regulatory genes. Various p53 recognition sequences have recently been identified. pOST2 contained two copies of a palindromic high-affinity DNA-binding sequence for p53; the other p53 recognition sequences included p53-binding fragments found in the human ribosomal gene cluster (pRGC) region and in the murine muscle creatine kinase promoter (pMCK). The purpose of this study was to compare the abilities of various p53 recognition sequences to mediate transcription in the presence of endogenously produced wild-type (wt) or mutant p53. Three p53-responsive chloramphenicol acetyltransferase (CAT) reporter constructs (pOST2, pRGC, and pMCK) that contain one or two copies of p53 recognition sequences upstream of a herpes thymidine kinase (TK) promoter and CAT reporter cDNA were constructed. Either a p53-responsive gene or a control reporter gene was transfected into human carcinoma cell lines (having various p53 mutations) either with or without a wt or mutant p53 expression vector. CAT activity was assayed to measure transactivation through the various p53-responsive elements. We showed that pOST2 had a greater ability to mediate transactivation by p53 than either pRGC or pMCK. p53 with a mutation at either codon 175 or 248 was unable to transactivate a reporter gene with pOST2, pRGC, or pMCK. We found it interesting that pOST2, but not pRGC or pMCK, was able to mediate transactivation in cell lines that produce codon 273-mutant p53. These findings suggest that various sensitivities of the different p53-responsive elements to specific mutant and wt p53s may be an important factor in the role of p53 as a transcriptional activator both under normal physiological conditions and during carcinogenesis.
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PMID:p53 transactivation through various p53-responsive elements. 864 24

sgk is a novel member of the serine/threonine protein kinase gene family that is transcriptionally regulated by serum and glucocorticoids in mammary epithelial cells. To functionally determine if the sgk promoter is regulated by the p53 tumor suppressor protein in mammary cells, a series of sgk promoter fragments with 5'-deletions were linked to the bacterial chloramphenicol acetyltransferase gene (sgk-CAT) and transiently co-transfected into nontumorigenic NMuMG or transformed Con8Hd6 mammary epithelial cells with p53 expression plasmids. Wild-type p53, but not mutant p53, strongly stimulated sgk promoter activity in both mammary epithelial cell lines. These effects were mediated by specific regions within the sgk promoter containing p53 DNA-binding sites. The sgk p53 sequence at-1380 to-1345 (site IV) was sufficient to confer p53-dependent transactivation to a heterologous promoter, and p53 was capable of binding to this sequence in vitro as assessed by gel shift analysis. In the nontumorigenic NMuMG epithelial cell line, cotransfection of wild-type p53 strongly stimulated the activities of both the sgk promoter and the well characterized p53-responsive p21/Waf1 promoter, whereas in Rat-2 fibroblasts, wild-type p53 repressed the basal activities of both promoters, revealing that sgk and p21/Waf1 are similarly regulated in a cell type-specific manner. Taken together, these results demonstrate that sgk is a new transcriptional target of p53 in mammary epithelial cells and represent the first example of a hormone-regulated protein kinase gene with a functionally defined p53 promoter recognition element.
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PMID:p53 stimulates promoter activity of the sgk. serum/glucocorticoid-inducible serine/threonine protein kinase gene in rodent mammary epithelial cells. 864 46

Cancer-related mutations of the p53 tumor suppressor gene are clustered in the four so-called 'hot spots', codons 175, 248, 273 and 281/282. By using recombination PCR in vitro mutagenesis, we introduced point mutations into the codon 273 of wild-type (wt) p53 (pC53-SN3) from Arg to His (pC53-273H [273H]), Asp (273D), Pro (273P), Lys (273K), Leu (273L) or Thr (273T), and compared their biological and biochemical activities with wt p53 and cancer-derived 175H, 248W and 273H/309S. Among them, 273H/309S, 273H and 273D as well as wt p53 transactivated the chloramphenicol acetyltransferase (CAT) gene placed downstream of the p53 binding consensus, while none of the other mutants including 273L did. Transcriptions from human c-fos and rat PCNA promoters were suppressed by wt p53 and 273D, while they were enhanced variously by all other mutants in Saos-2 and/or NIH3T3 cells. On the other hand, growth of human squamous carcinoma cell lines measured by the plating efficiency of G418-resistant colonies was enhanced by transfection of 175H, 248W, 273H/309S and 273P, while suppressed by not only wt p53, 273D and 273H but also 273L. Thus, 273H/309S enhanced cell growth in spite of its p53-specific transactivation activity, while 273L suppressed cell growth in spite of its complete loss of the p53-specific transactivation. We concluded that the sequence-specific transactivation of p53 is not always correlated with its growth inhibitory activity.
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PMID:The 273rd codon mutants of p53 show growth modulation activities not correlated with p53-specific transactivation activity. 864 76

The tumor suppressor p53 protein down-regulates in vitro the expression of several cellular and viral promoters. However, it is not clear whether this down-regulation reflects equivalent modulation of the activity of these promoters in vivo. Here, we propose a suitable system to assess the effect of p53 on gene expression in vivo: the pair of p53 antisense-transfected and parental HeLa cells. The low amount of free wild-type p53 in HeLa cells seems still sufficient for the repression of several promoters that might be derepressed in p53 antisense-transfected HeLa cells. We have used this system for the demonstration both in vivo and in vitro of the repression of the fibronectin (FN) gene promoter by wild-type p53. The protein and mRNA amounts for FN were increased in the p53 antisense-transfected HeLa clones. This was accompanied by the restoration of the FN network in these cells. FN promoter constructs fused to the chloramphenicol acetyltransferase reporter gene were specifically repressed by wild-type p53 in different cell lines. Integrin alpha 5 beta 1 clustering was changed in the sites of focal contacts, most probably representing its relocalization as a consequence of the increased amounts of fibronectin.
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PMID:Down-regulation of fibronectin gene expression by the p53 tumor suppressor protein. 873 72


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