The p53 gene and its homologue p73 are rarely mutated in neuroblastoma. In recent studies, we showed that overexpression of ΔNp73α, an isoform lacking the N-terminal transactivation (TA) domain, surprisingly induces p53 protein accumulation in the wild-type (wt) p53 human neuroblastoma line SH-SY5Y. As can be expected owing to its dominant-negative effect, ΔNp73α inhibits Waf1/p21 gene expression, but equally importantly, it upregulates BTG2TIS21/PC3, another p53 target gene. This effect is not observed in neuroblastoma cells that express a mutated p53. To better understand the ΔNp73-mediated transactivation of the BTG2TIS21/PC3 gene we performed luciferase assays with two reporter plasmids harboring long and short BTG2 promoter sequences in three human neuroblastoma cell lines and one breast cancer cell line. Our results demonstrate that BTG2TIS21/PC3 transactivation by ΔNp73α depends on both p53 status (as it is not observed in a p53–/– neuroblastoma cell line) and cellular context (as it occurs in a p53+/+ neuroblastoma cell line but not in a p53+/+ breast tumor cell line). The fact that ΔNp73α may either inhibit or stimulate wt-p53 transcriptional activity, depending on both the p53 target gene and the cellular context, was confirmed by real-time quantitative PCR. Moreover, transactivation of the BTG2TIS21/PC3 promoter requires a complete ΔNp73α C-terminus sequence as it is not observed with ΔNp73β, which lacks most of the C-terminal domain. We have previously shown that ΔNp73α is the only p73 isoform expressed in undifferentiated neuroblastoma tumors. In light of all these findings, we propose that ΔNp73α not only acts as an inhibitor of p53/TAp73 functions in neuroblastoma tumors, but also cooperates with wt-p53 in playing a physiological role through the activation of BTG2TIS21/PC3 gene expression.

In MCF-7 cells, TNFα induces a G1 arrest with an increased expression of p21/Waf1 , an activation of NF-κB and an accumulation of p53. NF-κB and p53 are two transcriptional factors known to activate p21/Waf1 gene expression. Here we show that p53 inhibition has no effect on p21/Waf1 mRNA accumulation following TNFα treatment. In contrast, inactivation of NF-κB inhibits p21/Waf1 expression without affecting G1 arrest. The fact that p21/Waf1 gene expression is still stimulated when p53 is inactivated strongly suggests that TNFα induces accumulation of an inactive form of p53 protein. This assumption was further supported by the following observations: (i) the p53 DNA-binding activity to its consensus sequence was not stimulated following TNFα treatment, (ii) phosphorylation at Ser-15, -20 or -392 was not detected in response to TNFα, (iii) the transcription rate of Ddb2 , another p53 target gene, was not stimulated by TNFα. Finally, the accumulation of p53 in the nuclei of TNFα-treated MCF-7 cells was concomitant with an increase in p53 mRNA level, suggesting a regulation at the transcription level.

The p53 protein is a tumor suppressor that protects the organism against malignant consequences of DNA damage. Interaction of p53 with numerous cellular or viral proteins regulates its functional activity either positively or negatively. An approach leading to identification of such protein interactions directly in a cell extract could be of help in the development of screening assays to search for drugs acting on p53 in its cellular environment, either by disrupting its association with inhibitory proteins or by increasing its affinity for activating proteins. We show that the homogeneous time-resolved fluorescence (HTRF) assay based on the time-resolved amplified cryptate emission (TRACE) technology allows identification of such an interaction by simply adding a mixture of two labeled monoclonal antibodies, directly in a cellular extract. We validate this assay by studying p53/SV40-LTAg interactions. The antibodies directed against genuine p53 and SV40-LTAg epitopes were labeled with europium cryptate (donor) and XL665, a crosslinked allophycocyanin (acceptor), respectively. We demonstrated that a nonradiative energy transfer occurs between labeled antibodies only when p53 interacts with SV40-LTag, which opens up the possibility of extending this approach to other p53 partners to search for drugs that restore p53 tumor-suppressor activity.

p53 is a tumor suppressor protein that induces apoptosis at least in part through its ability to act as a sequence-specific transactivator. This work reports that intron 1 of the mouse Fas death receptor gene contains a p53-responsive element (p53RE) that matches the p53 consensus sequence and that is located between nucleotides +1704 and +1723 from the transcription initiation site. This element is specifically bound by p53 and functions as a p53-dependent enhancer in mammalian or in yeast reporter gene assays. Contrary to bax, another known pro-apoptotic p53-target gene, both mouse and human FAS p53REs are still activated by the discriminatory p53 mutants Pro-175 and Ala-143, a class of mutants unable to induce apoptosis. We propose that p53-dependent up-regulation of Fas does not induce apoptosis per se but sensitizes the cell to other pro-apoptotic signal(s). The functional conservation of p53-dependent Fas up-regulation argues strongly in favor of its biological importance and suggests that murine models may be used to study further the in vivo role of Fas in the p53 response.

Background/Aims: Protein kinase CK2 (CK2) increases when cells are committed to proliferate, as in liver regeneration, This enzyme phosphorylates the tumour suppressor protein p53, whose expression controls the levels of many other cell cycle proteins. The aim of this study was to determine if CK2 was affected by p53.Methods: Male Sprague-Dawley rats (200-250 g) were subjected to either partial hepatectomy or laparotomy and the levels and subcellular distribution of p53 were studied, following the approach used earlier for CK2. The levels of both proteins were also studied in the human cell lines HL-60 (devoid of p53) and HepG2 (with normal p53 levels) and in fibroblasts from transgenic p53-deficient mice (p53-/-) or homozygous for wild-type p53 (p53+/+), Computer-assisted search was used to detect p53 consensus sequences in genes for CK2 submits, Binding of p53 protein to some of these sequences was assayed by electrophoretic mobility shift assay.Results: Rat liver p53 protein was present mainly in the fraction extracted from intact nuclei by nucleases (S1) and showed a transient increase, at 6 h post partial hepatectomy, as observed previously with nuclear CK2, The human CK2 alpha gene presents the consensus sequence for trans-activation by p53 and specific binding of p53 protein to some of these sequences was detected in vitro. Total CR2 alpha was higher in HepG2 than in HL-60 cells but total CK2 and its cytosolic/ nuclear distribution was similar in mice (p53+/+) fibroblasts and (p53-/-) fibroblasts.Conclusions: p53 is present in the nuclease-extracted S1 fraction from liver cells, as described for CK2, and undergoes similar changes at the beginning of rat liver regeneration. However, the data on cultured cells suggest that the expression of CK2 and its subcellular localization are p53-independent events.

The molecular basis of cirrhosis, the most frequent underlying liver disease in hepatocellular carcinoma, remains unclear. We investigated microsatellite instability at six different loci on chromosomes 2p, 3p, 5q, 9p, 13q and 17p, in DNA from 38 cirrhotic livers of viral (n=28) and nonviral (n=10) origin. Sixty percent of the patients exhibited microsatellite alterations in at least one chromosome locus. A striking feature was the close association between genomic instability and cirrhosis linked to hepatitis B viral infection (P<0.01). This high instability may be a clue to the etiology of cancer induced by the hepatitis B virus.

Mutations in the p53 tumor suppressor gene play an important role in the development of many common human malignancies. In nasopharyngeal carcinomas (NPC), p53 gene mutations were not detected in primary tumors, with one exception for a primary tumor displaying a p53 mutation at codon 280, whereas p53 mutations were identified in some metastatic and nude mouse-passaged NPC specimens. In the present report, 41 NPC primary tumors of the undifferentiated carcinoma nasopharyngeal type (UCNT; 21 from Hong Kong and 20 from Guangxi, southeastern China) were studied. Four point mutations that result in amino acid substitutions were identified by PCR amplification of exons 2-9 and direct DNA sequencing, combined with PCR-single-strand conformation polymorphism analysis. The 4 mutations detected were clustered within the DNA stretch from codon 175 to 177. Our data, taken together with those of others, suggest that mutation in p53 may occur in NPC at various points during tumorigenesis. Alternative mechanisms of p53 inactivation in NPC are also possible. (C) 1995 Wiley-Liss, Inc.

We report an original application of competitive reverse transcription-polymerase chain reaction (RT-PCR) for the quantification of MDR1 mRNA in clinical specimens by simultaneous reverse transcription and PCR amplification of cellular RNA with decreasing amounts of an internal standard. The competitor RNA shares the same MDR1 primer sequences as the cellular mRNA, but yields a different-sized PCR product. This allows resolution of the amplified cDNA fragments after agarose gel electrophoresis and ethidium bromide staining. The concentration of MDR1 mRNA is derived from the ratio between the intensities of the bands corresponding to the amplified products. We have used this assay to measure MDR1 expression in breast carcinomas and assessed the precision, sensitivity, and accuracy of the method. Competitive RT-PCR is a simple, highly specific, nonradioactive procedure for the quantification of MDR1 mRNA and is particularly suitable for use in the clinical laboratory.

Background/Aims: Immunohistochemical reactivity for p53 protein is; common in various human malignancies and often related to p53 gene mutation. However, in some tumor types, accumulation of wild-type p53 has been shown. previously, we analyzed 96 European hepatocellular carcinomas using immunohistochemistry and found that 31% of these tumors overexpressed p53 in the cell nucleus. the aim of the present study was to establish whether p53 positivity correlates with the presence of structural p53 gene abnormalities in European hepatocellular carcinoma. Methods: DNA from 20 tumors, 10 with strong immunostaining and 10 with undetectable staining for p53, was extracted from frozen sections, and the entire coding portion of the p53 gene was sequenced. Results: Five of the 10 tumors containing high levels of p53 protein showed missense point mutations. The remaining 5 tumors with high p53 levels showed the wild-type coding sequence. One of the 10 tumors containing undetectable levels of p53 protein had a 1-base pair deletion in the splice acceptor site of intron 4. Conclusions: The results strongly suggest that, in European hepatocellular carcinomas, stabilization of the p53 protein depends on factors other than p53 gene mutation, such as binding to other molecules of cellular or viral origin.

Human hepatocellular carcinomas (HCC) and hepatoblastomas (HB) from patients in France and Italy, respectively, which are both areas with a low incidence of HCC and a low dietary exposure to aflatoxin B1 (AFB1), were analysed for alterations of the p53 tumour suppressor gene. An abnormality in the p53 gene was detected in only one of the seven HCCs examined. Sequencing of the cDNA of this HCC revealed a G to T transversion at the first nucleotide of codon 245 that was not found in normal tissue, excluding the possibility of germinal transmission of this alteration. All tumours had the wild-type sequence at codon 249, which has been reported to be a mutational hot spot in the p53 gene in HCCs from high incidence areas, such as China and Southern Africa. Seven samples of HB, the most common hepatic tumour of children, were also tested for loss of heterozygosity (LOH) and mutations in the p53 gene. In contrast to what is observed in HCC of adulthood, for which environmental conditions are important etiological risk factors, HB probably stems uniquely from genetic disorders. PCR-direct sequencing of exons 4 to 8 and PCR-single strand conformation polymorphism (PCRSSCP) of exons 2, 3 and 9 revealed no mutations in the coding sequences examined. LOH was observed in one of the five informative cases when codon 72 and intron 6 were subjected to polymorphism analysis.

Late promoter activity measured before viral DNA replication results from a complex involvement of negative and positive cis-acting elements located both in the enhancer and in the 21-bp repeats. GC motifs located within the 21-bp repeats act in cooperation with sequences overlapping the early TATA box to down-regulate the late promoter activity. Analysis of insertion mutants indicates that the late promoter might be negatively regulated at least partially by the early promoter machinery. The GTI motif located within the enhancer as well as the GC motifs lose the ability to down-regulate the late promoter in the presence of T antigen. Results obtained with tsA58 protein indicate that two different domains of T antigen are involved in the negative autoregulation of the early promoter activity and in the release of the down-regulation of the late promoter by the GC motifs.

The DNA-directed DNA polymerase activity of avian myeloblastosis virus and Rauscher leukemia virus was not enhanced by SV40 DNA, whereas that of murine sarcoma virus preparations was. The latter stimulation is ascribed to an endonucleolytic DNase which occurs in a mycoplasma contaminant. This can be removed by differential velocity centrifugation. Deoxyribonuclease activity is not a property of oncorna viruses, and the possibility is raised that other nonpolymerase enzymatic activities associated with these viruses may be contaminants.