Mutations of the p53 tumor suppressor gene occur infrequently in Wilms' tumor

Mutations of the p53 tumor suppressor gene occur frequently in a variety of adult-onset tumors, including colon, breast, lung, and brain, yet are infrequently identified in pediatric malignancies. Wilms' tumor, a common solid tumor of childhood, can be associated with mutations of the WT1 gene. Alterations of the p53 gene have been shown to modulate the ability of WT1 to transactivate its targets. Although positive p53 immunostaining has been demonstrated in Wilms' tumors, the correlation to p53 gene mutations is not clear. We examined Wilms' tumor samples for p53 mutations utilizing polymerase chain reaction-single-strand conformation polymorphism analysis and single-strand DNA sequencing. Mutations in the coding region of the p53 gene were demonstrated in 2 of 21 (9.5%) Wilms' tumors. Each mutation yielded a substitution of amino acid residues. One mutation was located in exon 6 and the other in exon 7. Both mutations were found in tumors from patients with advanced stage disease. Focal anaplasia was demonstrated in one of these tumors. Our data suggest that although p53 mutations occur infrequently in Wilms' tumor, they may be associated with advanced disease.     

Midkine as a novel target gene for the Wilms' tumor suppressor gene (WT1)

Midkine (MK) is a heparin-binding growth factor which is strongly expressed during the midgestation period of mouse embryogenesis. Wilms' tumor is an embryonal kidney malignancy in infants, and WT1 has been identified as its tumor suppressor gene. The high expression level of MK in all Wilms' tumor specimens so far examined and the presence of two WT1 elements (5'-GCGGGGGCG-3') in the human MK promoter region led us to examine the possible role of the WT1 gene product in the regulation of MK gene expression. A gel shift assay verified the complex formation between the WT1 gene product and WT1 consensus sequence of MK gene. DNase1 footprint analysis also demonstrated that the downstream WT1 element was protected from DNase1 cleavage by the addition of the WT1 protein. The human MK promoter fused with the chloramphenicol acetyltransferase gene (phMK2.3kCAT) was co-transfected with an effector plasmid containing the WT1 gene into several cell lines. Transient transfection assays showed suppression of the MK promoter by WT1 co-transfection in recipient cells; deletion of the WT1 binding site abolished the suppression. The evidence reported in this study indicates that MK gene is a newly identified WT1 target gene.     

The two-exon gene of the human forkhead transcription factor FREAC-2 (FKHL6) is located at 6p25.3

Subcutaneous metastases from clear cell endometrial carcinoma are an uncommon event and tumor implantations are rarely found with diagnostic imaging techniques. The nodular form is the most frequent type of subcutaneous metastasis from genital system tumors, even though plaque-like and infiltrative forms have also been reported. We report the first case of subcutaneous metastasis from clear cell endometrial carcinoma whose progression from the early nodular to the lymphangitic infiltrative form was studied with computed tomography (CT). Differential diagnostic problems are discussed.     

Immunohistoselective sequencing (IHSS) of p53 tumor suppressor gene in human oesophageal precancerous lesions

Accumulation of p53 protein occurs in human oesophageal precancerous lesions and even in near-normal oesophageal epithelium. In some instances, p53 gene mutations have been detected. In many of the cases of p53 protein accumulation in early lesions, however, p53 mutations were not detected due to either the lack of mutation or the low abundance of cells with a mutation. In order to enrich p53 immunostain-positive cells for single strand conformation polymorphism (SSCP) analysis and DNA sequencing, an immunohisto-selective sequencing (IHSS) method was developed. Anti-p53 antibody-peroxidase stained oesophageal tissue sections were subjected to ultraviolet (UV) irradiation to damage the DNA in p53 immunostain-negative cells. The immunostain protected p53 immunostain-positive cells from the UV light and thus preserved the DNA in those cells for PCR amplification. Comparison of the SSCP results from sections with and without UV treatment showed that the IHSS method selectively enriched p53 immunostain-positive cells. With this method, we could analyse mutations in samples with as few as 30 p53 immunostain-positive cells per tissue section. Analysis was carried out on tissues with precancerous lesions from six surgically-resected oesophageal specimens and 13 oesophageal biopsies from symptom-free subjects. The results of mutation analysis for some of the samples were confirmed by microdissection to enrich the p53-positive cells. The mutations in tissues with precancerous lesions were compared with those in the corresponding squamous cell carcinomas. The IHSS method is shown to be a simple and effective way to analyse mutations in p53 immunostain-positive cells. IHSS may also be a general method for molecular analysis of biological specimens after immunohistochemical staining.     

Molecular surgery for human colorectal cancer with tumor suppressor p53 gene transfer

Recent advances in molecular biology have demonstrated that multistep genetic alterations are involved in the carcinogenesis of human colorectal cancer and that alteration of the p53 gene by mutation, deletion, or rearrangement is a major factor in this process. Human gene therapy has become a reality with the development of effective techniques for delivering the gene to the target cells. The efficacy of gene therapy for various types of genetic disease now being evaluated in clinical trials. These findings led us to develop a novel gene therapeutic strategy for human colorectal cancer that could replace the abnormal p53 gene using a recombinant, replication-defective adenoviral vector (termed Adp53). Infection with Adp53 induced rapid apoptotic cell death in DLD-1 and LoVo human colorectal cancer cell lines differing in their p53 status. Treatment with cisplatin following infection with Adp53 significantly suppressed the growth of WiDr colorectal cancer cells compared to single treatments alone. Thus restoration of wild-type p53 function exhibited an antitumor effect by inducing apoptosis as well as by markedly enhancing the effect of common chemotherapeutic agents in human colorectal cancer cells. In addition, Adp53 infection was antiangiogenic in SW620 human colorectal cancer cells. The application of this technology to human cancer therapy is now in progress. The article reviews recent highlights in this rapidly evolving field.     

Regulation of ornithine decarboxylase gene expression by the Wilms' tumor suppressor WT1

The importance of ornithine decarboxylase (ODC) to cell proliferation is underscored by the complex array of cell-specific mechanisms invoked to regulate its synthesis and activity. Misregulation of ODC has severe negative consequences on normal cell function, including the acquisition of tumorigenic growth properties by cells overexpressing ODC. We hypothesize that ODC gene expression is a candidate target for the anti-proliferative function of certain tumor suppressors. Here we show that the Wilms' tumor suppressor WT1 binds to multiple sites within the human ODC promoter, as determined by DNase I protection and methylation interference assays. The expression of WT1 in transfected HCT 116, NIH/3T3 and HepG2 cells represses activity of the ODC promoter controlling expression of a luciferase reporter gene. In contrast WT1 expression enhances ODC promoter activity in SV40-transfected HepG2 cells. Both the extent of modulation of ODC gene expression and the mediating WT1 binding elements are cell specific. Constructs expressing WT1 deletion mutants implicate two regions required for repressor function, as well as an intrinsic activation domain. Understanding the regulation of ODC gene expression by WT1 may provide valuable insights into the roles of both WT1 and ODC in development and tumorigenesis.     

Structures and functions of the tumor suppressor p53

The tumour suppressor p53 plays a crucial role in the cellular response to DNA damage. The p53 protein is able both to detect sites of DNA damage and to interact with DNA in a sequence-specific manner and function in the regulation of target gene expression. These two properties map to discrete functional domains of the protein, the C-terminus and the central core domain respectively. They are essential for integration of a normal cellular response to DNA damage, with initiation of either G1 cell cycle arrest or apoptosis. This review considers the domain structure of p53 in relation to the protein's various functions, together with the importance of tertiary structure and conformational flexibility. The precise regulation of p53 function remains to be established, although the protein is known to be phosphorylated/de-phosphorylated by a number of specific protein kinases/phosphatases. A recent discovery indicates that p53 may be activated by autoproteolysis and that proteolytic cleavage is induced by direct interaction with sites of DNA damage. This process is reminiscent of the bacterial Lex A system and would provide one mechanism for activation of p53 in response to cellular DNA damage.     

Inactivation of the p53 tumor suppressor gene via a novel Alu rearrangement

Inactivation of the p53 tumor suppressor gene is a common finding in human cancer. In most cases, inactivation is due to a point mutation in the gene, but rearrangement of the p53 gene is sometimes observed. We analyzed the inactivation of p53 in the human pancreas cancer cell line Hs766T, which harbors a structural alteration in the p53 gene. This inactivation was found to be the result of a complex deletion/insertion event involving at least two different Alu elements. The rearrangement eliminated exons 2-4 from the p53 gene, whereas a 175-bp Alu fragment was inserted between the breakpoints of the deletion. DNA sequence analysis of this Alu fragment revealed that it is identical to an Alu element in intron 1 of the p53 gene. This is the first report of p53 inactivation due to a rearrangement involving Alu elements. This type of inactivation may go unnoticed when only traditional methods to detect p53 alterations are used.     

Li-Fraumeni syndrome and the role of the p53 tumor suppressor gene in cancer susceptibility

Mutation of the tumor suppressor gene p53 is a molecular genetic event frequently observed in human cancer, and inactivating missense mutations usually are accompanied by the resultant overexpression of mutant p53 protein. In gynecologic cancers, p53 is also often altered; the frequency varies depending on types of cancers and where they develop. Further, human papillomavirus oncoproteins that inactivate p53 and Rb proteins play important roles in the development of several gynecologic cancers. Individuals who are heterozygous for germline mutations of the p53 gene are strongly predisposed to a variety of cancers. The identification of these individuals may have profound value in the future when therapies or chemopreventive agents specific for the p53 alteration are available. The role of p53 tumor suppressor gene in gynecologic cancers and heritable cancer susceptibility syndromes including Li-Fraumeni and Lynch II syndromes is an active and important area of study.     

Squamous cell carcinoma of the endometrium with human papillomavirus type 31 and without tumor suppressor gene p53 mutation

The extracellular conversion of lung surfactant from tubular myelin to the small vesicular form has previously been shown to require a serine-active enzyme called "surfactant convertase." In the present study, a 72kD serine-active enzyme previously identified in mouse lung alveolar lavage and having convertase activity was partially sequenced. Sixty-eight residues obtained from amino acid sequencing of this protein show that it is a new member of the mouse carboxylesterase family (EC 3.1.1.1). The 72kD lung protein also has esterase activity. A commercial esterase of the same family was able to reproduce surfactant convertase bioactivity in vitro, unlike several serine proteinases previously tested. We conclude that surfactant convertase is a carboxylesterase which mediates a biochemical step in the extracellular metabolism of surfactant.     

Role of the p53 tumor suppressor gene in the tumorigenicity of Burkitt's lymphoma cells

Burkitt's lymphoma (BL) cell lines carry a translocated c-myc gene and, in 60-80% of cases, exhibit mutations in the p53 tumor suppressor gene. We examined the potential role of the p53 gene in BL tumorigenicity using an in vitro assay that measures p53-dependent cell cycle arrest in the G1 phase of the cell cycle and an in vivo athymic murine model that detects differences in the tumorigenicity of BL cell lines. A highly significant inverse correlation was found between the ability of BL cells to arrest in G1 after irradiation and their tumorigenicity in athymic mice, consistent with the notion that loss of p53 function is associated with increased tumorigenicity. Inactivation of wild-type (wt) p53 function by expression of the human papillomavirus E6 protein in the AG876V BL cell line, which carries both wt and mutant p53 proteins, rendered the cell line significantly more tumorigenic in athymic mice. Transfection of the wt p53 gene into the p53 mutant and highly tumorigenic BL-41 cell line caused it to acquire wt p53 function and rendered it less tumorigenic in mice. In addition to confirming a role for the loss of p53 function in tumor progression, the data demonstrate that wt p53 protein can reduce BL tumorigenicity in vivo.