Interferon-gamma suppresses S100A4 transcription independently of apoptosis or cell cycle arrest.

The S100A4 protein has been associated with increased metastatic capacity of cancer cells, and recent studies have suggested a correlation between the expression level of S100A4 and the prognostic outcome for patients with various types of cancer. The knowledge about the mechanisms underlying the metastasis-promoting effects is still limited, and the aim of the present study was to elucidate signal transduction pathways involved in the regulation of S100A4. After treatment of human carcinoma cells with interferon-gamma (IFN-gamma), we observed downregulation of S100A4 both at mRNA and protein levels. The effect was not dependent on IFN-gamma-induced apoptosis or IFN-gamma-mediated cell cycle arrest. Moreover, IFN-gamma-mediated decrease in mRNA stability could not account for the observed decrease in S100A4 transcript level. Finally, microarray analysis suggests ISGF3G, ETV5, ZNF133 and CEBPG as possible candidate genes involved in IFN-gamma-mediated repression of S100A4.

Frequent promoter hypermethylation of the O6-Methylguanine-DNA Methyltransferase (MGMT) gene in testicular cancer.

Testicular germ cell tumours are classified into two major histological subgroups, seminomas and nonseminomas. All tumours display several recurrent chromosomal aberrations, but few target genes have been identified. Previous studies have shown that genome-wide hypermethylation of CpG islands is significantly more prevalent in nonseminomas than in seminomas. We have studied two potential target genes in testicular cancer. A series of 70 tumours were analysed for methylation of CpG sites in the O(6)-methylguanine-DNA methyltransferase (MGMT) gene promoter, and in exon 1alpha of the cyclin-dependent kinase inhibitor 2A gene (CDKN2A). In addition, eight microsatellite markers within and flanking these genes at chromosome arms 10q and 9p, respectively, were analysed for allelic imbalances. Allele alterations were frequently seen at 9p loci (47 out of 70, 67%), but none of the tumours (none out of 55) showed methylation of CDKN2A. On the other hand, a high frequency of MGMT promoter methylation (32 out of 69, 46%) was found, as well as allelic imbalances at 10q markers (50 out of 70, 71%). A significantly higher methylation frequency was found in nonseminomas (24 out of 35, 69%) compared to seminomas (eight out of 33, 24%) (P=0.0003, Fisher's exact test). Immunohistochemical analysis of the MGMT protein in a subgroup (n=20) of the testicular tumours supported the hypothesis of gene silencing being the functional consequence of the promoter methylation. In summary, our data suggest that inactivation of MGMT contributes to development of nonseminomatous testicular cancer.

Biallelic inactivation of TP53 rarely contributes to the development of malignant peripheral nerve sheath tumors.

About 10% of the patients with neurofibromatosis type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNSTs), accounting for half of all MPNST cases. Several nonrandom chromosomal aberrations have been found, but the target genes remain mostly unrecognized. Mutations in the NF1 and TP53 genes have been found in some MPNSTs, and recent data from mouse models support a synergistic effect of these two genes in the development of MPNST. In the present study, we have analyzed 16 MPNSTs, including 11 from patients with NF1 and 5 sporadic cases, for mutations in the coding sequence of the TP53 gene (exons 2-11). We applied denaturing gradient gel electrophoresis and modifications of this technique for analyses of 12 genomic fragments, followed by direct sequencing for identification of the mutated base(s). None of the MPNSTs revealed mutations. The detection of control mutants for each fragment analyzed, the high sensitivity of the technique, the detection of polymorphisms in some samples, and the high content of tumor tissue in the biopsies imply that false negatives are highly unlikely. Although we cannot exclude that deletions including large parts of the gene remain undetected by the mutation analyses, previous comparative genomic hybridization (CGH), cytogenetic banding analysis, and/or loss of heterozygosity studies on 14 of the cases included here had revealed 17p deletions in only three. We thus conclude that TP53 biallelic inactivation is rare in MPNST, and that the potential impact of an altered TP53 pathway on the malignant transformation of a neurofibroma into an MPNST may more frequently occur by changes in other components of that pathway.

Detection of mutations by single-strand conformation polymorphism (SSCP) analysis and SSCP-hybrid methods.

Single-strand conformation polymorphism (SSCP) analysis detects mutations based on the fact that single-nucleotide changes in DNA sequences alter the mobility of single-stranded DNA in nondenaturing gels. Four methods for detecting mutations based on SSCP are described here. (1) Traditional SSCP analysis is technically easy and can be used for screening large numbers of samples. SSCP-hybrid methods detect mutations based on either an SSCP effect or an altered component independent of the SSCP effect. (2) Dideoxy fingerprinting (ddF) involves PCR amplification of the target and creation of a set of dideoxy-terminated strands with the mutation. (3) Bi-directional dideoxy fingerprinting (Bi-ddF) involves production of two sets of dideoxy-terminated strands that are generated from two different primers. (4) Restriction endonuclease fingerprinting (REF) involves cleavage of the amplified target with five to six groups of restriction endonucleases.

Detection of mutations by denaturing gradient gel electrophoresis.

This unit describes the procedure for determining the melting profile for a given PCR-amplified sequence by perpendicular denaturing gradient gel electrophoresis (DGGE) and, using that information, for developing a screening assay based on either parallel DGGE, CDGE (Constant Denaturant Gel Electrophoresis), or TTGE (Temporal/Temperature Gradient Electrophoresis). Four support protocols describe techniques for pouring perpendicular and parallel denaturing gradient gels, constant denaturant gels, and temporal temperature gradient gels.

No association between radiosensitivity and TP53 status, G1 arrest or protein levels of p53, myc, ras or raf in human melanoma lines.

PURPOSE: First, to investigate whether TP53 status and/or radiation-induced G1 arrest are associated with radiosensitivity, and, second, to detect possible associations between protein levels of p53, myc, ras or raf and radiosensitivity and to investigate whether hypoxia-induced changes in the levels of these proteins are related to hypoxia-induced changes in radiosensitivity in human melanoma lines. MATERIALS AND METHODS: Radiosensitivity was assessed by clonogenic assays. TP53 status was investigated at the genomic level by constant denaturant gel electrophoresis and at the cDNA level by sequencing. G1 arrest was investigated by flow cytometric analysis of DNA. Protein expression of hypoxia-treated and untreated cells was assessed by flow cytometric measurements and Western blotting. RESULTS: Considerable differences in radiosensitivity were detected among melanoma lines with wild-type TP53. Only a fraction of the melanoma cells, differing between the lines, was arrested in G1. No association between the fraction of arrested cells and radiosensitivity was detected. Protein levels of p53, myc, ras or raf were not associated with radiosensitivity. Hypoxia-induced changes in p53, ras and raf levels were detected in all cell lines. Changes in the level of myc protein were detected for two of the four cell lines, while hypoxia-induced changes in radiosensitivity were observed only for one. CONCLUSIONS: Differences in radiosensitivity among melanoma lines cannot be elucidated by TP53 status, differences in G1 arrest or different levels of p53, myc, ras or raf proteins. Hypoxia-induced changes in p53, myc, ras or raf levels do not seem to be related to hypoxia-induced changes in radiosensitivity.

Therapy effect of either paclitaxel or cyclophosphamide combination treatment in patients with epithelial ovarian cancer and relation to TP53 gene status.

Cell death after treatment with chemotherapy is exerted by activation of apoptosis, and the p53 protein has been shown to actively participate in this process. This recent focus on TP53 status as a possible determinant of cancer therapy response has raised the question of whether or not mutations in the TP53 gene have an influence on paclitaxel therapy. The TP53 status has been analysed at the DNA level in tumours from 45 ovarian cancer patients randomized to treatment with paclitaxel and cisplatin or cyclophosphamide and cisplatin. Therapy response was obtained for 38 patients with clinically evaluable disease after initial surgery. The positive response rate to the paclitaxel/cisplatin therapy was 85% vs 61% for the patients who received the cyclophosphamide/cisplatin regimen. A significant difference in relapse-free survival in favour of paclitaxel/cisplatin chemotherapy was found (P = 0.001). A total of 33 tumour samples (73%) had detectable sequence alterations in the TP53 gene. When relapse-free survival was estimated for all patients with TP53 alterations in their tumours, a significant better outcome for the paclitaxel/cisplatin group was found compared with the patient group receiving cyclophosphamide and cisplatin therapy (P = 0.002). We did not observe an association between TP53 tumour status and prognosis for patients who received paclitaxel/cisplatin combination treatment, indicating that the effect of this therapy is not influenced by this parameter.

Specific P53 mutations are associated with de novo resistance to doxorubicin in breast cancer patients.

The mechanisms causing resistance to chemotherapeutic drugs in cancer patients are poorly understood. Recent evidence suggests that different forms of chemotherapy may exert their cytotoxic effects by inducing apoptosis. The tumor suppressor gene P53 has a pivotal role inducing apoptosis in response to cellular damage. In vitro investigations have shown intact p53 to play a critical role executing cell death in response to treatment with cytotoxic drugs like 5-fluorouracil, etoposide and doxorubicin. Recently, mutations in the P53 gene were found to confer resistance to anthracyclines in a mouse sarcoma tumor model, and overexpression of the p53 protein (which, in most cases, is due to a mutated gene) was found to be associated with lack of response to cisplatin-based chemotherapy in non-small cell lung cancer. Previous studies have shown mutations in the P53 gene or overexpression of the p53 protein to predict a poor prognosis, but also a beneficial effect of adjuvant radiotherapy or chemotherapy in breast cancer. In this study we present data linking specific mutations in the P53 gene to primary resistance to doxorubicin therapy and early relapse in breast cancer patients.

Functional analysis of Burkitt's lymphoma mutant c-Myc proteins.

The c-myc gene encodes a sequence-specific DNA binding protein that activates transcription of cellular genes. Transcription activation by Myc proteins is regulated by phosphorylation of serine and threonine residues within the transactivation domain and by complex formation with the retinoblastoma-related protein p107. In Burkitt's lymphoma, missense mutations within the c-Myc transactivation domain have been found with high frequency. It has been reported that mutant c-Myc proteins derived from Burkitt's lymphoma cell lines are resistant to inhibition by p107, thus providing a rationale for the increased oncogenic activity of these mutant c-Myc proteins. It has been suggested that these mutant c-Myc proteins resist down-modulation by p107 because they lack cyclin A-cdk2-dependent phosphorylation. Here, we have examined three different Burkitt's lymphoma mutant c-Myc proteins found in primary Burkitt's lymphomas and one mutant c-Myc protein detected in a Burkitt's lymphoma cell line. All four have an unaltered ability to activate transcription and are sensitive to inhibition of transactivation by p107. Furthermore, we provide evidence that down-modulation of c-Myc transactivation by p107 does not require phosphorylation of the c-Myc transactivation domain by cyclin A-cdk2. Our data indicate that escape from p107-induced suppression is not a general consequence of all Burkitt's lymphoma-associated c-Myc mutations, suggesting that other mechanisms exist to deregulate c-Myc function.

CDKN2A (p16INK4A) somatic and germline mutations.

The cell cycle is composed of a series of steps that can be negatively or positively regulated by various factors. A group of low-molecular-weight proteins have recently been identified that specifically inhibit the function of cyclin-dependent kinases in mammalian cells. Inactivation of the CDKN2A gene (also known as p16INK4A and MTS1) attracted considerable interest after it was mapped to 9p21, a locus for familial melanoma. In an effort to standardize the information regarding human CDKN2A mutations detected in cancers, a database with information of 146 point mutations has been created. Cancer type, origin of cells, specific mutation, amino acid change, literature citation, and other data are provided for each mutation entry. Studies of biochemical and biological functions of both wild-type and mutant proteins are central to our understanding of the role of p16INK4a mutations in tumorigenesis, a summary of these studies is also included in the present update.

Somatic spectrum of cancer-associated single basepair substitutions in the TP53 gene is determined mainly by endogenous mechanisms of mutation and by selection.

The spectrum of somatic TP53 single basepair substitutions detected in 955 cancers was compared with that of 2,224 different germline mutations in 279 different human genes (other than TP53), reported as the cause of inherited disease. This comparison reveals that, disregarding a relatively small subset (12%) of TP53 mutations that probably result from the action of exogenous mutagens, both the relative rates and the nearest-neighbor spectra of single basepair substitutions are similar in the two datasets. This spectral resemblance suggests that a substantial proportion of cancer-associated somatic TP53 mutations result from endogenous cellular mechanisms. The likelihood of clinical observation of a particular mutation type differs, however, between tumors and genetic diseases, when the chemical properties of the resulting amino acid substitutions are considered. Together with a sixfold higher observation likelihood for mutations at evolutionarily conserved residues, this finding argues that selection is a critical factor in determining which TP53 mutations are found to be associated with human cancer.

Database of p53 gene somatic mutations in human tumors and cell lines.

A data base is described in which over 2,500 mutations in the p53 gene of human tumors and tumor cell lines are compiled from a systematic search of reports published before 1 January 1994. Data from 1994 are being added intermittently, with a systematic search and update scheduled for December, 1994. The compilation has been deposited with the EMBL Data Library and is available in electronic form free of charge. This report contains a rationale for the compilation, a brief summary of the major findings and a description of the data base.

Germline mutations of the p53 tumor suppressor gene in children with osteosarcoma.

PURPOSE: We investigated the possibility that a significant proportion of children with osteosarcoma harbor germline mutations of the p53 tumor suppressor gene and, therefore, this subgroup of pediatric cancer patients should be considered for large-scale predictive testing. PATIENTS AND METHODS: Genomic DNA extracted from peripheral-blood leukocytes from 235 unselected children with osteosarcoma from 33 institutions were screened for the presence of germline p53 mutations using constant denaturant gel electrophoresis (CDGE). Exons 5 through 8 were evaluated in all patients and exon 2 and exon 9 were analyzed in 59 and 95 patients, respectively. Those samples that showed aberrant migration on CDGE were sequenced or analyzed by restriction enzyme digestion of polymerase chain reaction (PCR) products to confirm the nature of the gene alteration. RESULTS: In 18 samples, CDGE showed fragments of the p53 gene with altered electrophoretic mobilities compared with wild-type p53. DNA sequencing showed that 11 samples had an identical, previously described polymorphism. The other seven contained heterozygous p53 mutations located in exon 5 (n = 3), exon 6 (n = 1), exon 7 (n = 1), and exon 8 (n = 2). Six alterations were missense mutations and one was a nonsense mutation. Three of these patients had first-degree relatives with cancer. One of these three kindreds had a family history consistent with Li-Fraumeni syndrome (LFS). CONCLUSION: We identified germline p53 mutations in seven of 235 (3.0%) children with osteosarcoma. Four of these mutations were found in patients who did not have first-degree relatives with cancer. Although genetic transmission of the altered p53 gene could not be tested in this survey because of how it was designed, it is possible that predictive testing for p53 mutations could identify unaffected relatives of gene carriers who also have a high risk for the development of cancer. This study provides evidence for the importance of considering children with osteosarcoma for predictive testing for germline p53 mutations.

Screening for TP53 mutations in osteosarcomas using constant denaturant gel electrophoresis (CDGE).

We have previously developed conditions to screen for TP53 point mutations inside the conserved domains II-V of the gene by using constant denaturant gel electrophoresis (CDGE). The present study reports conditions for screening more of the codons in the frequently mutated region exon 5-8 and for detecting mutations in sequences encoding functional domains in the N- and C-terminal part of the protein. The ability of the CDGE technique to detect mutations was studied using controls with known sequence deviations. The resolution power of the technique to separate different types of mutations was tested by using seven different single base pair mutants all residing in a stretch of four base pairs. All mutants were separated from the wild type. The established CDGE screening strategy was then used to look for mutations in DNA from 28 osteosarcomas. Six (21.5%) of the samples were shown to have a TP53 mutation, and the exact characterization was performed by direct sequencing. All of these were within the frequently reported mutated region exon 5-8.

Detection of DNA variation in cancer.

Detection of DNA variation in cancer is central to the identification of relevant genes and mutations involved in the tumourigenic process. Diverse methods exist for such detection. One category of methods is for the detection of frequent sites for larger DNA alterations in cancer. Such areas may provide clues to the positioning of relevant genes, such as loss of heterozygosity (LOH) as in the case of tumour suppressor genes. Another category of methods is for the detection of single base mutations within specific genes. Frequently, such mutations may obliterate normal protein function. Among the most well-known are DGGE, SSCP, the HOT-method and direct sequencing. The methods for detection of DNA variation of these different levels are discussed. Two methods are presented in more detail. At the large-scale level, two-dimensional DNA fingerprinting has the potential of revealing the extent and location of altered DNA regions. This method is demonstrated using a panel of breast cancer patients. As an example of methods for the small-scale level, a recent development from DGGE, constant denaturant gel electrophoresis (CDGE) is demonstrated. This method has successfully been applied for the detection of mutations in a number of genes. Results with this method in studies of the RB1 gene are given, and its applicability as a screening tool for base mutations is discussed.

Screening for mutations in human HPRT cDNA using the polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis (CDGE).

Previously, we reported the modification of denaturing gradient gel electrophoresis called constant denaturant gel electrophoresis (CDGE). CDGE separates mutant fragments in specific melting domains. CDGE seems to be a useful tool in mutation detection. Since the hypoxanthine phosphoribosyltransferase (HPRT) gene is widely used as target locus for mutation studies in vitro and in vivo, we have examined the approach of analyzing human HPRT cDNA by polymerase chain reaction (PCR) and CDGE. All nine HPRT exons are included in a 716-bp cDNA fragment obtained by PCR using HPRT cDNA as template. When the full-length cDNA fragment was examined by CDGE, it was possible to detect mutations only in the last part of exon 8 and exon 9. However, digestion of the cDNA fragment with the restriction enzyme AvaI prior to CDGE enabled us to detect point mutations in most of exon 2, the beginning of exon 3, the last part of exon 8 and exon 9. With the use of two internal primer sets, including a GC-rich clamp on one of the primers in each pair, a region containing most of exon 3 through exon 6 was amplified and we were able to resolve fragments with point mutations in this region from wild-type DNA. The approach described here allows for rapid screening of point mutations in about two thirds of the human HPRT cDNA sequence. In a test of this approach, we were able to resolve 12 of 13 known mutants. The mutant panel included one single-base deletion, one two-base deletion and 11 single-base substitutions.

No alterations in exon 21 of the RB1 gene in sarcomas and carcinomas of the breast, colon, and lung.

Studies of mutant genotypes of the retinoblastoma susceptibility gene (RB1) in different solid tumors have mainly been concentrated on the demonstration of loss of heterozygosity (LOH) at both internal and external polymorphic sites. One reason for this is the complex organization of the gene. The p105RB protein has been shown to interact with both DNA and regulatory cellular proteins and oncoproteins. The amino acids encoded by exon 21 are implicated in several of these interactions. Both point mutations and intragenic deletions involving exon 21 have previously been reported in human tumors. We have examined RB1 exon 21 from a number of human tumor types where significant LOH in or around the RB1 gene has been reported. DNA from 78 primary tumors was amplified using the polymerase chain reaction (PCR) with primers covering exon 21, followed by constant denaturant gel electrophoresis (CDGE). The 78 tumors included 11 breast carcinomas, 30 nonsmall cell lung carcinomas, 6 colon carcinomas, and 31 sarcomas. The small cell lung cancer cell line NCI-H209, previously shown to harbour a point mutation in codon 706: TGT- greater than TTT (Cys- greater than Phe), was detected using CDGE. Apart from this control mutant cell line, we did not detect any mutations in the examined region in any of the tumors.

Screening for germ line TP53 mutations in breast cancer patients.

The constant denaturant gel electrophoresis technique was used to screen for TP53 germ line mutations in 237 women with breast carcinoma (167 unselected patients, 30 patients with at least one first-degree relative with breast cancer, and 40 women diagnosed with breast cancer before age 35). A germ line mutation at codon 181 was noted in one of the unselected patients and a codon 245 mutation in one of the early-onset patients. Both had a family history of breast cancer and other malignancies suggestive of Li-Fraumeni syndrome. The codon 245 mutation was also present in this patient's affected mother.

Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations.

At present, mutation of the p53 gene appears to be the most common genetic alteration found in human cancers. These mutations can occur within many different regions of the gene. We have developed a modification of denaturing gradient gel electrophoresis termed "constant denaturant gel electrophoresis" (CDGE), which provides a rapid and sensitive method to screen the four conserved regions within the p53 gene where the majority of p53 mutations have been reported. The sensitivity of CDGE was first tested with known p53 mutations in all four conserved regions. The CDGE technique was then used to screen 32 breast carcinomas that had been analyzed by immunohistochemical methods for altered p53 protein levels and whose DNA had already been shown to have loss of heterozygosity for a chromosome 17p marker. By immunostaining techniques, only 6 of the 32 tumors had elevated p53 expression. However, CDGE detected p53 mutations in 11 of the 32 tumors. DNA sequence analysis was performed to determine the nucleotide positions of these mutations in all 11 samples. Loss of heterozygosity for the pYNZ22 or p144D6 markers did not associate with either the loss of heterozygosity at the p53 locus or the mutations detected by CDGE. We conclude that CDGE is a rapid and effective technique to screen for p53 mutations.