Somatic point mutations in the p53 gene of human tumors and cell lines: updated compilation.

In 1994 we described a list of approximately 2500 point mutations in the p53 gene of human tumors and cell lines which we had compiled from the published literature and made available electronically through the file server at the EMBL Data Library. This database, updated twice a year, now contains records on 4496 published mutations (July 1995 release) and can be obtained from the EMBL Outstation-the European Bioinformatics Institute (EBI) through the network or on CD-ROM. This report describes the criteria for inclusion of data in this database, a description of the current format and a brief discussion of the current relevance of p53 mutation analysis to clinical and biological questions.

Homozygous deletion frequency and expression levels of the CDKN2 gene in human sarcomas--relationship to amplification and mRNA levels of CDK4 and CCND1.

Homozygous deletions of the putative tumour-suppressor gene CDKN2, which encodes an inhibitor of cdk4, have been detected in a high percentage of cancer cell lines of various histological types. In the present study, 109 human sarcomas were examined for homozygous deletions and for mRNA expression levels of the CDKN2 gene. Altogether, deletions were found in only eight (7%) of the cases, but, interestingly, in two (of eight) malignant Schwannomas and in two (of five) rhabdomyosarcomas.

Semiquantitative polymerase chain reaction for t(14;18) in follicular lymphomas: a colorimetric approach.

Background: Autologous bone marrow transplantation is increasingly being used in the management of several types of cancer, and to avoid reintroduction of malignant cells, bone marrow purging is often performed. In such cases, sensitive quantitation methods are needed both to assess the efficacy of the purging and for surveillance of patients in remission. Polymerase chain reaction (PCR) has the necessary sensitivity for this application, but it requires that the cancer cells can be recognized by a defined genetic abnormality.

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.

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.

Genome scanning of human breast carcinomas using micro- and minisatellite core probes.

We have analyzed tumor and lymphocyte DNA from six breast cancer patients by one- and two-dimensional DNA fingerprinting using micro- and minisatellite core probes to estimate the extent and nature of DNA alterations in tumors. Both approaches were compared regarding sensitivity in genome analysis. We find that the number of deletions and amplifications increases linearly with the number of restriction fragments analyzed using the two-dimensional approach, as compared with the number found using the more traditional one-dimensional method.

p53 abnormalities in different subtypes of human sarcomas.

In this report we examined p53 alterations at the DNA, mRNA, and protein levels on tissue from 39 patients with different subtypes of sarcoma. Loss of heterozygosity for the chromosome 17p region was found in 60, 63, and 33% of 10 informative osteosarcomas, 11 malignant fibrous histiocytomas, and 6 leiomyosarcomas, respectively. In addition, 2 of 10 tumors belonging to a heterogeneous group of soft tissue sarcomas showed loss of heterozygosity.

A TP53 mutation detected in cells established from an osteosarcoma, but not in the retinoblastoma of a patient with bilateral retinoblastoma and multiple primary osteosarcomas.

A patient with bilateral retinoblastoma and subsequent multiple primary osteosarcomas has been described previously. Osteosarcoma cell lines established from this patient were shown to express a shortened RB1 mRNA transcript and no detectable normal Rb protein. We now show that the osteosarcoma cell lines have lost one TP53 allele and contain a mutation in exon 8 codon 286 [GAA to AAA (Glu to Lys)] in the remaining allele.

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.

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.

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.

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.

Chromosome 13 alterations in osteosarcoma cell lines derived from a patient with previous retinoblastoma.

Various sublines of cells established from an osteosarcoma that developed in a patient (O.H.) with previous bilateral retinoblastoma were examined for different restriction fragment-length polymorphisms of chromosome 13q, as well as for rearrangements of the retinoblastoma gene using a cDNA probe.

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.

T-cell receptor tau delta +/CD3+4-8-T- cell acute lymphoblastic leukemias: a distinct subgroup of leukemias in children. A report of five cases.

In a 10-year study of T-cell acute lymphoblastic leukemias (T-ALL) in children, we have identified five cases expressing the T-cell receptor tau delta (TCR tau delta). The incidence (26%) of TCR tau delta+T-cell leukemias in our material was high. Clinically, the TCR tau delta+ leukemias represented a distinct subgroup of T-cell leukemias.

Constant denaturant gel electrophoresis, a modification of denaturing gradient gel electrophoresis, in mutation detection.

Denaturing gradient gel electrophoresis (DGGE) is increasingly being utilized in mutational detection, both in characterization of variations in genomic DNA and in the generation of mutational spectra after in vitro and in vivo mutagenesis. The basis for this electrophoretic separation technique is strand dissociation of DNA fragments in discrete, sequence-dependent melting domains followed by an abrupt decrease in mobility. We have modified the DGGE by using constant denaturant gels corresponding to the specific melting domains of certain DNA fragments.

Formation of active diphtheria toxin in vitro based on ligated fragments of cloned mutant genes.

An intact gene coding for wild type diphtheria toxin was reconstituted in vitro from fragments of cloned inactive diphtheria toxin mutants with defects in different parts of the gene. The reconstituted DNA template was amplified using the Taq DNA polymerase chain reaction, providing a virtually unlimited supply. The toxin was expressed in vitro by transcription from a T3 RNA polymerase promoter, followed by translation of the mRNA in a rabbit reticulocyte lysate system.

Detection of base mutations in genomic DNA using denaturing gradient gel electrophoresis (DGGE) followed by transfer and hybridization with gene-specific probes.

It has been shown that minor differences, such as single-base-pair substitutions between otherwise identical DNA fragments can result in altered melting behavior detectable by denaturing gradient gel electrophoresis (DGGE). Sequence variations in only a small DNA region within one locus can be detected using the previously described procedures. We have developed a method for the efficient Southern transfer of genomic DNA fragments from the denaturing gradient gels in order to be able to analyze larger regions in several loci for variation.

Chromosome 13 instability and esterase D expression in an osteosarcoma cell line.

In order to assess the involvement of the 13q14 region in the development of osteosarcoma, both osteosarcoma tumor cells and normal tissue from a retinoblastoma patient previously used in restriction fragment length polymorphism studies, and sarcoma cells and normal fibroblasts from other tumor patients, have been investigated with respect to esterase D (E.C. 3.