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1.
J Neurooncol ; 52(3): 227-35, 2001 May.
Article in English | MEDLINE | ID: mdl-11519852

ABSTRACT

Cancers of the central nervous system are the most common solid tumors of childhood. Although somatic alterations of the p53 tumor suppressor gene have been implicated in brain tumorigenesis, the role of germline p53 mutations in the development of childhood brain tumors has not been well defined. As a component of an ongoing extensive study of the epidemiology of childhood brain tumors, we prospectively examined the germline and tumor p53 gene status in 85 children without a family history of cancer who were diagnosed with a sporadic malignant central nervous system tumor. Using PCR/single-strand conformational polymorphism analysis and direct DNA sequencing, 85 children were screened for the presence of constitutional p53 sequence alterations in exons 2 and 4 through 11. No mutations were identified. Commonly reported sequence polymorphisms were observed at codon 72, as well as in 2 other previously described nucleotide residues. Forty-four brain tumor samples were available for analysis and of these 40 were paired with peripheral blood. Once again, no p53 mutations were found. Of the 5 germline samples with the 2 common polymorphisms, only one had a paired tumor sample for comparison and the tumor contained the same alteration as the germline. Of note, one tumor, a PNET of the cerebellum (medulloblastoma), showed loss of heterozygosity at codon 72. We can conclude that the frequency of germline and somatic p53 mutations in sporadic childhood brain tumors is very low, probably less than 1%, and there is no need to screen these patients routinely for their germline p53 status. However, the potential significance of LOH at codon 72 remains to be elucidated.


Subject(s)
Brain Neoplasms/genetics , Genes, p53 , Germ-Line Mutation , Brain Neoplasms/blood , Brain Neoplasms/epidemiology , Cerebellar Neoplasms/blood , Cerebellar Neoplasms/genetics , Child , Child, Preschool , Codon/genetics , DNA/blood , DNA/genetics , DNA Mutational Analysis , DNA, Neoplasm/genetics , Ganglioglioma/blood , Ganglioglioma/epidemiology , Ganglioglioma/genetics , Genotype , Glioma/blood , Glioma/epidemiology , Glioma/genetics , Humans , Infant , Loss of Heterozygosity , Medulloblastoma/blood , Medulloblastoma/genetics , Neuroectodermal Tumors, Primitive/blood , Neuroectodermal Tumors, Primitive/epidemiology , Neuroectodermal Tumors, Primitive/genetics , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Prospective Studies
2.
Oncogene ; 20(33): 4441-9, 2001 Jul 27.
Article in English | MEDLINE | ID: mdl-11494139

ABSTRACT

Inactivation of wild-type p53 tumor suppressor function is the primary mechanism of tumor initiation in Li-Fraumeni syndrome (LFS) individuals with germline p53 mutations. Tumors derived from LFS patients frequently retain the normal p53 allele, suggesting that alternative mechanisms in addition to gene deletion must be involved in inactivating wild-type p53 protein. DNA tumor viruses, such as SV40, target p53 for inactivation through the action of viral oncoproteins. We studied the probands from two unrelated LFS families, each of whom presented with multiple malignant neoplasms. Patient 1 developed an embryonal rhabdomyosarcoma (RMS) and a choroid plexus carcinoma (CPC), while patient 2 developed a CPC and subsequently presented with both an osteosarcoma (OS) and renal cell carcinoma (RCC). We utilized DNA sequence analysis and immunohistochemistry to determine p53 gene status in the germline and tumors, as well as evidence for SV40 T-antigen oncoprotein expression. Each patient harbored a heterozygous germline p53 mutation at codons 175 and 273, respectively. In patient 1, the normal p53 gene was lost while the mutant p53 allele was reduced to homozygosity in the RMS. Both normal and mutant genes were maintained in the CPC. In patient 2, normal and mutant p53 alleles were retained in both the CPC and RCC. Both specific PCR and immunostaining detected SV40 T-antigen in both CPCs and the RCC. In addition to chromosomal alterations, epigenetic mechanisms may disrupt p53 function during tumorigenesis. In two LFS patients, we found SV40 DNA sequences and viral T-antigen expression that could account for inactivation of the normal p53 protein. Inactivation of p53 or other tumor suppressors by viral proteins may contribute to tumor formation in specific tissues of genetically susceptible individuals.


Subject(s)
Antigens, Polyomavirus Transforming/metabolism , Cell Transformation, Viral , DNA, Neoplasm/genetics , DNA, Viral/isolation & purification , Gene Expression Regulation, Neoplastic , Li-Fraumeni Syndrome/virology , Neoplasm Proteins/metabolism , Papillomavirus Infections/virology , Simian virus 40/physiology , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Virus Infections/virology , Antigens, Polyomavirus Transforming/genetics , Carcinoma/genetics , Carcinoma/metabolism , Carcinoma/virology , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/metabolism , Carcinoma, Renal Cell/virology , Cell Transformation, Neoplastic , Choroid Plexus Neoplasms/genetics , Choroid Plexus Neoplasms/metabolism , Choroid Plexus Neoplasms/virology , Codon/genetics , DNA, Viral/genetics , Facial Neoplasms/genetics , Facial Neoplasms/metabolism , Facial Neoplasms/virology , Fatal Outcome , Female , Genes, p53 , Genetic Predisposition to Disease , Humans , Infant , Kidney Neoplasms/genetics , Kidney Neoplasms/metabolism , Kidney Neoplasms/virology , Li-Fraumeni Syndrome/genetics , Li-Fraumeni Syndrome/metabolism , Male , Neoplasm Proteins/genetics , Organ Specificity , Osteosarcoma/genetics , Osteosarcoma/metabolism , Osteosarcoma/virology , Papillomavirus Infections/genetics , Papillomavirus Infections/metabolism , Pedigree , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Reproducibility of Results , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/metabolism , Rhabdomyosarcoma/virology , Simian virus 40/genetics , Simian virus 40/isolation & purification , Skull Neoplasms/genetics , Skull Neoplasms/metabolism , Skull Neoplasms/virology , Temporal Bone , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/metabolism
3.
Cancer Genet Cytogenet ; 123(1): 65-8, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11120338

ABSTRACT

Germline mutations of the TP53 tumor suppressor gene account for the predisposition to cancer observed in many Li-Fraumeni syndrome (LFS) families. A causative genetic factor in the remaining families that harbor no TP53 mutations remains to be elucidated. The PTEN phosphatase tumor suppressor gene is mutated in human cancers observed in LFS. There also exists some phenotypic overlap in the occurrence of cancers in LFS and Cowden's disease (CD), for which germline PTEN mutations are believed to be responsible. We hypothesized that PTEN may be altered in some TP53-wild-type LFS families. We examined LFS primary patient lymphocytes for PTEN alterations using SSCP and sequence analysis. A novel intronic deletion was found in two unrelated individuals, adjacent to the splice acceptor site of PTEN exon 4. Based on an in vitro mRNA processing assay this alteration is predicted to be a polymorphism. The in vivo effects of this proximal splice site deletion are unknown and a genetic cause for the cancers in these families remains to be elucidated. Germline mutations of PTEN were not detected in other families, suggesting that alterations of this tumor suppressor gene do not account for the cancers observed in the subset of LFS individuals with wild-type germline TP53.


Subject(s)
Introns/genetics , Li-Fraumeni Syndrome/genetics , Phosphoric Monoester Hydrolases/genetics , RNA, Messenger/genetics , Tumor Suppressor Proteins , Base Sequence , DNA/chemistry , DNA/genetics , DNA Mutational Analysis , Family Health , Female , Germ-Line Mutation , Humans , Li-Fraumeni Syndrome/pathology , Male , PTEN Phosphohydrolase , Pedigree , Polymorphism, Single-Stranded Conformational , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , Sequence Deletion
4.
Leuk Res ; 23(10): 871-80, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10573131

ABSTRACT

The role of p53 as a determinant of sensitivity of ten childhood acute lymphoblastic leukemia (ALL) cell lines to Adriamycin (ADR) was investigated. ADR-sensitive cell lines were found to have wild-type (wt) p53, whereas resistant cell lines contained point mutations in the gene. The basal level of wt p53 protein in sensitive cells was lower than that of mutant p53 in resistant cells, however, after ADR treatment a 6- to 20-fold dose-dependent increase in wt p53 was observed, whereas mutant p53 increased only twofold. The percentage of apoptotic cells in ADR-sensitive lines with wt p53 ranged from 43 to 93% following ADR treatment, whereas that in resistant lines with mutant p53 was only 8-13%. The ratio of constitutive levels of Bax/Bcl-2 was significantly higher in cells containing wt p53 than in cells with mutant p53. These results suggest that p53 gene status and the ability of p53 to induce apoptosis may be determinants of sensitivity to ADR in childhood ALL cells.


Subject(s)
Antineoplastic Agents/pharmacology , Doxorubicin/pharmacology , Drug Resistance, Neoplasm/genetics , Genes, p53 , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Antineoplastic Agents/therapeutic use , Child , Child, Preschool , Doxorubicin/therapeutic use , Gene Expression Regulation, Neoplastic , Humans , Tumor Cells, Cultured
6.
Blood ; 87(10): 4376-81, 1996 May 15.
Article in English | MEDLINE | ID: mdl-8639798

ABSTRACT

We investigated the frequency of p53 mutations in 19 pediatric cases of therapy-related leukemia or myelodysplastic syndrome. Eleven children presented with acute myeloid leukemia, one with mixed-lineage leukemia, two with acute lymphoblastic leukemia, and five with myelodysplasia at times ranging from 11 months to 9 years after a primary cancer diagnosis. The primary cancers, which included 11 solid tumors and eight leukemias, were treated with various combinations of DNA topoisomerase II inhibitors, alkylating agents, or irradiation. Leukemic or myelodysplastic marrows were screened for possible mutations by single-strand conformation polymorphism (SSCP) analysis of p53 exons 4 to 8. The only observed mutation was an inherited 2-basepair deletion at codon 209 in exon 6 that would shift the open reading frame, create a premature termination codon, and foreshorten the resultant protein. Prior therapy in this patient included DNA topoisomerase II inhibitors, alkylating agents, and irradiation. The secondary leukemia presented as myelodysplasia with monosomies of chromosomes 5 and 7 and abnormalities of chromosome 17. Although the primary cancer was an embryonal rhabdomyosarcoma and there was a family history of cancer, the case did not fulfill the clinical criteria for Li-Fraumeni syndrome. This study suggests that germline p53 mutations may predispose some children to therapy-related leukemia and myelodysplasia, but that p53 mutations otherwise are infrequent in this setting.


Subject(s)
Genes, p53 , Leukemia, Radiation-Induced/genetics , Myelodysplastic Syndromes/genetics , Neoplasms, Second Primary/genetics , Adolescent , Antimetabolites, Antineoplastic/adverse effects , Antimetabolites, Antineoplastic/therapeutic use , Antineoplastic Agents, Alkylating/adverse effects , Antineoplastic Agents, Alkylating/therapeutic use , Base Sequence , Bone Marrow/pathology , Child , Child, Preschool , Codon/genetics , Combined Modality Therapy , DNA, Neoplasm/genetics , Female , Genetic Predisposition to Disease , Humans , Infant , Leukemia/drug therapy , Leukemia/radiotherapy , Leukemia, Radiation-Induced/etiology , Li-Fraumeni Syndrome/genetics , Male , Molecular Sequence Data , Monosomy , Myelodysplastic Syndromes/etiology , Neoplasms/drug therapy , Neoplasms/radiotherapy , Neoplasms, Second Primary/etiology , Polymorphism, Single-Stranded Conformational , Radiotherapy/adverse effects , Rhabdomyosarcoma/drug therapy , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/radiotherapy , Soft Tissue Neoplasms/drug therapy , Soft Tissue Neoplasms/genetics , Soft Tissue Neoplasms/radiotherapy , Topoisomerase II Inhibitors , Translocation, Genetic
7.
J Clin Invest ; 95(4): 1606-11, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7706467

ABSTRACT

We investigated the possibility that a proportion of children with sporadic rhabdomyosarcoma (RMS) carry constitutional mutations of the p53 tumor suppressor gene. 33 patients with sporadic RMS at two large outpatient pediatric oncology clinics submitted blood samples. Genomic DNA was extracted from peripheral blood leukocytes and PCR was used to amplify exons 2-11 of the p53 gene. Amplified genomic DNA was screened for the presence of germline p53 mutations using single-strand conformation polymorphism (SSCP) analysis. The DNA sequence of those samples that showed aberrant migration of bands on SSCP analysis was determined to identify the precise nature of the gene mutations. Patient records were reviewed to assess clinical correlates of the mutant p53 carrier state. Heterozygous constitutional mutations were detected in 3/33 patient samples screened. Two of these missense mutations are located in exon 7 and one in exon 8 of the p53 gene. The presence of mutations was not correlated with tumor histology, stage, or site. However, an association between young age at diagnosis and presence of a constitutional p53 mutation was noted: 3/13 children under the age of 3 yr at diagnosis carried mutations, whereas none of 20 children over 3 yr of age at diagnosis harbored a detectable constitutional mutation. These results in children with RMS corroborates previous findings in other clinical settings suggesting that the mutant p53 carrier state may predispose individuals to malignancy at an early age. Although this study did not assess whether the mutations were preexisting or new germline alterations, assessment of close relatives of RMS patients for cancer risk and predictive genetic testing may be indicated.


Subject(s)
Point Mutation , Rhabdomyosarcoma/genetics , Tumor Suppressor Protein p53/genetics , Adolescent , Age Factors , Base Sequence , Child , Child, Preschool , Disease Susceptibility , Exons/genetics , Female , Humans , Infant , Lymphocytes , Male , Molecular Sequence Data , Polymerase Chain Reaction , Polymorphism, Genetic , Rhabdomyosarcoma/classification , Rhabdomyosarcoma/diagnosis , Sequence Analysis, DNA
8.
Cancer Res ; 54(8): 2077-9, 1994 Apr 15.
Article in English | MEDLINE | ID: mdl-8174107

ABSTRACT

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.


Subject(s)
Genes, Wilms Tumor , Genes, p53 , Kidney Neoplasms/genetics , Point Mutation , Wilms Tumor/genetics , Adult , Aged , Aged, 80 and over , Amino Acid Sequence , Base Sequence , Child , Child, Preschool , Codon , Exons , Female , Gene Expression , Humans , Kidney Neoplasms/drug therapy , Kidney Neoplasms/pathology , Male , Middle Aged , Neoplasm Staging , Wilms Tumor/drug therapy , Wilms Tumor/pathology
9.
J Biol Chem ; 265(30): 18192-9, 1990 Oct 25.
Article in English | MEDLINE | ID: mdl-2120224

ABSTRACT

Histidase (histidine ammonia-lyase, EC 4.3.1.3) catalyzes the deamination of histidine to urocanic acid. Apart from phenylalanine ammonia-lyase, which is not expressed in animals, histidase is the only enzyme known to have a dehydroalanine residue in its active site. The amino site precursor and the mechanism of formation of dehydroalanine are not known. As an initial step to determining the precursor of dehydroalanine in histidase, we have isolated a functional cDNA clone for histidase from a rat liver cDNA library using an affinity-purified antiserum. The 2.2-kilobase cDNA has a 1,971-base pair open reading frame coding for a 657-amino acid polypeptide with a predicted molecular mass of 72,165 Da. The cDNA has a rare polyadenylation signal (AAUACA) that appears to inefficiently direct polyadenylation in transfected COS monkey kidney cells. Conversion of this sequence to the consensus polyadenylation signal (AAUAAA) resulted in increased levels of stable mRNA. COS cells transfected with a histidase expression vector produce active histidase. The formation of active histidase in cells that have no endogenous histidase activity suggests either that the requisite modifying enzyme is present in these cells or that the dehydroalanine residue forms by an autocatalytic mechanism. Rat histidase was found to have 41 and 43% amino acid identity to Pseudomonas putida and Bacillus subtilis histidases, respectively. Phenylalanine ammonia-lyases from parsley, kidney bean, and two yeast strains were also found to have approximately 20% amino acid identity to rat histidase. On the basis of the similarity of function of histidase and phenylalanine ammonia-lyase, dehydroalanine at the active sites, and the sequence conservation over a large evolutionary distance (mammals, bacteria, yeast, and plants), we propose that the genes for histidase and phenylalanine ammonia-lyase have diverged from a common ancestral gene, of which the most conserved regions are likely to be involved in catalysis or dehydroalanine formation.


Subject(s)
Histidine Ammonia-Lyase/genetics , Amino Acid Sequence , Animals , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Base Sequence , Blotting, Northern , Cell Line , Cloning, Molecular , DNA/genetics , Fungal Proteins/genetics , Gene Expression Regulation, Enzymologic , Molecular Sequence Data , Molecular Weight , Phenylalanine Ammonia-Lyase/genetics , Plant Proteins/genetics , Poly A/genetics , Pseudomonas/genetics , Rats , Regulatory Sequences, Nucleic Acid , Restriction Mapping
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