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1.
J Invest Dermatol ; 114(2): 277-80, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10651986

ABSTRACT

The PTEN/MMAC1 gene on chromosome 10q23 encodes a lipid phosphatase with tumor-suppressive properties. Germline PTEN/MMAC1 mutations have been implicated as the predisposing factor in Cowden disease and other hamartoma syndromes, and somatic mutations and deletions have been identified in a wide range of human cancers, including 30-40% of metastatic melanoma cell lines. To study further the possible role of PTEN/MMAC1 in the pathogenesis and progression of malignant melanoma, we examined uncultured specimens from 16 primary and 61 metastatic tumors from 67 patients. Denaturing gradient gel electrophoresis was used to analyze systematically the coding region of PTEN/MMAC1 and revealed mutations in four of the metastatic samples (7%). Sequence analysis of the mutants identified a 1 bp frameshift insertion, a 2 bp frameshift deletion, an 11 bp frameshift deletion, and a single base substitution resulting in the generation of a premature stop codon. Analysis of two intragenic polymorphisms showed allelic loss in three of eight informative primary tumors (38%) and in 18 of 31 metastatic tumors (58%). One of the mutant cases showed allelic loss, suggesting that both PTEN/MMAC1 alleles were inactivated in this tumor. Altogether, these results suggest that mutation and deletion of PTEN/MMAC1 may contribute to the development and progression of malignant melanoma.


Subject(s)
Melanoma/pathology , Melanoma/secondary , Phosphoric Monoester Hydrolases/genetics , Tumor Suppressor Proteins , Biopsy , Genes, Tumor Suppressor , Germ-Line Mutation , Humans , Loss of Heterozygosity , PTEN Phosphohydrolase , Polymerase Chain Reaction , Polymorphism, Genetic
2.
Oncogene ; 18(9): 1777-80, 1999 Mar 04.
Article in English | MEDLINE | ID: mdl-10208439

ABSTRACT

Mutations in LKB1/STK11, a gene mapping to chromosome 19p13.3 and encoding a widely expressed serine/threonine kinase, were recently identified as the cause of Peutz-Jeghers syndrome. Despite the hamartomatous polyps and increased cancer risk associated with this syndrome, somatic alterations in LKB1/STK11 have not been identified in human tumours. Prompted by another feature of the syndrome, lentigines of the lips and oral mucosa, we evaluated the status of LKB1/STK11 expression, deletion, and mutation in cell lines and tumour samples from 35 patients with sporadic malignant melanoma. Two somatic mutations were identified, a nonsense mutation (Glu170Stop) causing exon skipping and intron retention, and a missense mutation (Asp194Tyr) affecting an invariant residue in the catalytic subunit of LKB1/STK11. Our data suggest that LKB1/STK11 may contribute to tumorigenesis in a small fraction of malignant melanomas.


Subject(s)
Mutation , Peutz-Jeghers Syndrome/enzymology , Peutz-Jeghers Syndrome/genetics , Protein Serine-Threonine Kinases/genetics , AMP-Activated Protein Kinase Kinases , Humans , Melanoma , Tumor Cells, Cultured
3.
Blood ; 92(9): 3018-24, 1998 Nov 01.
Article in English | MEDLINE | ID: mdl-9787134

ABSTRACT

Fas (APO-1/CD95) is a cell-surface receptor involved in cell death signaling. Germline mutations in the Fas gene have been associated with autoimmune lymphoproliferative syndrome, and somatic Fas mutations have been found in multiple myeloma. We have examined the entire coding region and all splice sites of the Fas gene in 150 cases of non-Hodgkin's lymphoma. Overall, mutations were identified in 16 of the tumors (11%). Missense mutations within the death domain of the receptor were associated with retention of the wild-type allele, indicating a dominant-negative mechanism, whereas missense mutations outside the death domain were associated with allelic loss. Fas mutations were identified in 3 (60%) MALT-type lymphomas, 9 (21%) diffuse large B-cell lymphomas, 2 (6%) follicle center cell lymphomas, 1 (50%) anaplastic large cell lymphoma, and 1 unusual case of B-cell chronic lymphocytic leukemia with a marked tropism for skin. Among the 16 patients with somatic Fas mutations, 15 showed extranodal disease at presentation, and 6 relapsed in extranodal areas. Ten of 13 evaluable patients showed features suggestive of autoreactive disease. Our data indicate that somatic disruption of Fas may play a role in the pathogenesis of some lymphomas, and suggest a link between Fas mutation, cancer and autoimmunity.


Subject(s)
Autoimmunity/genetics , DNA, Neoplasm/genetics , Lymphoma, Non-Hodgkin/genetics , Neoplasm Proteins/genetics , fas Receptor/genetics , Adult , Aged , Aged, 80 and over , Alleles , Amino Acid Substitution , Apoptosis , Autoimmune Diseases/complications , Autoimmune Diseases/genetics , Codon/genetics , DNA Mutational Analysis , Female , Genetic Predisposition to Disease , Humans , Lymphoma, Non-Hodgkin/classification , Lymphoma, Non-Hodgkin/immunology , Lymphoma, Non-Hodgkin/pathology , Male , Middle Aged , Mutation, Missense , Neoplasm Proteins/physiology , Paraneoplastic Syndromes/etiology , Paraneoplastic Syndromes/immunology , Paraneoplastic Syndromes/pathology , Polymorphism, Genetic , Protein Structure, Tertiary , RNA Splicing/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sjogren's Syndrome/complications , Sjogren's Syndrome/genetics , Thyroiditis, Autoimmune/complications , Thyroiditis, Autoimmune/genetics , fas Receptor/physiology
4.
Nucleic Acids Res ; 26(6): 1548-9, 1998 Mar 15.
Article in English | MEDLINE | ID: mdl-9490806

ABSTRACT

Denaturing gradient gel electrophoresis (DGGE) in combination with PCR and 'GC-clamping' has proven highly efficient as a method for detection of DNA sequence differences. Due to strand dissociation phenomena, however, its use has been limited to the analysis of sequences with a relatively low content of GC pairs. This paper describes how treatment of template DNA with sodium bisulphite drastically lowers the melting temperature of very GC-rich sequences and renders them amenable to DGGE analysis. We demonstrate the use of bisulphite DGGE for rapid and efficient detection of mutations in the p16(INK4/CDKN2) tumour suppressor gene.


Subject(s)
DNA Mutational Analysis/methods , DNA/chemistry , DNA/genetics , Electrophoresis/methods , Mutation , Base Composition , Base Sequence , DNA/isolation & purification , DNA Primers/genetics , Evaluation Studies as Topic , Genes, p16 , Humans , Nucleic Acid Denaturation , Polymerase Chain Reaction , Sulfites
5.
Cancer Res ; 57(17): 3660-3, 1997 Sep 01.
Article in English | MEDLINE | ID: mdl-9288767

ABSTRACT

The MMAC1/PTEN gene, located at 10q23.3, is a candidate tumor suppressor commonly mutated in glioma. We have studied the pattern of deletion, mutation, and expression of MMAC1/PTEN in 35 unrelated melanoma cell lines. Nine (26%) of the cell lines showed partial or complete homozygous deletion of the MMAC1/PTEN gene, and another six (17%) harbored a mutation in combination with loss of the second allele. Mutations could also be demonstrated in uncultured tumor specimens from which the cell lines had been established, and cell lines derived from two different metastases from one individual carried the same missense mutation. Collectively, these findings suggest that disruption of MMAC1/PTEN by allelic loss or mutation may contribute to the pathogenesis or neoplastic evolution in a large proportion of malignant melanomas.


Subject(s)
Chromosomes, Human, Pair 10/genetics , Gene Deletion , Genes, Tumor Suppressor/genetics , Melanoma/genetics , Mutation , Phosphoric Monoester Hydrolases , Protein Tyrosine Phosphatases/genetics , Skin Neoplasms/genetics , Tumor Suppressor Proteins , Base Sequence , Exons/genetics , Humans , Molecular Sequence Data , PTEN Phosphohydrolase , Polymerase Chain Reaction , Tumor Cells, Cultured
6.
Int J Cancer ; 72(5): 780-3, 1997 Sep 04.
Article in English | MEDLINE | ID: mdl-9311594

ABSTRACT

The cyclin-dependent kinase 4 (CDK4) is a key component in regulation of the mammalian cell cycle. The recent discovery of a common missense mutation (Arg24Cys) in both sporadic and familial forms of malignant melanoma strongly supports the candidacy of CDK4 as a proto-oncogene. To study further the role of CDK4 in melanoma pathogenesis, we have established a method based on polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE) to scan the CDK4 gene for point mutations. By analyzing the entire coding sequence of the CDK4 gene in 56 sporadic metastatic malignant melanomas, we identified a novel missense mutation, Asn41Ser. This mutation was also found in the germline of the patient who had no family history of melanoma. Analysis of a tumor-derived cell line demonstrated equal expression of the mutant and wild-type CDK4 alleles, together with lack of functional p16. Our findings suggest that an oncogenic mechanism of the CDK4-Asn41Ser variant would be different from the CDK4-Arg24Cys variant. Altogether, our data demonstrate that point mutation of CDK4 is a rare event in melanoma pathogenesis.


Subject(s)
Cyclin-Dependent Kinases/genetics , DNA Mutational Analysis/methods , Melanoma/genetics , Mutation , Proto-Oncogene Proteins , Asparagine/genetics , Biopsy , Cyclin-Dependent Kinase 4 , Electrophoresis, Polyacrylamide Gel , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Proto-Oncogene Mas , Serine/genetics , Tumor Cells, Cultured
7.
Hum Mutat ; 9(4): 348-55, 1997.
Article in English | MEDLINE | ID: mdl-9101296

ABSTRACT

We present a simple nonradioactive assay based on polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE), which allows comprehensive mutation scanning of the entire coding sequence and splice junctions of the p53 gene in one analytical step. The key features of the present method are (1) all fragments can be amplified using one common PCR protocol; (2) all fragments can be scanned for mutations on a single "broad-range" denaturing gradient gel; and (3) all fragments were tailored by the attachment of appropriate GC clamps and otherwise manipulated to consist of only two melting domains in order to maximize resolution of mutations by DGGE. The entire procedure starting with a sample of genomic DNA can be completed within 6 hr and has the potential to detect any sequence variation, irrespective of its location in the p53 gene. The mutation detection sensitivity was demonstrated by the analysis of 26 constructed control mutations distributed over the whole of the p53 gene. We have applied the method to genetic diagnosis in 43 cases of colorectal cancer. Overall, a point mutation, microdeletion, or microinsertion was found in 26 (61%) of the tumors. In addition to missense and frameshift mutations within exons 4-8, a 20-bp insertion in exon 11 was found in one sample, illustrating the importance of comprehensive gene scanning for reliable diagnosis.


Subject(s)
Colorectal Neoplasms/diagnosis , Electrophoresis, Polyacrylamide Gel/methods , Genes, p53/genetics , Mutation , Polymerase Chain Reaction/methods , Aged , Aged, 80 and over , Algorithms , Base Sequence , Colorectal Neoplasms/genetics , DNA, Neoplasm/analysis , DNA, Neoplasm/chemistry , Female , Humans , Male , Middle Aged , Molecular Sequence Data , Nucleic Acid Denaturation , RNA Splicing/genetics , Rectal Neoplasms/diagnosis , Rectal Neoplasms/genetics
8.
Nucleic Acids Res ; 21(18): 4272-80, 1993 Sep 11.
Article in English | MEDLINE | ID: mdl-8414982

ABSTRACT

We have significantly improved a method originally developed by Liang and Pardee [Science 257 (1992) 967-971] to display a broad spectrum of expressed genes and to detect differences in expression between different cell types. We have analysed various aspects of the technique and have modified it for both, the application to fast and efficient identification of genes and the use with automatic analysis systems. Based on the mathematical background we have devised the appropriate number of optimal PCR primers. We have also introduced nondenaturating gels for separating double stranded fragments as single bands. By applying the method to regenerating mouse liver, we have identified, out of a total of 38,000 bands, about 70 fragments where the expression of the corresponding genes seems to be differentially regulated at different time points. Application of the method to an automatic DNA sequencer was successfully done. Thus, we have confirmed the usefulness and increased the power of the RNA display technique, which we named differential display reverse transcription PCR (DDRT-PCR), and have extended the range of its application.


Subject(s)
Polymerase Chain Reaction/methods , RNA, Messenger/analysis , Animals , Base Sequence , DNA, Single-Stranded , Gene Expression , Liver Regeneration/genetics , Mice , Molecular Sequence Data
9.
Cancer Immunol Immunother ; 31(2): 93-8, 1990.
Article in English | MEDLINE | ID: mdl-2108803

ABSTRACT

In this study we have treated three malignant (TGrIII) and two pre-malignant (TGrII) urothelial cell lines with recombinant human interferon gamma (rHu-INF gamma). The malignant cells (HCV29-T112C1, Hu1703He and T24) were inhibited in growth by more than 50% after treatment with 100-1000 units of rHu-INF gamma/ml for 4 days as compared to untreated controls. The growth of the pre-malignant cell lines (HCV29 and Hu609) was not influenced to the same extent in the presence of rHu-INF gamma in the culture medium. Treatment with rHu-INF gamma increased the expression of monomorphic human leukocyte antigens (HLA) A,B,C as well as beta 2-microglobulin in all the cell lines tested, as demonstrated using a quantitative immunofluorescence assay. The tumourigenic cell lines increased their expression of HLA in a dose-dependent way, whereas treatment of the non-tumourigenic cells with higher concentrations of rHu-INF gamma than 10 units/ml, did not increase the HLA-A,B,C expression further. None of the cell lines expressed HLA-DR unless treated with rHu-INF gamma. No correlation between tumourigenicity and the dose of rHu-INF gamma required for "de novo" induction of HLA-DR could be demonstrated. After removal of rHu-INF gamma from the medium, the expression of HLA-DR gradually decreased in less than 14 days, indicating that the expression of HLA-DR is not constitutive but dependent upon the presence of rHu-INF gamma. We conclude that human urothelial cells grown in vitro are sensitive to the anti-proliferative and major-histocompatibility-complex-modulating effects of rHu-INF gamma, and that malignant urothelial cells are more sensitive than pre-malignant cells. Finally, our data indicate a possible role for rHu-INF gamma in the management of human bladder cancer.


Subject(s)
HLA-A Antigens/analysis , HLA-B Antigens/analysis , HLA-C Antigens/analysis , HLA-DR Antigens/analysis , Interferon-gamma/pharmacology , Urinary Bladder Neoplasms/immunology , Animals , Cell Division/drug effects , Humans , Rabbits , Recombinant Proteins , Tumor Cells, Cultured , Urinary Bladder Neoplasms/pathology , beta 2-Microglobulin/analysis
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