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1/4. Tissue-specific expression of SV40 in tumors associated with the li-fraumeni syndrome.

    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.
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2/4. Burkitt-like lymphoma in an English child: characterisation of tumour biopsy cells and of the derived tumour cell line.

    An eight year old English boy presented with an abdominal undifferentiated 'Burkitt-like' lymphoma. lymphoma cells from ascitic fluid were cultured on a human embryo fibroblast feeder layer and, after a short lag period, a cell line (DH-BL) was established which, like the original tumour, was both negative for the Epstein-Barr nuclear antigen (EBNA) and expressed a monoclonal pattern of surface immunoglobulin (alpha lambda). DH-BL also possessed the Burkitt-related 8:14 chromosome translocation in all metaphases analysed; no other chromosomal abnormalities were present. The cell surface phenotype of the original biopsy cells and the cultured tumour cells in early passage were investigated using a panel of monoclonal antibodies to B lineage-associated antigens. These antibodies had recently been used to characterise African 'endemic' Burkitt's lymphoma (BL) biopsy cells and their derived cell lines. The cell surface phenotype of this English EBNA negative Burkitt-like lymphoma biopsy was indistinguishable from that previously shown by biopsies of EBNA positive endemic BLs. It therefore appears that both the endemic and sporadic forms of BL, as illustrated by this case, may be derived from the same subset of progenitor cells.
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3/4. Single amino acid substitution (58Pro-->Ser) in HTLV-I tax results in loss of ras cooperative focus formation in rat embryo fibroblasts.

    Two tax genes cloned from a healthy HTLV-I carrier in whom the viral genome is clonally integrated into peripheral CD4 CD8 cells showed a considerable difference in ras cooperative focus forming ability in rat embryo fibroblasts (REF). sequence analysis revealed differences in two codons of the two genes. SH-1tax and SH-2tax. Studies using recombinants between these two tax genes showed that the deficiency in ability of SH-1tax to cooperate with ras in focus formation in REF was caused by a 58Pro-->Ser substitution. This amino acid substitution did not affect other tax functions such as colony formation in soft agar, focus formation in Rat-1 cells, immortalization of REF, and transcriptional activation through the CREB/ATF and NFkB/rel pathways. These results suggest that the domain of tax required for cooperative focus formation with ras in primary rat fibroblasts may be different from those required for other tax functions.
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4/4. Preimplantation genetic diagnosis of marfan syndrome with the use of fluorescent polymerase chain reaction and the Automated Laser fluorescence DNA Sequencer.

    OBJECTIVE: To develop and apply clinical preimplantation genetic diagnosis (PGD) for marfan syndrome. DESIGN: Case report. SETTING: Centers for medical genetics and reproductive medicine in university hospitals. PATIENT(S): One couple in which the husband was affected with marfan syndrome. INTERVENTION(S): The couple underwent three intracytoplasmic sperm injection cycles. MAIN OUTCOME MEASURE(S): The correct diagnosis was obtained for embryos in three PGD cycles. RESULT(S): Although all the PGD cycles were followed by ET, no pregnancy ensued. CONCLUSION(S): This assay can provide a reliable and accurate preimplantation diagnosis of marfan syndrome.
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