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Biomedical subjects

E Hovig

Publications and source records attributed to E Hovig.

48 records · Page 3Linked to original sources
PubMed1992

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. Consequently, the osteosarcoma cell lines have no normal Rb protein and no normal p53 protein. Neither constitutional DNA nor DNA extracted from a retinoblastoma of the left eye of the patient contained the TP53 mutation, suggesting that the TP53 mutation in the osteosarcoma cells may represent a tumor-promoting mutation, which confers a selective growth advantage. If both RB1 and TP53 are involved in the initiation of osteosarcoma, the mechanisms for development of the retinoblastoma and osteosarcoma tumors are different.

Base Sequence↗
PubMed1992

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.

Animals↗
PubMed1991

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.

Base Sequence↗
PubMed1991

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. Mean age at onset of disease, 1.8 years, was remarkably low for mature T-cell leukemias. White blood cell counts were high, lymph node enlargements were discrete, and no mediastinal tumors were seen. Four of five TCR tau delta+ leukemias carried rearrangements of the C tau 2 gene, and transcribed the T-early alpha genetic element.

Antigens, CD↗
PubMed1991

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. The independently established sublines were used to help separate primary and secondary events taking place in tumorigenesis of the osteosarcoma of this patient. Information from the present DNA analysis, taken together with data from cytogenetic and enzymatic studies on chromosome 13 in the cell lines, revealed both common and distinct genetic changes on chromosome 13q. The common changes may indicate the nature of the first and second mutational events in the development of the osteosarcoma. The first, constitutional cancer predisposing mutation seemed to be a base mutation or a small deletion/insertion, and the second event involved a deletion of a larger part of the long arm of chromosome 13. The distinct genetic changes included other deletion and duplication events of chromosome 13q. The existence of multiple sublines with different genetic constitutions provides improved possibilities for gaining insight into the nature of the genetic lesions leading to tumor formation, as these may reflect the clonal variation present in the primary tumor. We also demonstrate the difficulty of inferring from single tumor cell isolates to properties of the primary tumor.

Chromosome Aberrations↗
PubMed1991

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. This leads to increased resolution of mutants as fragments differing in as little as 1 base pair migrate with a consistently different mobility through the whole gel allowing separations of several centimeters. By using a set of constant denaturant gels it is also possible to obtain a better approximation of the location of the different mutations as each denaturant concentration will correspond to specific melting domains. We have used this technique to separate 6 out of 7 exon-3 hypoxanthine phosphoribosyltransferase (HPRT) mutants while using conventional DGGE we were only able to separate 3.

Animals↗
PubMed1989

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. Diphtheria toxin synthesised in this manner was shown to be as toxic as natural toxin and behaved like natural toxin in every way tested. This can serve as a general approach for study of toxic proteins, opening up the possibility of in vitro gene manipulation. Reconstitution of the wild type gene from fragments followed by polymerase chain reaction amplification bypasses the hazards of cloning protein toxins.

Animals↗
PubMed1988

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. The gels were made using polyacrylamide containing 2% low-geling-temperature agarose (LGT). The polyacrylamide gel (PAG) was crosslinked with a reversible crosslinker, and after electrophoresis the crosslinks were cleaved, the structure of the gel being maintained by the agarose. After this treatment of the denaturing gels, more than 90% of the DNA fragments could be transferred to nylon membranes by alkaline transfer, while electroblotting transferred only 10% of the DNA. Hybridization with gene-specific probes was then performed. We have used this technique to identify an RFLP in the COL1A2 gene in a human genomic DNA sample. The transfer technique described should make the use of DGGE more widely applicable since the genomic DNA fragments separated on one gel can be screened with several different probes, both cDNA and genomic probes.

Base Sequence↗
PubMed1987

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. 3.1.1.1) expression and chromosome pattern. In spite of an increased number of apparently normal chromosomes #13, a 50% reduction in esterase D activity in osteosarcoma cells from the retinoblastoma patient was observed. This suggests that loss of the RB1 gene or an OSRC gene closely linked to the ESD and RB1 gene loci is involved in the development of the osteosarcoma tumor. No reduction in esterase D expression was seen in four other sarcoma cell lines.

Carboxylesterase↗
PubMedDate not supplied

CA 125: the end of the beginning.

CA 125, a high-molecular-weight mucin, was first defined in 1981 by the monoclonal antibody OC125. Until recently, it has defied many attempts to purify it from a variety of sources, although many research groups have successfully raised antibodies that bind to CA 125. Nevertheless, CA 125 has demonstrated its considerable value as a marker in monitoring patients with ovarian cancer. This year, two research groups have succeeded in cloning the high-molecular-weight mucin CA 125. Their findings are summarized and the significance discussed in light of existing data from the human genome.

CA-125 Antigen↗
PubMedDate not supplied

Interleukin-1 alpha and basic fibroblast growth factor induction of matrix metalloproteinases and their inhibitors in osteosarcoma cells is modulated by the metastasis associated protein CAPL.

BACKGROUND: Several recent investigations have shown that the expression of the CAPL protein seems to be of importance in the metastatic potential in some types of cancer. However, the mechanisms behind this and other biological functions of CAPL are still largely unknown. The aim of the present work was to investigate whether CAPL could affect the expression of candidate proteolytic facilitators of the metastatic process, i.e. matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). MATERIALS AND METHODS: A highly metastatic osteosarcoma cell-line with a high expression of CAPL was transfected with either a vector containing a ribozyme against this transcript, or with the vector alone as a control. The expression of MMPs and TIMPs was investigated with ELISA and gelatin zymography. RESULTS: The cell-line with a low CAPL expression (III-14) responded to bFGF treatment by an increased synthesis of MMP-1 and MMP-9 and to Il-1 alpha treatment by an increased synthesis of MMP-9. In contrast, the cell-line with a high CAPL expression (pH beta-1) did not respond with an altered expression of these MMPs. Neither of these two cell-lines responded with an altered expression of MMP-2. bFGF treatment resulted in an increased expression of TIMP-1 in both cell-lines, while Il-1 alpha treatment resulted in a decreased production of TIMP-1 in pH beta-1 cells, and III-14 cells were unaffected. CONCLUSIONS: The CAPL protein expressed in cell-cultures appear to block the MMP induction by bFGF and Il-1 alpha. However, the induction of TIMP-1 by bFGF must proceed through a pathway different from the MMP induced pathway, i.e. a pathway unaffected by CAPL. In addition, CAPL appeared to act in synergy with Il-1 alpha to reduce the synthesis of TIMP-1.

Bone Neoplasms↗