[Diagnosing predisposition to tumor development by genetic typing].
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Genetic Markers↗
Biomedical subjects
G Thomas
Publications and source records attributed to G Thomas.
[Profile of p53 mutations and abnormal expression of P53 protein in 2 forms of esophageal cancer].
AIMS AND METHODS: p53 tumor suppressor gene is involved in the development of esophageal cancer. However, its role is not precisely defined in the two types of cancer, squamous cell carcinoma and adenocarcinoma developed in Barrett's esophagus. The aim of this work was to compare the frequency and type of mutation of the p53 gene and the expression of the p53 protein in a series of 21 squamous cell carcinomas and 27 adenocarcinomas of the esophagus in a single institution. p53 protein accumulation was assessed by immunohistochemistry with the monoclonal antibody PAb 1801. The mutations of exons 5 to 8 of p53 gene were detected by denaturating gradient gel electrophoresis, and sequenced. RESULTS: A mutation of the p53 gene and/or an accumulation of the p53 protein were found in 85% of squamous cell carcinomas and 92% of adenocarcinomas, respectively. The profile of mutations differed with the type of tumor; large predominance of transitions on CpG dinucleotides in adenocarcinomas suggesting an endogenous mechanism, high percentage of transversions in squamous cell carcinomas suggesting a direct effect of carcinogens present in tobacco and alcohol. CONCLUSION: Mutation of the p53 gene is a very frequent event in the two types of esophageal cancer. The mechanism responsible for these mutations is different in squamous cell carcinomas and adenocarcinomas developed in Barrett's esophagus.
Adenocarcinoma↗
The role of radiotherapy in the management of pelvic recurrence of rectal cancer.
Unresectable pelvic recurrence from carcinoma of the rectum becomes invariably symptomatic. While radiotherapy remains the most common antineoplastic modality used for palliation of symptoms, the optimal radiation dose and fractionation remains undefined. A systematic review of the literature was performed to determine the most effective dose fractionation schedule for the relief of symptoms in patients with pelvic recurrence. An expert panel reviewed and interpreted the data, with a special focus on indications, effectiveness, optimal dose fractionation, and toxicity of radiotherapy in this context. Only retrospective data (level V evidence) were available on this issue and were reviewed. Pain relief was the major indication for treatment, although bleeding and mucous discharge were also seen as indications for radiotherapy. Initial pain relief appeared to be achievable in 70-90% of patients. The median duration of pain relief was approximately three months, 23-50% of patients had symptom control at six months. The value of "local control" as a meaningful additional endpoint was discussed. There were no significant differences observable in initial symptom response and the proportion maintaining a response at six months, within the range of doses employed, comparing "lower" versus "higher" doses (using 45-50 Gy as the dividing dose). Toxicity was usually evaluated qualitatively and was deemed acceptable. The expert panel agreed that pelvic radiotherapy has a definite value in the relief of symptoms in patients with pelvic recurrence from rectal carcinoma. The optimal dose fractionation in this context could not be determined in view of the quality of the data available. Well designed, randomized studies with clinically relevant study arms and endpoints are necessary to define an optimal dose fractionation against which alternative strategies can be compared.
Humans↗
[Importance of the DIRVISH technique for detecting microdeletions].
The screening of genomic microdeletions is a difficult challenge when these deletions are too small for an efficient FISH analysis, and to large for a classic molecular detection. The DIRVISH technique appears as an attractive alternative in this situation.
Chromosomes, Human↗
S-nitrosylated tissue-type plasminogen activator protects against myocardial ischemia/reperfusion injury in cats: role of the endothelium.
S-Nitrosylated tissue plasminogen activator (tPA) is formed by S-nitrosylation of the clinically important agent tPA by nitric oxide, thus conferring nitric oxide donor properties to the molecule. Cats were subjected to 90 min of myocardial ischemia and 270 min of reperfusion and were treated with either tPA or S-nitrosylated tPA 10 min before reperfusion. S-Nitrosylated tPA-treated cats demonstrated marked attenuation of cardiac necrosis after myocardial ischemia/reperfusion, compared with cats receiving only tPA (13 +/- 3% vs. 28 +/- 3%, P < .01). Relaxation of ischemic/reperfused left anterior descending coronary artery rings in response to the endothelium-dependent dilators acetylcholine and A23187 was greater in the S-nitrosylated tPA-treated group, compared with the cats receiving only tPA, indicating that coronary vascular endothelial function was preserved by S-nitrosylated tPA. S-Nitrosylated tPA also resulted in markedly reduced adherence of neutrophils to the coronary vascular endothelium, compared with nonnitrosylated tPA (P < .01). Immunohistochemical localization of P-selectin in the ischemic region was also significantly reduced by S-nitrosylated tPA, compared with the control group (P < .01). These data indicate that S-nitrosylated tPA is a cardioprotective agent, likely exerting its effect by site-specific nitric oxide donation resulting in inhibition of neutrophil-endothelium interaction via a P-selectin-dependent mechanism.
Animals↗
Localization of TEC to 9q22.3-q31 by fluorescence in situ hybridization.
The TEC gene encodes for a novel orphan nuclear receptor. Recently, it has been shown to be involved in the recurrent t(9;22) translocation observed in extraskeletal myxoid chondrosarcoma, in a fusion gene with the EWS gene. We report her on its precise localization on chromosome 9 by fluorescence in situ hybridization.
Chondrosarcoma↗
EWS/FLI-1 antagonists induce growth inhibition of Ewing tumor cells in vitro.
Among primitive neuroectodermal tumors, Ewing tumors are characterized by a gene rearrangement recombining the 5'-EWS gene portion with one of two ETS-related proto-oncogenes, FLI-1 or ERG, in roughly 90 and 10% of cases, respectively. We attempted to antagonize EWS/FLI-1 function in Ewing tumor cell lines. As a control, a cell line derived from another small round cell tumor of neuroectodermal origin, neuroblastoma, was used. This cell line was found to express almost identical patterns of ETS proteins except for EWS/FLI-1 and a novel, ELF-related gene product. Stable expression of antisense EWS/FLI-1 cDNA resulted in decreased endogenous EWS/FLI-1 RNA levels and growth reduction of ET cells but not of neuroblastoma cells. DNA-binding FLI-1 derivatives localizing to the nucleus in which the 5'-EWS regulatory domain was replaced by bacterial beta-galactosidase dominantly modulated transcriptional transactivation from a degenerate ETS-binding motif. Transient transfection of these dominant-negative recombinants resulted in a significant decrease in the relative number of mitoses in four Ewing tumor cell lines tested but not in the neuroblastoma cell line. The presented evidence for modulation of tumor cell proliferation by EWS/FLI-1 antagonists suggests a causal role for EWS/FLI-1-mediated gene activation in the malignant transformation of the enigmatic Ewing tumor-precursor cell.
Blotting, Northern↗
[Quality assurance in heart surgery: 8 years experience with a "feedback-control" system in Heidelberg].
An important aspect of quality assurance in cardiac surgery covers the epidemiological analysis of patient data. After an 8 year period of clinical experience with quality assurance, we summarize and evaluate current concepts and actual experiences regarding a special type of database application and organisation ("feedback-control-system") for quality assurance. It had been developed to meet and solve the problems related to the data acquisition process, that are typically present in the clinical routine of quality assurance. In 1988 the "feedback-control-system" was designed and implemented in the Department of Cardiac Surgery at Heidelberg University. Since then it had been continuously improved and adapted to satisfy current needs in cardiac surgery. More than 1500 items are now recorded routinely per patient. At present, detailed information of more than 10,000 patients is available for the specific methods of analysis in the field of quality assurance. The basic concept included 1. the integration of the data acquisition in the daily clinical routine, 2. the evaluation and improvement of collected data material by means of "output-functions", that require previously recorded reliable data (that is automatically computer generated operation reports, letters, statistics, accounting etc.), and 3. to ensure that the medical and non-medical staff members participate in the advantages and the responsibilities of the data-base system for quality assurance. Analyses of perioperative risks and results, early discovery of trends, identification of special subpopulations receiving special types of treatment in cardiac surgery etc. have now become a regularly performed tool in clinical routine. This includes the availability of "problem profiles", "trend analysis", the use of simple concluding statistics as well as the calculation of multivariable models. This internal quality assurance is completed by "multicentric" comparisons with further hospitals already using the same data-base system (external quality assurance). Within 8 years, the feedback-control-system has become a reliable and valuable tool for quality assurance in daily routine. The high acceptance of the database system is related to the advantages it provides for every participant. We conclude that the concept of data evaluation and improvement by means of "output functions" and "integration of data acquisition in clinical routines" has proved to be efficient in everyday practice. The sensitivity and specifity to such a feedback controlled system as a tool for measuring surgical quality, however, still remains a matter requiring further research.
Cardiac Surgical Procedures↗
Incidence of germ-line p53 mutations in patients with gliomas.
Epidemiological studies on intracranial tumors have suggested that the observed familial aggregation of a proportion of gliomas may be due to inherited predisposition to their development. In the Li-Fraumeni syndrome (LFS) associated with germ-line mutations of the p53 gene, nervous-system tumors are observed with increased frequency. However, the contribution of germ-line p53 mutation to the incidence of brain tumors has not been investigated. In order to address this point, we have performed 2 independent investigations. First, we have examined an unselected series of brain tumors. Whenever the presence of a p53 mutation in the tumor was observed, the possible germ-line origin of the mutation was investigated. Germ-line p53 mutations were also analyzed in constitutional DNA of patients with gliomas that had been selected for an unusual personal or familial history of cancer. Germ-line p53 mutations were detected in 1 out of 80 unselected cases and in 3 out of 15 selected cases (20%). We conclude that germ-line p53 mutation may contribute to a small fraction of gliomas that develop in the general population. The presence of a personal or familial history of cancer in a patient with glioma should prompt the search for a germ-line p53 mutation. However, the low frequency of p53 germ-line mutation suggests that alterations of this gene may not account for most familial cases of gliomas.
Amino Acid Sequence↗
Intracellular trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail.
Human furin catalyzes the proteolytic maturation of many proproteins in the exocytic and endocytic secretory pathways by cleavage at the C-terminal side of the consensus sequence-ArgXaaLys/ArgArg decreases -. Both the trans-Golgi network (TGN) concentration and intracellular routing of furin require sequences in its 56 amino acid cytoplasmic tail. Here, we show that the furin cytoplasmic tail contains multiple trafficking signals. Localization to the TGN requires a cluster of acidic amino acids that, together with a pair of serine residues, forms a casein kinase II (CK II) phosphorylation site. We show that CK II efficiently phosphorylates these serines in vitro, and using a permeabilized cell system we provide evidence that CK II is the in vivo furin kinase. Analysis by mass spectrometry shows that, in vivo, furin exists as di-, mono- and non-phosphorylated forms. Finally, employing (i) furin constructs that mimic either non-phosphorylated or phosphorylated furin and (ii) the phosphatase inhibitor tautomycin, we show that the phosphorylation state of the furin cytoplasmic tail modulates retrieval of the endoprotease to the TGN. Thus, routing of furin is a two-tiered process combining a set of trafficking signals comprised of the primary amino acid sequence of the tail with its phosphorylation state.
Amino Acid Sequence↗
The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain.
The immunosuppressive agent rapamycin induces inactivation of p70s6k with no effect on other mitogen-activated kinases. Here we have employed a combination of techniques, including mass spectrometry, to demonstrate that this effect is associated with selective dephosphorylation of three previously unidentified p70s6k phosphorylation sites: T229, T389 and S404. T229 resides at a conserved position in the catalytic domain, whose phosphorylation is essential for the activation of other mitogen-induced kinases. However, the principal target of rapamycin-induced p70s6k inactivation is T389, which is located in an unusual hydrophobic sequence outside the catalytic domain. Mutation of T389 to alanine ablates kinase activity, whereas mutation to glutamic acid confers constitutive kinase activity and rapamycin resistance. The importance of this site and its surrounding motif to kinase function is emphasized by its presence in a large number of protein kinases of the second messenger family and its conservation in putative p70s6k homologues from as distantly related organisms as yeast and plants.
3T3 Cells↗
Assignment of the beta B1 crystallin gene (CRYBB1) to human chromosome 22 and mouse chromosome 5.
By using primers complementary to the rat beta B1 crystallin gene sequence, we amplified exons 5 and 6 of the orthologous human gene (CRYBB1). The amplified human segments displayed greater than 88% sequence homology to the corresponding rat and bovine sequences. CRYBB1 was assigned to the group 5 region in 22q11.2-q12.1 by hybridizing the exon 6 PCR product to somatic cell hybrids containing defined portions of human chromosome 22. The exon 5 and exon 6 PCR products of CRYBB1 were used to localize, by interspecific backcross mapping, the mouse gene (Crybb1) to the central portion of chromosome 5. Three other beta crystallin genes (beta B2(-1), beta B3, and beta A4) have previously been mapped to the same regions in human and mouse. We demonstrate that the beta B1 and beta A4 crystallin genes are very closely linked in the two species. These assignments complete the mapping and identification of the human and mouse homologues of the major beta crystallins genes that are expressed in the bovine lens.
Animals↗
Rapamycin, wortmannin, and the methylxanthine SQ20006 inactivate p70s6k by inducing dephosphorylation of the same subset of sites.
Activation of p70s6k in cells stimulated with serum correlates with the phosphorylation of seven sites. Pretreatment of Swiss 3T3 cells with the immunosuppressant rapamycin blocks phosphorylation of four of these sites (Thr229, Thr389, Ser404, and Ser411), whereas phosphorylation proceeds in the remaining three sites (Ser418, Thr421, and Ser424). If rapamycin is added postserum stimulation, the pattern of phosphorylation is qualitatively similar except that Ser411 is still highly phosphorylated. The inhibitory effect of rapamycin on serum-induced p70s6k activation and the phosphorylation of Thr229, Thr389, Ser404, and Ser411 is rescued by FK506, providing further evidence that the inhibitory effect is exerted through a complex of rapamycin-FKBP12. Wortmannin treatment pre- or post-serum stimulation inhibits phosphorylation of the same set of sites as rapamycin, supporting the argument that both agents act on the same pathway. Likewise, methylxanthine phosphodiesterase inhibitors block p70s6k activation and phosphorylation of the same set of sites as wortmannin and rapamycin. However, other agents that raise intracellular cAMP levels have no inhibitory effect, leading to the hypothesis that the inhibitory actions of methylxanthines on p70s6k activity are not through activating protein kinase A but through inhibition of an upstream kinase. Together the results indicate that there are two kinase signaling pathways that must converge to activate p70s6k and that only one of these pathways is sensitive to rapamycin, wortmannin, and methylxanthine inhibition.
3T3 Cells↗
Structure and promoter characterization of the human stromelysin-3 gene.
In the present study, we have isolated the human stromelysin-3 (ST3) gene which encodes a matrix metalloproteinase (MMP) expressed in fibroblastic cells of tissues associated with intense remodeling. The gene was found to span 11.5 kilobases (kb) including 8 exons and 7 introns. The genomic organization of ST3 gene exons is well conserved compared to other members of the MMP family, except for the 3 last exons corresponding to the hemopexin-like domain and to a long 3'-untranslated region. The transcription initiation site was located 31 nucleotides downstream of a TATA box. Analysis of 1.4 kb of 5'-flanking DNA sequence in the ST3 gene promoter revealed the presence of putative regulatory elements, but no consensus sequence for AP1-binding site in contrast to other MMP promoters. However, a specific cis-acting retinoic acid responsive element of the DR1 type was identified in the proximal region (-385) of the ST3 gene promoter. Transient transfection experiments demonstrated that a minimal promotor activity could be modulated by various sequences within the 3.4 kb of 5'-flanking region, and that the ST3 promoter was transactivated by retinoic acid receptors in the presence of retinoic acid. These findings indicate that the human ST3 gene promoter is characterized by structural and functional features which differ from those previously described in other MMP promoters, and further supports the possibility that ST3 gene expression is controlled by specific factors during tissue remodeling.
Animals↗
Frequent loss of heterozygosity on chromosome 9, and low incidence of mutations of cyclin-dependent kinase inhibitors p15 (MTS2) and p16 (MTS1) genes in gliomas.
Four cyclin-dependent kinase inhibitors called p15, p16, p21 and p27 have been identified in mammals. Because these proteins participate in the control of cell cycle, they are potential targets for somatic mutations during carcinogenesis. In order to document the prevalence of p15 and p16 alterations in gliomas, we looked for loss of heterozygosity of chromosome 9p where these genes are localized. Allelic losses were observed in 31 of 44 investigated cases. In all cases they involved the p15/p16 locus. We then looked for mutations in the p16 and p15 genes in 46 gliomas. A total of three DNA variants were observed which were all present in the matched constitutional DNA. They may be unrelated to tumor development. A single somatic mutation was detected. It involved a C to G substitution in codon 93 of p16 and is predicted to change a threonine into an arginine. Taken together, these data indicate that inactivation by point mutation of these two cyclin-dependent kinase inhibitors is uncommon in glial tumor carcinogenesis, but that there may be a tumor suppressor gene on 9p in the vicinity of p16 and p15 genes.
Base Sequence↗
Physical mapping of 30 CA repeats on human chromosome 22.
We report the physical mapping of 30 microsatellite markers specific for chromosome 22 by PCR amplification of DNA from hybrids that divide the long arm into 27 subregions. This work permits further refining of the genetic linkage ordering previously published.
Base Sequence↗
High resolution genetic map of the adenomatous polyposis coli gene (APC) region.
Familial adenomatous polyposis coli (APC) is a dominantly inherited colorectal cancer susceptibility disease caused by mutation in a gene called APC and located on chromosome 5q21. Presymptomatic diagnosis of this condition is recommended because it enables restriction of the efficient but demanding prevention program to those relatives that are genetically affected. The large size of the APC gene makes the direct search for the causal alteration difficult to implement in routine diagnostic laboratories. Because APC appears to be genetically homogeneous with alteration in a single locus causing the disease, cosegregation analysis may represent an alternative efficient method for presymptomatic diagnosis. However, the reliability of the risk estimation by linkage analysis in APC families is hampered by the lack of a short range genetic map of the APC locus. A combined approach including genotyping of 65 APC families, analysis of the CEPH database, and complementary typing of both APC and CEPH families has made it possible to derive the following genetic map: Centromere-[D5S82-D5S49]-0.02-D5S122-0.01-D5S136 -0.01-D5S135-0.02-[APC-D5S346-MCC]-0.04-[D5S81-D5S6 4]-Telomere. This order, which differs from previously proposed genetic maps, is fully compatible with recent physical mapping data. These data should contribute to increase the reliability of the presymptomatic test for APC.
Adenomatous Polyposis Coli↗
Sedation in fibreoptic bronchoscopy. Patients must be monitored.
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Procedural Sedation↗