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PubMed1999

Expression of p53 and retinoblastoma gene in high-grade nodal peripheral T-cell lymphomas: immunohistochemical and molecular findings suggesting different pathogenetic pathways and possible clinical implications.

The expression of p53 and the retinoblastoma gene has been investigated by immunohistochemical and molecular analysis in 45 cases of nodal peripheral T-cell lymphoma with high-grade histology. Most cases (73.3 per cent) were primary nodal lymphomas without any extra-nodal site involvement. Most of them (75.6 per cent) were histologically classified as pleomorphic, small, medium, and large cell type. Immunohistochemistry detected p53 in nine cases (20 per cent). In each of these cases, the polymerase chain reaction (PCR)/heteroduplex analysis did not show the presence of mutations, this finding being consistent with an alteration of the p53 functional pathway, in the presence of a wild-type protein. The retinoblastoma gene product was detected by immunohistochemistry in 35 cases (77.8 per cent) and not detected in ten cases (22.2 per cent). In the latter cases, the reverse transcription (RT)-PCR analysis showed the presence of a specific retinoblastoma gene transcript in six cases and was negative in the remaining four cases. The immunohistochemical and molecular findings seem to be consistent with abnormalities of retinoblastoma gene expression at either the transcriptional or the post-transcriptional level. Since all nine p53-positive cases by immunohistochemical analysis were also retinoblastoma gene product-positive, and all ten retinoblastoma gene product-negative cases were also p53-negative, two different and mutually exclusive pathways of possible pathogenetic significance may be suggested, the former involving abnormalities of the functional pathway of p53 in the absence of mutations and the latter abnormalities of retinoblastoma gene expression at the transcriptional and/or post-transcriptional level. Finally, the clinico-pathological correlations showed that p53 immunohistochemical expression is significantly associated with a poorer response to intensive chemotherapy.

Adolescent↗
PubMed1994

Transgenic mice provide genetic evidence that transforming growth factor alpha promotes skin tumorigenesis via H-ras-dependent and H-ras-independent pathways.

The epidermal growth factor receptor (EGFR), which mediates the mitogenic activity of transforming growth factor alpha (TGF-alpha), has been shown to activate Ras in cultured cells through well-defined intermediary proteins. To examine the in vivo relationship between EGFR and Ras, chemical carcinogenesis of TGF-alpha transgenic mouse skin was chosen as an experimental model. Transgenic mice overexpressing TGF-alpha in a wide variety of epithelial tissues by virtue of a metallothionein promoter demonstrate a multitude of premalignant and neoplastic lesions but not spontaneous skin tumors. Transgenic skin was initiated with a single dose of 7,12-dimethylbenz[a]anthracene (DMBA), shown previously to induce, in concert with a tumor promoter, murine papillomas that consistently contain specific H-ras mutations. Virtually all DMBA-treated TGF-alpha transgenic mice, but not treated control animals, developed hyperplasias, papillomas, sebaceous adenomas, and more infrequently, sebaceous and squamous cell carcinomas. Therefore, TGF-alpha functions as an autonomous tumor promoter in DMBA-initiated transgenic skin. Skin tumors could be separated into two mutually exclusive genetic classes. In tumors harboring mutant H-ras, TGF-alpha transgene expression was relatively low and essentially unchanged relative to untreated skin; however, only 42% of skin tumors contained mutations in H-ras. Conversely, in most tumors with wild-type H-ras, transgenic TGF-alpha transcripts were enhanced 10- to 20-fold. These results suggest that strong constitutive EGFR stimulation, through TGF-alpha transgene overexpression, can substitute functionally for mutational activation of H-ras in skin tumorigenesis. Moreover, because H-ras mutational activation could not induce skin tumors without TGF-alpha transgene activity, simultaneous stimulation of an EGFR-mediated H-Ras-independent pathway appears to be required for tumor development as well.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2004

Kinetic studies and site-directed mutagenesis of Escherichia coli agmatinase. A role for Glu274 in binding and correct positioning of the substrate guanidinium group.

The interaction of Escherichia coli agmatinase (EC 3.5.3.11) with the substrate guanidinium group was investigated by kinetic and site-directed mutagenesis studies. Putrescine and guanidinium ions (Gdn+) were slope-linear, competitive inhibitors with respect to agmatine and their bindings to the enzyme were not mutually exclusive. By site-directed mutagenesis, the E274A variant exhibiting about 1-2% of wild-type activity was obtained. Mutation produced a moderate, but significant, increase in the Km value for agmatine (from 1.1 +/- 0.2 mM to 6.3 +/- 0.3 mM) and the Ki value for competitive inhibition by Gdn+ (from 15.0 +/- 0.1 mM to 44.2 +/- 2.1 mM), but the Ki value for putrescine inhibition (2.8 +/- 0.2 mM) was not altered. The tryptophan fluorescence properties (lambdamax = 342 nm) and circular dichroism spectra were not significantly altered by the Glu274 --> Ala mutation. The dimeric structure of the enzyme was also maintained. We conclude that Glu274 is involved in binding and positioning of the guanidinium moiety of the substrate for efficient catalysis. A kinetic mechanism involving rapid equilibrium random release of products is proposed for E. coli agmatinase.

Binding Sites↗
PubMed1995

p53 and human papilloma virus type 16 in cervical intraepithelial neoplasia and carcinoma.

A series of cervical biopsies and excision specimens representing normal cervical epithelium, metaplastic epithelium, cervical intraepithelial neoplasia and cervical carcinoma were examined for the presence of p53 in relation to the human papillomavirus type 16 (HPV 16). The results show that p53 accumulation in premalignant cervical lesions is almost identical to the low levels detected in normal endo- and ectocervical epithelia, reserve cells, immature and mature squamous metaplastic epithelium. p53 levels were low and seem to be independent of the grade in cervical intraepithelial neoplasia (CIN). However, carcinomas of the cervix contained high levels of immunohistochemically detectable p53. Apparently, a p53 mutation is not an early factor in cervical carcinogenesis. Furthermore, our studies demonstrate that alterations in p53 levels and the presence of HPV 16 are not mutually exclusive markers of cervical tumorigenesis. This contrasts with several reports in the literature and underlines the observation that p53 expression is probably an inadequate prognosticator for estimating progression or regression of CIN lesions.

DNA, Viral↗
PubMed2007

The natural history of a combined defect in MSH6 and MUTYH in a HNPCC family.

In the inherited syndromes, MUTYH-associated polyposis (MAP) and hereditary nonpolyposis colorectal cancer (HNPCC), somatic mutations occur due to loss of the caretaker function that base-repair (BER) and mismatch repair (MMR) genes have, respectively. Recently, we identified a large branch from a MSH6 HNPCC family in which 19 family members are heterozygous or compound heterozygous for MUTYH germ line mutations. MSH6/MUTYH heterozygote mutation carriers display a predominant HNPCC molecular tumour phenotype, with microsatellite instability and underrepresentation of G>T transversions. A single unique patient is carrier of the MSH6 germline mutation and is compound heterozygote for MUTYH. Unexpectedly, this patient has an extremely mild clinical phenotype with sofar only few adenomas at age 56. Four out of five adenomas show characteristic G>T transversions in APC and/or KRAS2, as seen in MUTYH associated polyposis. No second hit of MSH6 is apparent in any of the adenomas, due to retained MSH6 nuclear expression and a lack of microsatellite instability. Although this concerns only one case, we argue that the chance to find an additional one is extremely small and currently a mouse model with this genotype combination is not available. Moreover, the patients brother who is also compound heterozygous for MUTYH but lacks the MSH6 germline mutation presented with a full blown polyposis coli. In conclusion, these data would support the notion that abrogation of both MSH6 DNA mismatch repair and base repair might be mutually exclusive in humans.

Adult↗
PubMedDate not supplied

Analysis of non-linearities in mutation frequency curves.

Mutation frequency curves for ultraviolet light and other mutagens often exhibit non-linear, as well as linear components. A common pattern observed for UV-induced reversion of auxotrophy in yeast is a biphasic, linear-quadratic (or higher order) response. The non-linear component in such a biphasic frequency curve can arise in two distinct, but non-mutually exclusive, ways: (i) as a result of the existence of two-hit processes in the molecular mechanism(s) of mutagenesis; and (ii) as a result of the possible stochastic dependence of mutation and killing, such that the probability of clone formation by the mutant cells differs from that of the non-mutant cells in the population. We describe here a simple mathematical method for distinguishing between these two sources of non-linearity. It is based on the calculation of a quantity that we call 'apparent survival.' This is given, for any mutagen dose chi, by the ratio of the mutant yield to the corresponding linear component of mutation frequency. If the apparent survival rises to values greater than unity before declining at high doses, then there must exist positive two-hit (or higher order) components in the mutational mechanism. If the final slope of the apparent survival curve differs from that of the measured survival curve, then there also exists some degree of stochastic dependence between mutation and killing.

Dose-Response Relationship, Radiation↗
PubMed2007

Structural analysis of Bub3 interactions in the mitotic spindle checkpoint.

The Mad3/BubR1, Mad2, Bub1, and Bub3 proteins are gatekeepers for the transition from metaphase to anaphase. Mad3 from Saccharomyces cerevisiae has homology to Bub1 but lacks a corresponding C-terminal kinase domain. Mad3 forms a stable heterodimer with Bub3. Negative-stain electron microscopy shows that Mad3 is an extended molecule (approximately 200 A long), whereas Bub3 is globular. The Gle2-binding-sequence (GLEBS) motifs found in Mad3 and Bub1 are necessary and sufficient for interaction with Bub3. The calorimetrically determined dissociation constants for GLEBS-motif peptides and Bub3 are approximately 5 microM. Crystal structures of these peptides with Bub3 show that the interactions for Mad3 and Bub1 are similar and mutually exclusive. In both structures, the GLEBS peptide snakes along the top surface of the beta-propeller, forming an extensive interface. Mutations in either protein that disrupt the interface cause checkpoint deficiency and chromosome instability. We propose that the structure imposed on the GLEBS segment by its association with Bub3 enables recruitment to unattached kinetochores.

Cell Cycle Proteins↗
PubMed1975

Simultaneous ribosomal resistance to trichodermin and anisomycin in Saccharomyces cerevisiae mutants.

A spontaneous mutant of Saccharomyces cerevisiae resistant to trichodermin has been isolated. It displays cross resistance both in vivo and in vitro to a number of sesquiterpene antibiotics (fusarenon X, trichothecin and verrucarin A) and to the chemically unrelated antibiotic anisomycin. The mutation conferring resistance to anisomycin and trichodermin is expressed in the 60-S subunit of the yeast 80-S ribosome. Mutant ribosomes bind [-14C]trichodermin much less efficiently than wild type ribosomes, suggesting that resistance may be due, at least in part, to this property. However, both types of ribosomes bind [-3H] anisomycin equally. These results suggest that anisomycin and trichodermin have different binding sites on the 60-S subunit of eukaryotic ribosomes, even though previous results have shown that both antibiotics bind to mutually exclusive sites.

Anti-Bacterial Agents↗
PubMedDate not supplied

Mutation L210W of HIV-1 reverse transcriptase in patients receiving combination therapy. Incidence, association with other mutations, and effects on the structure of mutated reverse transcriptase.

Mutation L210W of HIV-1 reverse transcriptase (RT) is one of the six main mutations that confer in vivo resistance to zidovudine. Surprisingly, this mutation has received scant appraisal and its contribution to the genotypic resistance to nucleoside analogs is not well understood. The aim of this study was: (1) to study the frequency of mutation L210W in a large collection of HIV-1 sequences (2,049 samples, including 395 DNA and 1,654 RNA sequences) from patients receiving combination therapy, and (2) to analyze its association with the other mutations that confer resistance to zidovudine. A mutation at codon 210 (mainly L210W) was found in 647 (32%) of the 2,049 sequences analyzed. Only 43 (<7%) of these 647 genomes were also mutated at codon 70 (p < 10(-5)). In contrast, 98% of these 647 sequences were also mutated at codon 215 (essentially T215Y/F), and 94% at codon 41 (mainly M41L). These data showing a close association between L210W, T215Y/F, and M41L, and a mutual exclusion between K70R and L210W, were confirmed by analyzing the sequences stored in the HIV-1 sequences available through the Stanford HIV RT and Protease Database. Follow-up studies demonstrated that L210W appeared always after T215Y/F. This observation is consistent with crystallographic studies which suggested that the aromatic side chain of Trp 210 could stabilize the interaction of Phe/Tyr215 with the dNTP-binding pocket. This molecular cross-talk between amino acid chains occurs nearby the conserved Asp113 residue. Since the lateral chain of Arg70 may also interact with Asp113, this is likely to create a sterical hindrance around this residue. Thus, the R-->K reversion of codon 70 may represent a compensatory mechanism allowing a functional rearrangement of the dNTP-binding pocket in the mutated RT.

Amino Acid Sequence↗
PubMed1998

No evidence for functional inactivation of wild-type p53 protein by MDM2 overexpression in gastric carcinogenesis.

Inactivation of wild-type p53 during gastric carcinogenesis is usually caused by mutations within exons 5-8 of the p53 gene leading to mutated, usually immunohistochemically detectable p53 proteins. However, functional inactivation of wild-type p53, mimicking mutational inactivation, may also result from binding to overexpressed MDM2 protein. While these two mechanisms of p53 inactivation are considered to be mutually exclusive, no data exist as to whether MDM2 overexpression occurs during gastric carcinogenesis. MDM2 protein overexpression was therefore studied in relation to p53 protein accumulation in gastric carcinogenesis. Forty-five paraffin-embedded gastrectomy specimens from early gastric carcinomas were examined for the presence of chronic active gastritis, chronic atrophic gastritis, subtypes of intestinal metaplasia, and dysplasia. The Lauren type was reassessed for all early carcinomas. p53 protein accumulation was examined using the monoclonal antibody DO-7. MDM2 protein overexpression was assessed with the monoclonal antibody SMP-14. Complete absence of nuclear p53 protein accumulation was observed in chronic active gastritis, chronic atrophic gastritis, and intestinal metaplasia, irrespective of the subtype. In gastric dysplasia (one mild, two moderate, one severe), only severe dysplasia was p53-positive. Intestinal-type (n = 20) and diffuse-type early gastric carcinoma (n = 25) were p53-positive in 70 and 52 per cent of the cases, respectively. MDM2 protein overexpression was not observed during gastric carcinogenesis, either in the p53-positive or in the p53-negative cases. In conclusion, it appears that functional inactivation of wild-type p53 by MDM2 protein overexpression plays no role in (early) gastric carcinogenesis.

Chronic Disease↗
PubMed1994

DNA double-strand breaks induced by low energy protons in V79 cells.

The initial production of DNA double-strand breaks (dsb) was determined in V79 Chinese hamster cells irradiated with proton beams of 3.24, 1.50 and 0.88 MeV, corresponding to values of unrestricted LET evaluated at the cell midplane of 10.9, 20.0 and 30.5 keV/micron, respectively. X-rays were used for comparison. Dsb were measured with the low speed sedimentation technique in neutral sucrose gradients. The initial yield of dsb rose linearly with the dose and did not significantly depend on the proton LET, in contrast with the results obtained in previous studies for cell inactivation and mutation induction. Also, no significant differences for dsb induction were found between protons and X-rays. Two possible explanations, not necessarily mutually exclusive, are proposed: (1) dsb are not the only lesions involved in cellular effects; and (2) the initial number of dsb is not the only important parameter since a fundamental role is played by the degree of clustering, i.e. the association of dsb with other dsb or other types of damage.

Animals↗
PubMed2000

Genetic profile of gliosarcomas.

There are distinct genetic pathways leading to the glioblastoma, the most malignant astrocytic brain tumor. Primary (de novo) glioblastomas develop in older patients and are characterized by epidermal growth factor (EGF) receptor amplification/overexpression, p16 deletion, and PTEN mutations, whereas secondary glioblastomas that progressed from low-grade or anaplastic astrocytoma develop in younger patients and frequently contain p53 mutations. In this study, we assessed the genetic profile of gliosarcoma, a rare glioblastoma variant characterized by a biphasic tissue pattern with alternating areas displaying glial and mesenchymal differentiation. Single-strand conformation polymorphism followed by direct DNA sequencing revealed p53 mutations in five of 19 gliosarcomas (26%) and PTEN mutations in seven cases (37%). Homozygous p16 deletion was detected by differential polymerase chain reaction in seven (37%) gliosarcomas. The overall incidence of alterations in the Rb pathway (p16 deletion, CDK4 amplification, or loss of pRb immunoreactivity) was 53%, and these changes were mutually exclusive. Coamplification of CDK4 and MDM2 was detected in one gliosarcoma. None of the gliosarcomas showed amplification or overexpression of the EGF receptor. Thus gliosarcomas exhibit a genetic profile similar to that of primary (de novo) glioblastomas, except for the absence of EGFR amplification/overexpression. Identical PTEN mutations in the gliomatous and sarcomatous tumor components were found in two cases. Other biopsies contained p16 deletions, an identical p53 mutation, or coamplification of MDM2 and CDK4 in both tumor areas. This strongly supports the concept of a monoclonal origin of gliosarcomas and an evolution of the sarcomatous component due to aberrant mesenchymal differentiation in a highly malignant astrocytic neoplasm.

Adult↗
PubMed2000

Update of genetics in colorectal carcinomas: genomic instability and somatic evolution.

INTRODUCTION: Currently, there are two contrasting viewpoints on what drives the process of carcinogenesis. The genomic (DNA or chromosomal) instability model contends that an increased mutation rate early in carcinogenesis is necessary for the multistage process, while the somatic evolution model postulates that normal mutation rate with selective advantage and clonal expansion is sufficient to cause cancer. METHODS: Evidence from colorectal carcinoma (CRC) for and against the two models are compared and contrasted. RESULTS: With the exception of hereditary non-polyposis colorectal carcinoma (HNPCC) where DNA instability attributable to mismatch repair deficiency is clearly demonstrated, the majority of CRC appear to progress through the selection of a series of mutations without the need of first acquiring a mutator phenotype. Aneuploidy or chromosomal instability is more likely to be a consequence of non-random selection of mutations in genes residing on the chromosome rather than the direct cause of cancer. Nevertheless, aneuploidy and/or DNA alterations can lead to secondary instability, hence, contributing to the phenotypes associated with carcinoma. CONCLUSIONS: Present knowledge, thus, points to multiple, mutually non-exclusive pathways for different cancer populations, further emphasising tumour heterogeneity.

Adenomatous Polyposis Coli↗
PubMed2006

An apparent pseudo-exon acts both as an alternative exon that leads to nonsense-mediated decay and as a zero-length exon.

Pseudo-exons are intronic sequences that are flanked by apparent consensus splice sites but that are not observed in spliced mRNAs. Pseudo-exons are often difficult to activate by mutation and have typically been viewed as a conceptual challenge to our understanding of how the spliceosome discriminates between authentic and cryptic splice sites. We have analyzed an apparent pseudo-exon located downstream of mutually exclusive exons 2 and 3 of the rat alpha-tropomyosin (TM) gene. The TM pseudo-exon is conserved among mammals and has a conserved profile of predicted splicing enhancers and silencers that is more typical of a genuine exon than a pseudo-exon. Splicing of the pseudo-exon is fully activated for splicing to exon 3 by a number of simple mutations. Splicing of the pseudo-exon to exon 3 is predicted to lead to nonsense-mediated decay (NMD). In contrast, when "prespliced" to exon 2 it follows a "zero length exon" splicing pathway in which a newly generated 5' splice site at the junction with exon 2 is spliced to exon 4. We propose that a subset of apparent pseudo-exons, as exemplified here, are actually authentic alternative exons whose inclusion leads to NMD.

Alternative Splicing↗
PubMed2003

More extensive genetic alterations in unmutated than in hypermutated cases of chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (CLL) is not a uniform disease entity; approximately half of the CLL cases have undergone immunoglobulin V(H) gene hypermutation, whereas the other half display unmutated V(H) genes. We investigated genome changes in 12 hypermutated cases (M-CLL) and 22 unmutated cases (UM-CLL) by use of comparative genomic hybridization, G-banding, and multicolor fluorescence in situ hybridization (m-FISH) after optimal mitogen stimulation and FISH analysis of typical CLL aberrations: 11q deletion, 13q deletion, and trisomy 12. Very high frequencies of aberrations were found in both groups: 82% in UM-CLL and 83% in M-CLL. Deletions of 11q and 13q were equally distributed in M-CLL and UM-CLL. However, larger aberrations detectable by CGH, trisomy 12, and complex aberrations were less frequent in M-CLL than in UM-CLL. These observations led to a hypothesis that unmutated and mutated CLL have different biological Backgrounds, given that large and/or complex chromosomal aberrations and hypermutation of the CLL progenitor cells tend to be mutually exclusive.

Chromosome Aberrations↗
PubMed2000

Structural basis of SNT PTB domain interactions with distinct neurotrophic receptors.

SNT adaptor proteins transduce activation of fibroblast growth factor receptors (FGFRs) and neurotrophin receptors (TRKs) to common signaling targets. The SNT-1 phosphotyrosine binding (PTB) domain recognizes activated TRKs at a canonical NPXpY motif and, atypically, binds to nonphosphorylated FGFRs in a region lacking tyrosine or asparagine. Here, using NMR and mutational analyses, we show that the PTB domain utilizes distinct sets of amino acid residues to interact with FGFRs or TRKs in a mutually exclusive manner. The FGFR1 peptide wraps around the beta sandwich structure of the PTB domain, and its binding is possibly regulated by conformational change of a unique C-terminal beta strand in the protein. Our results suggest mechanisms by which SNTs serve as molecular switches to mediate the essential interplay between FGFR and TRK signaling during neuronal differentiation.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Insights into the mitochondrial signaling pathway: what lessons for chemotherapy?

Mitochondria are potent integrators/coordinators of apoptosis signaling pathways. Indeed, under physiological conditions, the initiation of apoptosis leads to the accumulation of second messengers that converge on mitochondria. In response, these organelles undergo a membrane permeabilization, presumably due to the opening of protein channels, culminating in the release of proapoptotic proteins into the cytosol. Under pathological conditions, a failure of mitochondrial membrane permeabilization (MMP) can result in an inhibition of apoptosis and enhanced resistance to chemotherapy. Several non-mutually exclusive mechanisms may account for a defect in the execution or regulation of MMP. These include (i) alterations in gene transcription, (ii) gene mutations resulting in protein inactivation, and (iii) defects of intracellular localization. This may concern structural proteins of the permeability transition pore complex, as well as MMP regulatory proteins, such as Bax/Bcl-2 family members, p53, and cyclophilin D. Analysis of these mechanisms should improve our understanding of the basic function of mitochondria in apoptosis and help elaborate new strategies to correct MMP failure from a therapeutic perspective.

Animals↗
PubMed2005

CpG island promoter hypermethylation of a novel Ras-effector gene RASSF2A is an early event in colon carcinogenesis and correlates inversely with K-ras mutations.

We report in silico identification and characterisation of a novel member of the ras association domain family 1 (RASSF1)/NORE1 family, namely, RASSF2, located at chromosomal region 20p13. It has three isoforms, all contain a ras association domain in the C-terminus. The longest isoform RASSF2A contains a 5' CpG island. RASSF2A was cloned from a brain cDNA library and directly sequenced, confirming the genomic gene structure. In previous reports, we and others have demonstrated that RASSF1A is epigenetically inactivated in a variety of cancers, including sporadic colorectal cancer (CRC). In the present report, we analysed the methylation status of RASSF2A promoter region CpG island in sporadic CRC and compared it to K-ras mutation status. RASSF2A promoter region CpG island was hypermethylated in a majority of colorectal tumour cell lines (89%) and in primary colorectal tumours (70%), while DNA from matched normal mucosa was found to be unmethylated (tumour-specific methylation). RASSF2A expression was reactivated in methylated tumour cell lines after treatment with 5-aza 2-deoxycytidine. RASSF2A methylation is an early event, detectable in 7/8 colon adenomas. Furthermore, 75% of colorectal tumours with RASSF2A methylation had no K-ras mutations (codons, 12 and 13) (P=0.048), Fisher's exact test). Our data demonstrate that RASSF2A is frequently inactivated in CRCs by CpG island promoter hypermethylation, and that epigenetic (RASSF2A) and genetic (K-ras) changes are mutually exclusive and provide alternative pathways for affecting Ras signalling.

Base Sequence↗