Correlation between chromosome 5q deletions and different mechanisms of c-myc overexpression in human colorectal cancer.
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Adenomatous Polyposis Coli↗
Biomedical subjects
A Viel
Publications and source records attributed to A Viel.
Three genes under different developmental control encode elongation factor 1-alpha in Xenopus laevis.
We have cloned cDNAs encoding two variants of the elongation factor for protein synthesis in Xenopus laevis, called EF-1 alpha. One of these (42Sp50) is expressed exclusively in immature oocytes. It is one of two protein components of a 42S RNP particle that is very abundant in previtellogenic oocytes. The 42S RNP particle consists of various tRNAs, 5S RNA, 42Sp50 and a 5S RNA binding protein (42Sp43). A major function served by 42Sp50 appears to be the storage of tRNAs for later use in oogenesis and early embryogenesis. The second EF-1 alpha variant (EF-1 alpha O) is expressed mainly in oocytes but transiently in early embryogenesis as well. Its mRNA cannot be detected after neurulation in somatic cells. EF-1 alpha O is closely related to a third EF-1 alpha (EF-1 alpha S), discovered originally by Krieg et al. (1). EF-1 alpha S is expressed at low levels in oocytes but actively in somatic cells. The latter two proteins are very similar to known eukaryotic EF-1 alpha from other organisms and presumably function in their respective cell types to support protein synthesis.
Amino Acid Sequence↗
Effects of ionizing radiations on proteins. Evidence of non-random fragmentations and a caution in the use of the method for determination of molecular mass.
We have reinvestigated the use of ionizing radiations to measure the molecular mass of water-soluble or membrane proteins. The test was performed by using the most straightforward aspect of the technique, which consists of SDS/PAGE analysis of the protein-fragmentation process. We found that exposure of purified standard proteins to increasing doses of ionizing radiation causes progressive fragmentation of the native protein into defined peptide patterns. The coloured band corresponding to the intact protein was measured on the SDS gel as a function of dose to determine the dose (D37.t) corresponding to 37% of the initial amount of unfragmented protein deposited on the gel. This led to a calibration curve between 1/D37.t and the known molecular mass of the standard proteins whose best fit gave Mr = 1.77 x 10(6)/D37.t at -78 degrees C, i.e. 35% higher than the generally accepted value at that temperature obtained from inactivation studies. However, we have to conclude that this method is useless to determine the state of aggregation of a protein, since, for all the oligomers tested, the best fit was obtained by using the protomeric molecular mass, suggesting that there is no energy transfer between promoters. Furthermore, SDS greatly increases the fragmentation rate of proteins, which suggests additional calibration problems for membrane proteins in detergent or in the lipid bilayer. But the main drawback of the technique arises from our observation that some proteins behaved anomalously, leading to very large errors in the apparent target size as compared with true molecular mass (up to 100%). It is thus unreliable to apply the radiation method for absolute molecular-mass determination. We then focused on the novel finding that discrete fragmentation of proteins occurs at preferential sites, and this was studied in more detail with aspartate transcarbamylase. N-Terminal sequencing of several radiolysis fragments of the catalytic chain of the enzyme revealed that breaks along the polypeptide chains are localized close to the C-terminal end. Examination of the three-dimensional structure of aspartate transcarbamylase suggests that radiolysis sites (fragile bonds) might be localized in connecting loops.
Dose-Response Relationship, Radiation↗
Ca2(+)-induced conformational changes and location of Ca2+ transport sites in sarcoplasmic reticulum Ca2(+)-ATPase as detected by the use of proteolytic enzyme (V8).
Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureus produced appreciable amounts of a Ca2(+)-ATPase fragment (p85) in the presence of Ca2+ (E1 conformation of the enzyme), along with many other peptide fragments that were also formed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (E2 conformation). p85 was formed as a carboxyl-terminal cleavage product of Ca2(+)-ATPase by a split of the peptide bond between Glu-231 and Ile-232. Other conformation-dependent V8 splits were localized to the "hinge" region, involved in ATP binding, between the middle and COOH-terminal one-third of the Ca2(+)-ATPase polypeptide chain. Representative split products in this region (p48,p31) were identified as NH2-terminal and COOH-terminal cleavage products of p85. In the membrane p85 probably remains associated with its complementary NH2-terminal fragment(s) and retains the capacity to bind Ca2+ as evidenced by resistance to V8 degradation in Ca2+ and ability to become phosphorylated by ATP. However, the hydrolysis rate of the phosphorylated enzyme is reduced, indicating that peptide cleavage at Glu-231 interferes with Ca2+ transport steps after phosphorylation. Binding of Ca2+ to V8 and tryptic fragments of Ca2(+)-ATPase was studied on the basis of Ca2(+)-induced changes in electrophoretic mobility and 45Ca2+ autoradiography after transfer of peptides to Immobilon membranes. These data indicate binding by the NH2-terminal 1-198 amino acid residues (corresponding to the tryptic A2 fragment) and the COOH-terminal 715-1001 amino acid residues (corresponding to p31). By contrast the central portion of Ca2(+)-ATPase, including the NH2-terminal portion of p85, is devoid of Ca2+ binding. These results question an earlier proposition that Ca2(+)-binding is located to the "stalk" region of Ca2(+)-ATPase (Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H.) (1986) Cell 44, 597-607) but are in agreement with recent data obtained by oligonucleotide-directed mutagenesis of Ca2(+)-ATPase (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). These different studies suggest that Ca2+ translocation sites may have an intramembranous location and are formed predominantly by the carboxyl-terminal part of the Ca2(+)-ATPase polypeptide chain.
Animals↗
c-myc overexpression is a tumor-specific phenomenon in a subset of human colorectal carcinomas.
The transcriptional activity of the c-myc proto-oncogene was examined in 25 primary human colorectal carcinomas and their corresponding normal mucosae. The purpose was to determine whether the elevated levels of c-myc expression, frequently detected in this type of tumor, might be the consequence of alterations in the cell growth rate or the effect of a real transcriptional deregulation of the gene. In about 44% of the tumors the elevated c-myc expression was consequent to the enhanced growth rate of the neoplastic tissue, as estimated by the expression of the S-phase-specific histone H3 gene. In the other 56%, c-myc overexpression did not entirely depend on the proliferative activity of the neoplastic population. In this latter group, c-myc deregulation did not reside in structural modifications of the putative regulatory regions of the gene. Therefore, c-myc overexpression, at least in a subset of colorectal cancer, seems to be consequent to alterations in transregulative phenomena exerted on the c-myc gene by other genetic loci.
Adenocarcinoma↗
Elongation factor 1 alpha (EF-1 alpha) is concentrated in the Balbiani body and accumulates coordinately with the ribosomes during oogenesis of Xenopus laevis.
In Xenopus laevis oocytes two distinct systems catalyze the mRNA-dependent binding of aminoacyl tRNA to the A site of ribosomes. These systems are elongation factor 1 alpha (EF-1 alpha) and the 42S nucleoprotein particle. This particle is also implicated in the long-term storage of 5S RNA and aminoacyl tRNA during early oogenesis. We report here that the ribosomes and the storage particles are distributed uniformly in the cytoplasm of previtellogenic (stage I) oocytes. In contrast, EF-1 alpha is concentrated in a small region of the cytoplasm, known as the mitochondrial mass or Balbiani body. When the Balbiani body disperses in early vitellogenic oocytes (stage II), EF-1 alpha becomes evenly distributed in the cytoplasm. The main phase of EF-1 alpha accumulation follows the disappearance of the 42S particles (stage II), but coincides with the main phase of ribosome accumulation (stages III and IV).
Animals↗
Pathogenesis of malignant lymphomas in intravenous drug-abuser, HIV-infected patients.
To study the pathogenesis of malignant lymphomas (ML) in intravenous drug-abuser HIV-infected patients, we analyzed 19 cases of reactive lymphadenopathy (LAS) and 10 cases of ML. Clonality and differences in characteristics of these lymphoproliferative disorders were investigated by immunohistochemical and Ig and TCR gene rearrangement analyses. Rearrangements at the c-myc locus and presence of HIV and EBV viral genomes were also investigated. Four out of the five non-Hodgkin's lymphomas (NHL) analyzed were high-grade extranodal ML and were found to derive from precursor B cells. Monoclonal cell expansions were also detected in 2 cases of LAS. These cell expansions also consisted of precursor B cells. HIV genome was not detected in any of the samples tested and was therefore considered not to be involved as an etiological agent in these lymphoproliferative disorders. EBV genome was present in a clonal episomal form in the five Hodgkin's disease (HD) specimens tested. This finding suggested that a clonal cell population harboring the EBV viral genome must be present in HDs, pointing to a possible etiological relationship between EBV and HD in HIV-infected patients.
Antigens, Differentiation, T-Lymphocyte↗
[Is the progesterone test a generalizable method of screening for neoplastic and preneoplastic lesions of the endometrium?].
A "progesterone test" has recently been proposed as a tentative method to detect early neoplastic and pre-neoplastic lesions of the uterus endometrium. At the time progesterone is stopped, a "deprivation" metrorragia demonstrates a remanent hyperoestrogenism, which represents a well-known risk factor for this type of lesion. The method has been evaluated in private practice; 389 patients were offered the test: 87% accepted. Metrorragias were observed in 28% of the cases; however, no neoplastic nor pre-neoplastic lesions could be detected in this group of patients. Moreover, one patient presented with an advanced endometrial cancer one year after a negative test. The reliability of such a test clearly needs further evaluation.
Aged↗
Sensitivity pattern of normal and Ha-ras transformed NIH3T3 fibroblasts to antineoplastic drugs.
Ha-ras-transformed NIH3T3 fibroblasts were compared with the parental cell line to investigate the influence of the Ha-ras oncogene on cellular chemosensitivity to antineoplastic drugs. Four NIH3T3 cell clones independently transformed by the Ha-ras oncogene, activated by mutation or overexpression, were analyzed: 3 clones were obtained by transfection of NIH3T3 cells with a mutation-activated Ha-ras gene and 1 clone by transfection of a large copy number of the normal Ha-ras proto-oncogene. Chemosensitivity of the transformed clones and of the parental cell line was analyzed when cells were in the same condition of proliferative activity and cell cycle phase distribution. No significant differences in chemosensitivity were observed between transformed and untransformed cell lines to doxorubicin, VP-16, cis-platinum or mitomycin C. Therefore, data suggest that activated Ha-ras oncogenes have no role in sensitivity to these antineoplastic agents.
Animals↗
The immunocross test. An immunoelectrophoretic method based on the difference between the isoelectric points of antigens and immunoglobulins.
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Animals↗
Size exclusion chromatography and universal calibration of gel columns.
We have tested the proposal (M. Potschka (1987) Anal. Biochem. 162, 47-64) that the elution position of macromolecules by gel chromatography is better correlated with the viscosity-based Stokes radius (R eta) than with the Stokes radius (RS) calculated from the frictional coefficient. By the use of different gel matrices (agarose, Sephadex, TSK silica gel columns) we found that the elution of dextran fractions and reduced proteins, denatured with guanidinium hydrochloride, agreed with their R eta, using water-soluble, globular proteins for gel calibration. However, the elution of large sodium dodecyl sulfate-protein complexes and other elongated macromolecules (fibrinogen, tropomyosin) was systematically retarded while polyethylene glycol and polyethylene glycol detergents eluted earlier than water-soluble, globular protein as a function of R eta. The same was the case for bacteriorhodopsin, solubilized by C12E8 or Triton X-100. It is concluded that for steric reasons size exclusion chromatography is more sensitive than hydrodynamic measurements to the detailed conformation of macromolecules (rods and random coils) and that for this reason gels with inert pores cannot be universally calibrated for all kinds of macromolecules.
Calibration↗
In K562 leukemia cells treated with doxorubicin and hemin, a decrease in c-myc mRNA expression correlates with loss of self-renewal capability but not with erythroid differentiation.
The decrease in c-myc mRNA expression occurring in leukemia cell lines induced to differentiate is supposed to be an early event of the commitment to the differentiation program. Alternatively, the decrease in c-myc mRNA expression could be simply a consequence of loss of the self-renewal capability characteristic of the terminal differentiated phenotypes. In an attempt to clarify these hypotheses, we analysed comparatively the kinetics of variations in c-myc mRNA expression, hemoglobin synthesis, DNA and RNA syntheses, cell cycle kinetics and self-renewal capability in normal and hemin-treated K562 leukemia cells exposed for different periods of time to the antitumoral antibiotic doxorubicin. Times of exposure to doxorubicin were either 2 h, which resulted in reversible induction of hemoglobin synthesis without significant cytostatic effects, or continuously for more than 5 days, which resulted in an irreversible induction of hemoglobin synthesis and in the complete and irreversible loss of self-renewal activity. Comparative analysis of the experimental data indicated that the decrease in c-myc mRNA expression correlated with the loss of replicative activity, possibly due to an irreversible cytostatic effect of the long exposure to doxorubicin, but not with the commitment to the differentiation programs.
Blotting, Northern↗
N-myc activation by proviral insertion in MCF 247-induced murine T-cell lymphomas.
N-myc proto-oncogene rearrangement was found in three out of six AKR murine T-cell lymphomas induced by the highly oncogenic MCF 247 MuLV. Molecular analyses showed that structural modification of the proto-oncogene in all three lymphomas was in the consequence of MCF 247 proviral integration within the gene III exon. All integrated proviruses have the same transcriptional orientation as the N-myc gene. As a consequence of proviral insertion, the N-myc gene becomes transcriptionally active, producing an abnormal mRNA. These findings suggest a possible causative role of such an integrative event in murine T-cell lymphomagenesis.
Amino Acid Sequence↗
Activation by point mutation of Ki-ras gene occurring in transfected human normal DNA.
A mutation-activated human Ki-ras gene was detected in NIH 3T3 cells transfected with high molecular weight DNA extracted from peripheral blood leukocytes of a healthy blood donor. A deoxyguanosine at position 35 of the first exon was substituted by deoxythymidine. Nevertheless, cloning and sequencing of seven independent Ki-ras first exons, isolated from the same human genomic DNA used to transfect NIH 3T3 cells, failed to reveal the expected point mutation. Since transfected DNA is susceptible to mutagenesis in mammalian cells, we hypothesize that a base substitution occurred during the transfection assay.
Animals↗
Nuclear oncogene amplification or rearrangement is not involved in human colorectal malignancies.
We have examined 44 cases of human colonic and rectal carcinomas for structural rearrangement and amplification of c-myc, N-myc, L-myc, c-myb and p53 oncogenes. DNA hybridization showed evidence of c-myc amplification in only one of the samples tested. In addition, the same tumour also showed a rearrangement immediately 3' to the c-myc locus. No rearrangement could be found at the c-myc locus in the other 43 cases. Moreover, our molecular analysis of N-myc, L-myc, c-myb and p53 genes indicated no relevant alteration of the copy number and/or genomic structure of these nuclear oncogenes. Thus, at least in human colorectal malignancies, it is unlikely that nuclear oncogene structural alterations and/or amplification plays a major role in tumour induction or progression.
Adenocarcinoma↗
Thesaurin a, the major protein of Xenopus laevis previtellogenic oocytes, present in the 42 S particles, is homologous to elongation factor EF-1 alpha.
We have purified in SDS X.laevis thesaurin a (Mr 50,000) which is part of the 42 S storage particles. Its N-terminal amino acid is blocked and several peptides obtained by V8 protease treatment were purified and sequenced. As expected from one of the functional roles of the 42 S particles (tRNA binding, protection against deacylation and exchange with the ribosome), the amino acid sequence of thesaurin a was found to be closely related to that of the elongation factor EF-1 alpha. We suggest that all three proteins involved in 5 S RNA and tRNA storage in previtellogenic oocytes, TFIIIA, thesaurin a and thesaurin b, have a dual function: storage and a role in transcription or in protein synthesis.
Amino Acid Sequence↗
Ha-ras-1 restriction fragment length polymorphism and susceptibility to colon adenocarcinoma.
It is not yet clear whether some polymorphic variants of the Ha-ras-1 gene confer genetic predisposition to cancer. However, recent data on myelodysplasia and lung cancer are controversial. To clarify this point, 62 colorectal adenocarcinoma patients were examined for the Ha-ras-1 gene restriction fragment length polymorphism and results were compared with those of 108 healthy blood donors. No Ha-ras-1 polymorphic variants specifically associated with the cancer patients were detected. However, a specific genotype was significantly more frequent in the healthy donors than in the cancer patients (16% versus 5%), suggesting an interaction between the two alleles of the gene.
Adenocarcinoma↗
The photosensitizing activity of haematoporphyrin on mollicutes.
The photosensitizing activity of haematoporphyrin (HP) on Mycoplasma hominis and Acholeplasma laidlawii was studied as a function of the phase of growth and the amount of sterols in the cell membrane. Less HP was bound to cells when the membrane had a high sterol content. Both strains in the exponential but not in the stationary phase of growth were sensitive to HP treatment (above 1 microgram ml-1) in the dark. Visible light irradiation of HP-loaded cells caused in all cases a decrease of cell survival, with concomitant changes in the pattern of membrane proteins that suggested protein-protein cross-linking, and the appearance of ultrastructural alterations (rounded and lysed cells); the photosensitivity was indirectly related to the sterol content of the cell membrane. On the whole, our findings suggest that the cell membrane is a major target for HP photosensitization of mycoplasma cells.
Acholeplasma laidlawii↗