Regulation of Unr expression by 5'- and 3'-untranslated regions of its mRNA through modulation of stability and IRES mediated translation.

Unr (upstream of N-ras) is a cytoplasmic RNA-binding protein that can act as a regulator of mRNA stability and IRES-mediated translation. Unr, a member of the cold-shock domain (CSD) protein super-family, is ubiquitously expressed, with variable abundance, in different tissues or during embryonic development. Prokaryotic and eukaryotic cold-shock protein expression is highly regulated at both the transcriptional and post-transcriptional levels.

Unr, a cytoplasmic RNA-binding protein with cold-shock domains, is involved in control of apoptosis in ES and HuH7 cells.

Unr (upstream of N-ras) is a cytoplasmic RNA-binding protein involved in the regulation of messenger RNA stability and internal initiation of translation. We have used Unr-deficient murine embryonic stem (ES) cells to analyse Unr role in cell proliferation and response to stress. Disruption of both unr gene copies had no effect on ES cell proliferation.

Characterization of prmt7alpha and beta isozymes from Chinese hamster cells sensitive and resistant to topoisomerase II inhibitors.

By selection of genetic suppressor elements (GSEs) conferring resistance to topoisomerase II inhibitors in Chinese hamster cells (DC-3F), we identified a gene encoding two proteins of 78 and 82 kDa which belong to the protein arginine methyltransferase (PRMT) family. Down-regulation of these enzymes (named PRMT7alpha and beta), either induced by an antisense GSE or as observed in the 9-OH-ellipticine (9-OH-E) resistant mutant DC-3F/9-OH-E, was responsible for cell resistance to various DNA damaging agents. Alternative splicing alterations in the 5'-terminal region and changes of the polyadenylation site of PRMT7 mRNAs were observed in these resistant mutant cells.

Characterization of promoter elements involved in the down-regulation of topoisomerase IIalpha expression in a drug-resistant cell line.

Reduced expression of topoisomerase II is one of the mechanisms observed in cell lines and clinical samples that are resistant to topoisomerase II-targeting agents. The Chinese hamster lung cell line DC-3F/9-OH-E made resistant to 9-OH ellipticine and cross-resistant to other topoisomerase II inhibitors has previously been shown to express lower level of topoisomerase IIalpha isoform, than the parental DC-3F cell line. We have shown here that topoisomerase IIalpha promoter activity is lower in the resistant cell line.

Identification of new drug sensitivity genes using genetic suppressor elements: protein arginine N-methyltransferase mediates cell sensitivity to DNA-damaging agents.

Genetic suppressor elements (GSEs) are cDNA fragments encoding either truncated proteins, acting as dominant-negative mutants, or inhibitory antisense RNA segments counteracting with the gene from which they are derived. To identify genes controlling the cell response to cytotoxic agents, a normalized retroviral library of randomly fragmented cDNAs from Chinese hamster cell line DC-3F was screened for GSEs conferring resistance to the topoisomerase II inhibitor 9-OH-ellipticine. From 218 cDNA fragments isolated, 11 functional GSEs, corresponding to at least 8 independent genes, were selected.

Bleomycin resistance in mammalian cells expressing a genetic suppressor element derived from the SRPK1 gene.

The genetic suppressor element (GSE) approach allows identification of genes essential for certain cell phenotypes. To identify genes controlling the cell response to cytotoxic agents, a normalized retroviral library of randomly fragmented cDNAs from the Chinese hamster cell line DC-3F was screened for GSEs conferring resistance to bleomycin. One of these GSEs, GSE(BLM), conferring an approximately 2-fold bleomycin resistance in DC-3F cells, displayed 98% identity with an amino acid sequence located in the functional domain of human SRPK1.

Cellular resistance to the antitumor DNA topoisomerase II inhibitor S16020-2: importance of the N-[2(Dimethylamino)ethyl]carbamoyl side chain.

The new olivacine derivative S16020-2 (NSC-659687) is a DNA topoisomerase II inhibitor endowed with a remarkable antitumor activity against various experimental tumors. In vitro physicochemical properties of this compound, in particular its interaction with DNA and DNA topoisomerase II, were very similar to those of ellipticine derivatives, except for a strictly ATP-dependent mechanism of cleavable complex induction. From the Chinese hamster lung fibroblast cell line DC-3F, a subline resistant to S16020-2, named DC-3F/S16, was selected by adding stepwise increasing concentrations of the drug to the cell growth medium.

Nuclear translocation of a leukocyte elastase Inhibitor/Elastase complex during staurosporine-induced apoptosis: role in the generation of nuclear L-DNase II activity.

Using L1210 murine leukemia cells, we have previously shown that in response to treatment with drugs having different targets, apoptotic cell death occurs through at least two different signaling pathways. Here, we present evidence that nuclear extracts from staurosporine-treated cells elicit DNase II activity that is not detected in nuclear extracts from cisplatin-treated cells. This activity correlates with the accumulation of two nuclear proteins (70 and 30 kDa) which are detected by an anti-L-DNase II antibody.

Transfection of 9-hydroxyellipticine-resistant Chinese hamster fibroblasts with human topoisomerase IIalpha cDNA: selective restoration of the sensitivity to DNA religation inhibitors.

In the Chinese hamster lung cell line DC-3F/9-OH-E, selected for resistance to 9-OH-ellipticine and cross-resistant to other topoisomerase II inhibitors, the amount of topoisomerase IIalpha is 4-5-fold lower than in the parental DC-3F cells, whereas topoisomerase IIbeta is undetectable. Cloning and sequencing of topoisomerase IIalpha cDNAs from DC-3F and DC-3F/9-OH-E cells revealed an allele polymorphism, one allele differing from the other by the presence of seven silent mutations and three mutations in the noncoding region. In addition, the mutated allele contains three missense mutations located close to the ATP binding site (Thr371Ser) or to the catalytic site (Ala751Gly; Ile863Thr).

Cysteine control over glutathione homeostasis in Chinese hamster fibroblasts overexpressing a gamma-glutamylcysteine synthetase activity.

Gamma-glutamylcysteine synthetase (GCS) catalyses the first step of glutathione (GSH) biosynthesis and is considered to be the rate-limiting step of this pathway. In several experimental systems, GCS overexpression has been associated with GSH pool expansion and drug resistance. In this report, we describe a mutant line of Chinese hamster fibroblasts that overexpress this activity by 4-5 times, due to the amplification of the gene encoding the catalytic subunit of GCS.

Alteration in p53 pathway and defect in apoptosis contribute independently to cisplatin-resistance.

The accumulation of molecular genetic defects selected during the adaptation process in the development of cisplatin-resistance was studied using progressive cisplatin-resistant variants (L1210/DDP2, L1210/DDP5, L1210/DDP10) derived from a murine leukemia cell line (L1210/0). Of these cell lines, only the most resistant L1210/DDP10 was cross-resistant to etoposide and deficient in apoptosis induced by these two drugs, indicating that resistance to DNA-damaging agents correlates with a defect in apoptosis. This defect was tightly associated with the loss of a Ca2+/Mg2+-dependent nuclear endonuclease activity present in the less cisplatin-resistant cells.

S16020-2, a new highly cytotoxic antitumor olivacine derivative: DNA interaction and DNA topoisomerase II inhibition.

S16020-2 (NSC-659687) is a new olivacine derivative that is highly cytotoxic in vitro and displays remarkable antitumor activity against various experimental tumors, especially some solid tumor models. Its antitumor activity is notably higher than that of 2-methyl-9-hydroxy-ellipticinium (NMHE) and comparable to that of doxorubicin HCl, although with a different tumor specificity. S16020-2 is being tested in phase I clinical trials.

[Cytotoxicity and interaction of amsacrine derivatives with topoisomerase II: role of the 1' substitute on the aniline nucleus].

Amsacrine is an intercalating planar polycyclic aromatic molecule that displays antitumor activity. The cytotoxicity of this compound is related to its interaction with topoisomerase II. The substituent at position 1' on the aniline is thought to be essential to the formation of the topoisomerase II-DNA cleavable complex and hence the cytotoxicity of the drug.

Role of topoisomerase II beta in the resistance of 9-OH-ellipticine-resistant Chinese hamster fibroblasts to topoisomerase II inhibitors.

In the Chinese hamster lung cell line DC-3F/9-OH-E, made resistant to 9-OH-ellipticine and cross-resistant to other topoisomerase II inhibitors, the amount of topoisomerase II alpha is 4-5-fold lower than in the parental DC-3F cells. A mutation in position 1710 of topoisomerase II beta cDNA, generating a stop codon, completely abolishes the expression of this isoform in DC-3F/9-OH-E cells. To analyze the contribution of the loss of topoisomerase II beta to the resistance phenotype, DC-3F/9-OH-E cells were cotransfected with two plasmids, one conferring the resistance to G418, the other carrying the topoisomerase II beta cDNA.

[P53 mutations, asset or disadvantage for cancer chemotherapy].

The "guardian of the genome" p53 is an essential modulator of the cellular response to cytotoxic agents. After introduction of DNA damages, the p53 protein prevents the cells to divide, either transiently by arresting their progression at the G1/S transition, or definitely by inducing apoptosis. In approximately half of the human tumors, mutations result in profound alterations of the p53 protein properties.

Expression of the activated p185erbB2 tyrosine kinase in human epithelial cells leads to MAP kinase activation but does not confer oncogenicity.

Amplification of the c-erbB2 gene and overexpression of p185erbB2 is found in approximately one-third of primary breast and ovarian cancers and also in some colon carcinomas. Moreover, a single point mutation in erbB2(V 664 E) confers transforming potential to erbB2 in NIH3T3 cells, even when expressed at low levels. To examine the transformation potential of erbB2 or erbB2(V-E) in colon epithelial cells, we have transfected a nontumorigenic clone of SW 613-S cells with either wild-type p185erbB2 or mutated p185erbB2(V-E).

Epidermal growth factor receptor signaling cascade as target for tyrphostin (RG 50864) in epithelial cells. Paradoxical effects on mitogen-activated protein kinase kinase and mitogen-activated protein kinase activities.

Tyrphostins are synthetic compounds that have been described as in vitro inhibitors of epidermal growth factor receptor (EGF-R) tyrosine kinase activity. The inhibitory effect of tyrphostins in intact cells has been shown only after prolonged treatment. However, these compounds appear to be readily incorporated, which suggests that tyrphostin acts indirectly on EGF-R.

Induction of pgp3 expression and reversion of the multidrug resistance phenotype in 9-OH-ellipticine-resistant Chinese hamster lung fibroblasts transfected with the MYC oncogene.

Chinese hamster lung cells resistant to the DNA topoisomerase II inhibitor 9-OH-ellipticine (DC-3F/9-OH-E) are cross resistant to various drugs through the expression of the MDR phenotype. The myc oncogene was approximately 10-fold amplified and 20-fold overexpressed in parental DC-3F cells as compared with DC-3F/9-HO-E cells. Transfection of the resistant cells with a mouse c-myc gene did not alter the resistance to topoisomerase II inhibitors and, in cells with a low multidrug (MDR) expression, reversed this phenotype.

Genistein analogues: effects on epidermal growth factor receptor tyrosine kinase and on stress-activated pathways.

Two genistein analogues (MD831 and MD833) have been synthesized and analyzed for their biological properties and their mechanism of action im comparison to genistein either in vitro or in intact cells. We showed that, in vitro, one of these compounds (MD831) inhibits the tyrosine kinase activity associated with the epidermal growth factor receptor (EGFR) as efficiently as genistein. However, treatment of A431 cells with these compounds did not result in any significant modification of EGFR tyrosine phosphorylation.