Alain Jacquemin-Sablon

Reduced DNA topoisomerase II activity and drug-stimulated DNA cleavage in 9-hydroxyellipticine resistant cells☆

We have isolated a Chinese hamster lung cell line resistant to 9-hydroxyellipticine (DC-3F/9-OH-E) which is also cross-resistant to topoisomerase II inhibitors such as amsacrine and etoposide. In this work we have studied quantitatively both DNA topoisomerase II activity by decatenation of kinetoplast DNA and drug-stimulated DNA cleavage of pBR 322. DNA topoisomerase II activity of DC-3F/9-OH-E nuclear extract was reduced by 3.5-fold as compared to that from DC-3F (sensitive parent) nuclear extract. We also found that DC-3F/9-OH-E nuclear extracts have a reduced capacity to induce in vitro topoisomerase II-mediated DNA cleavage upon stimulation by etoposide and amsacrine (7- and 10-fold respectively). Besides, mixing nuclear extracts from both sensitive and resistant cells indicates that either the enzyme in resistant cells is modified or a modulating factor is associated to it. Our results suggest that the resistance of the DC-3F/9-OH-E cell line to topoisomerase II inhibitors might be due to both a reduced amount of the enzyme and its reduced ability to form the cleavable complex in the presence of drugs.

Uptake and cytofluorescence localization of ellipticine derivatives in sensitive and resistant chinese hamster lung cells

Uptake of two ellipticine derivatives, 2-N-methyl-ellipticinium (NME) and 2-N-methyl-9-hydroxy-ellipticinium, by sensitive and resistant Chinese hamster lung cells was studied. The results show that uptake and retention of these molecules by both types of cells were identical, thus indicating that the resistance to ellipticines, in this system, is not related to an impaired permeability of the cells to the drugs. However, influx and efflux kinetics, as well as experiments at increasing external concns, showed that both drugs accumulate within the cells in different ways. A cellular overconcentration of the drugs, which does not require an energy-dependent process, is observed. Fluorescence microscopy showed that, in both sensitive and resistant cells, NME is mainly, if not entirely, located in the cytoplasm.

Cloning and characterization of full-length cDNAs coding for the DNA topoisomerase II β from Chinese hamster lung cells sensitive and resistant to 9-OH-ellipticine☆

DNA topoisomerase II beta cDNAs from Chinese hamster lung cells sensitive (DC-3F) and resistant to 9-OH-ellipticine (DC-3F/9O-HE) were isolated. In the sensitive cells, the sequence defines an open reading frame of 4839 nucleotides, and extends over a 323 nucleotides untranslated region up to the putative polyadenylation site. The deduced amino acid sequence predicts a protein with 1612 amino acids in length and a calculated molecular mass of approx. 182 kDa. The cDNAs from the resistant cells only differs by one mutation at position length and a calculated molecular mass of approx. 182 kDa. The cDNAs from the resistant cells only differs by one mutation at position 1710 which converts a Trp codon (TGG) to a stop codon (TGA). This mutation accounts for the loss of DNA topoisomerase II beta in the 9-OH-ellipticine resistant cells.

Lambda-repressed mutants of bacteriophage T5. I. Isolation and genetical characterization.

Abstract We have isolated mutants of T5 whose growth is inhibited in wild-type lambda lysogens, and which we refer to as “lambda-repressed” or “ lr ” mutants. These mutants grow normally in nonlysogenic strains, as well as in strains in which the lambda gene rex is defective or deleted. The results of recombination and complementation tests indicate that lr mutations occur in gene I of the FST-DNA. Amber mutants on the left side, but not on the right side, of the lr -1 locus are also partially inhibited in Su + lysogens, suggesting that lr mutants can only be found in part of gene I. Evidence is presented that the lr gene I product is synthesized in lambda lysogens, and is partially active. The possible mechanisms of this growth inhibition are discussed.

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. Evidence is presented that p53-dependent function (a) is lost not only in the apoptosis defective L1210/DDP10 cells, but also in the apoptosis susceptible L1210/DDP5 cells; (b) is unrelated to drug-induced cell cycle pertubations. These results suggest that deficiency in the p53 pathway and resistance to DNA-damaging agents due to a defect in apoptosis are independent events.

Effect of membrane potential on the cellular uptake of 2-N-methyl-ellipticinium by L1210 cells

Some quaternary ammonium derivatives of ellipticine are active antitumor drugs on both experimental and human tumors. Because of their positive charge, the cellular uptake of these molecules is expected to be influenced by the electric membrane potential. Experimental variations of the potential were produced by changing the external potassium concentration and the potassium permeability by the addition of valinomycin. Using the fluorescent lipophilic cationic dye 3,3-dihexyloxacarbocyanine iodide, the L1210 cell membrane potential was estimated at -35 mV by flow cytometric analysis, and the same technique was then used to study the effects of the membrane potential variations on 2-N-methyl-ellipticinium (NME) cellular uptake. Our results show that indeed NME uptake depends on the cell membrane potential, which might then influence its pharmacological properties.

Activation of the mitogen-activated protein kinase cascade by tyrphostin (RG 50864)

Tyrphostins are synthetic compounds which have been described as in vitro inhibitors of epidermal growth factor (EGF)-receptor tyrosine kinase activity. In NIH3T3 cells, stimulation of EGF-receptor tyrosine kinase leads to an increase of intracellular protein phosphorylations, among them the phosphorylation of mitogen-activated protein (MAP) kinase and the S6 kinases p90rsk and p70S6K. Phosphorylation of these proteins, either on tyrosine or serine/threonine residues or on both residues increases their protein kinase activity. Unexpectedly, treatment of NIH3T3 cells with both tyrphostin (RG 50864) and EGF results in an increase in the level of tyrosine phosphorylation of the MAP kinase. During this treatment, we also observed an increase in MAP kinase and S6 kinase p90rsk activities. Tyrphostin treatment diminishes the level of c-fos mRNA but has no effect on c-myc mRNA expression nor on S6 kinase p70S6K activity. Mitogenic signalling induced by EGF in NIH3T3 cells was blocked by tyrphostin, suggesting that the target(s) for this event may be elements downstream from the MAP kinase or independent of this signal transduction.

Binding of the Escherichia coli UvrAB proteins to the DNA mono- and diadducts of cis-[N-2-amino-N-2-methylamino-2,2,1-bicycloheptane]dichloroplatinum(II ) and cisplatin. Analysis of the factors controlling recognition and proof of monoadduct-mediated UvrB-DNA cross-linking.

The interactions of the Escherichia coli endonuclease UvrAB proteins with the DNA mono- and diadducts of both the cis-racemic exo-[N-2-amino-N-2-methylamino-2,2,1-bicycloheptane]dichloroplatinum(II) (complex 1) and cisplatin (cis-diamminedichloroplatinum(II) (cis-DDP)), have been studied. Complex 1 reacts faster with DNA than cis-DDP and gives monoadducts with a longer lifetime (8 h 20 min chelation t compared with 2 h 40 min for cis-DDP). Using pSP65 plasmid [3H]DNA, the filter binding assay was associated with the analysis of the nucleoprotein complexes to characterize the UvrAB recognition of the platinum adducts and to demonstrate the occurrence of platinum-mediated DNA-protein cross-linking. First, it is shown that the UvrAB proteins recognize the complex 1 mono- and diadducts with a higher affinity than those of cis-DDP. Fifteen times more cis-DDP adducts per plasmid are required than complex 1 adducts, to lead to similar UvrAB binding. However, the UvrAB proteins recognize monoadducts and diadducts of each complex with a similar affinity. Second, it is shown that UvrB is the protein involved in the nucleoprotein complexes formed from mono- and diadducts of complex 1 and cis-DDP. This protein is also partly cross-linked to DNA with a similar efficiency by monoadducts derived from complex 1 and cis-DDP. However, as UvrB has a greater affinity for the DNA adducts of complex 1 than for those of cis-DDP, more UvrB-platinum-DNA cross-links are formed with complex 1 than with cis-DDP. This study, using a bacterial repair system as a model, points to a possible strategy for making new cytotoxic platinum complexes for mammalian cells.

Inhibition by two lavendustins of the tyrosine kinase activity of pp60F527 in vitro and in intact cells

The mutant pp60F527 protein possesses an activated protein-tyrosine kinase (PTK) activity correlated with a transforming activity. We have studied the inhibition of the pp60F527 PTK activity by two EGF-R tyrosine kinase inhibitors, lavendustin A and one of its derivatives, lavendustin C6. In vitro, both molecules were non-competitive inhibitors for the ATP binding site and uncompetitive inhibitors for the peptide binding site. The determined IC50S of the inhibition of pp60F527 kinase activity were 18 microM for lavendustin A and 5 microM for lavendustin C6, as determined on the exogenous substrate enolase, showing that lavendustin C6 was more potent than lavendustin A. Lavendustin C6, but not lavendustin A, inhibited the tyrosine phosphorylation of pp60F527 cellular substrates (the GAP-associated p190, pp125FAK and cortactin) in intact cells. However, this in situ inhibitory effect did not result in a reversion of the morphological changes induced by pp60F527 in cells. On the other hand, lavendustin C6 and lavendustin A exerted antiproliferative effects on cells, suggesting that inhibition of cellular targets related or not to the kinase was also possible.

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. We studied the effects of a tyrphostin derivative, RG 50864, without preincubation in intact epithelial cells. We selected two human cell lines differing in degree of expression of the p185erbB2 protein, which is closely related to EGF-R. We showed that tyrphostin (RG 50864) had no effect on EGF-dependent EGF-R tyrosine phosphorylation in the parental cell line. On the contrary, it prolonged the EGF-dependent EGF-R and p185erbB2(V-E) tyrosine phosphorylation in p185erbB2(V-E)-expressing cells. Because tyrphostin has been shown to be an inhibitor of p185erbB2 and EGF-R in vitro, this finding indicates that the tyrphostin effect on p185erbB2(V-E) and EGF-R was the result of an indirect mechanism in transfected cells. Tyrphostin treatment alone led to the activation of mitogen-activated protein (MAP) kinase kinase or MAP kinase or extracellular signal-regulated kinase kinase (MEK), suggesting that one of the tyrphostin targets was upstream of MEK1. MAP kinase, however, was not activated after tyrphostin treatment. This finding indicates that tyrphostin had another target in intact cells because MEK1 activation by tyrphostin alone did not correlate with MAP kinase activation. In the two cell lines, tyrphostin modified the time course of EGF-dependent MEK and MAP kinase activation. We conclude that whereas tyrphostins were designed to inhibit EGF-R tyrosine kinase activity, under our conditions EGF-R is not a physiological target for tyrphostin, nor is one of its related protein tyrosine kinases, p185erbB2(V-E). On the contrary, our results show that tyrphostin targets are multiple, leading to complex effects on receptor signaling in these epithelial cells.