Transcriptional Activation and Cell Cycle Block Are the Keys for 5-Fluorouracil Induced Up-Regulation of Human Thymidylate Synthase Expression

International audienceBackground: 5-fluorouracil, a commonly used chemotherapeutic agent, up-regulates expression of human thymidylate synthase (hTS). Several different regulatory mechanisms have been proposed to mediate this up-regulation in distinct cell lines, but their specific contributions in a single cell line have not been investigated to date. We have established the relative contributions of these previously proposed regulatory mechanisms in the ovarian cancer cell line 2008 and the corresponding cisplatin-resistant and 5-FU cross-resistant-subline C13*. Methodology/Principal Findings: Using RNA polymerase II inhibitor DRB treated cell cultures, we showed that 70-80% of up-regulation of hTS results from transcriptional activation of TYMS mRNA. Moreover, we report that 5-FU compromises the cell cycle by blocking the 2008 and C13* cell lines in the S phase. As previous work has established that TYMS mRNA is synthesized in the S and G 1 phase and hTS is localized in the nuclei during S and G 2-M phase, the observed cell cycle changes are also expected to affect the intracellular regulation of hTS. Our data also suggest that the inhibition of the catalytic activity of hTS and the up-regulation of the hTS protein level are not causally linked, as the inactivated ternary complex, formed by hTS, deoxyuridine monophosphate and methylenetetrahydrofolate, was detected already 3 hours after 5-FU exposure, whereas substantial increase in global TS levels was detected only after 24 hours. Conclusions/Significance: Altogether, our data indicate that constitutive TYMS mRNA transcription, cell cycle-induced hTS regulation and hTS enzyme stability are the three key mechanisms responsible for 5-fluorouracil induced up-regulation of human thymidylate synthase expression in the two ovarian cancer cell lines studied. As these three independent regulatory phenomena occur in a precise order, our work provides a feasible rationale for earlier observed synergistic combinations of 5-FU with other drugs and may suggest novel therapeutic strategies. Cop. 2012 Ligabue et al

The 1,10-phenanthroline ligand enhances the antiproliferative activity of dna-intercalating thiourea-pd(Ii) and-pt(ii) complexes against cisplatin-sensitive and-resistant human ovarian cancer cell lines

Ovarian cancer is the most lethal gynecological malignancy, often because of the frequent insurgence of chemoresistance to the drugs currently used. Thus, new therapeutical agents are needed. We tested the toxicity of 16 new DNA-intercalating agents to cisplatin (cDDP)-sensitive human ovarian carcinoma cell lines and their resistant counterparts. The compounds were the complexes of Pt(II) or Pd(II) with bipyridyl (bipy) and phenanthrolyl (phen) and with four dierent thiourea ancillary ligands. Within each of the four series of complexes characterized by the same thiourea ligand, the Pd(phen) drugs invariably showed the highest anti-proliferative ecacy. This paralleled both a higher intracellular drug accumulation and a more ecient DNA intercalation than all the other metal-bidentate ligand combinations. The consequent inhibition of topoisomerase II activity led to the greatest inhibition of DNA metabolism, evidenced by the inhibition of the expression of the folate cycle enzymes and a marked perturbation of cell-cycle distribution in both cell lines. These findings indicate that the particular interaction of Pd(II) with phenanthroline confers the best pharmacokinetic and pharmacodynamic properties that make this class of DNA intercalators remarkable inhibitors, even of the resistant cell growth

Effetto delle poliammine sulla regolazione della proteina cinasi C.

Effetto delle poliamine sulla regolazione della proteina cinasi C. Nel tentativo di contribuire al chiarimento del ruolo delle poliamine sui processi di fosforilazione intracellulari, in questa fase dell’attività di ricerca abbiamo preso in esame gli effetti delle poliamine su diversi aspetti della regolazione della proteina cinasi C (PKC), serina/treonina cinasi che si presenta in diverse forme isoenzimatiche, alcune Ca++/diacilglicerolo (DAG)/fosfolipide dipendenti, ed altre con attività indipendente dal Ca++. Dal momento che l’associazione della PKC alle membrane è il prerequisito essenziale per la risposta fisiologica dell’enzima, particolare interesse è stato rivolto allo studio dell’effetto delle poliamine sul processo di associazione della PKC alle membrane. A questo scopo abbiamo utilizzato un sistema sperimentale costituito da PKC parzialmente purificata da cervello di ratto e da liposomi, costituiti da fosfolipidi acidi, soprattutto acido fosfatidico (PA) e fosfatidilserina (PS). La PKC associata all membrane è stata saggiata quantitativamente come recettore degli esteri del forbolo cioè misurando il legame dell’enzima attivato ad un estere del forbolo radioattivo (3H-PDBu) con cui l’enzima interagisce con una stechiometria di 1:1. Esperimenti eseguiti con liposomi di composizione definita hanno permesso di stabilire che nelle condizioni in cui prevale la formazione del complesso caratterizzato da una stechiometria di legame di 3/4 molecole di PL acido/molecola di SPM si ha un’inibizione nella formazione del complesso attivo. In questo caso infatti sembra che la SPM si disponga parallelamente alla superficie del liposoma impedendo l’ulteriore legame dell’enzima. Al contrario, nelle condizioni in cui prevale la formazione del complesso caratterizzato da una molecola di SPM/molecola di PS, la poliamina sembra legarsi perpendicolarmente alla superficie delle vescicole, e mediante i gruppi amminici non coinvolti nell’interazione con i fosfolipidi legare i siti anionici dell’enzima, favorendo l’interazione della PKC con i liposomi

Differential induction of spermidine/spermine N1-acetyltransferase in cisplatin-sensitive and -resistant human breast cancer cells by N1, N12-bis(ethyl)spermine.

The cisplatin-resistant human breast cancer MCF7/CH cells have been shown to be also cross-resistant to the spermine analog N1-N12-bisethylspermine (BESPM) in comparison to their -sensitive counterparts, MCF7 cells. This cross-resistance was, at least in part, ascribable to the reduced induction of the polyamine key catabolic enzyme SSAT, which is one of the main target of this drug. As a consequence of this different induction of SSAT activity, the resistant cell line was less depleted of its polyamine content than the parental line, accounting for its better survival to the analog treatment

Intracellular quantitative detection of human thymidylate synthase engagement with an unconventional inhibitor using tetracysteine-diarsenical-probe technology

Demonstrating a candidate drug's interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new model can be developed into a test for high-throughput screening studies of intracellular target-protein/small-molecule binding

Effect of spermine on irreversible insertion of protein kinase C into phospholipid vesicles.

Nelle condizioni in cui prevale la formazione del complesso caratterizzato da una molecola di SPM/molecola di PS, la poliamina sembra legarsi perpendicolarmente alla superficie delle vescicole, e mediante i gruppi amminici non coinvolti nell’interazione con i fosfolipidi legare i siti anionici dell’enzima, favorendo l’interazione della PKC con i liposomi. Questa disposizione sembra essere confermata anche da esperimenti di correlazione eteronucleare bi-dimensionale con la spettroscopia di risonanza magnetica nucleare. Poichè’ stato dimostrato che la PKC può esistere in 2 stati di associazione con la membrana, uno reversibile e Ca++-dipendente ed uno irreversibile Ca++-indipendente, avente le caratteristiche di una proteina intrinseca di membrana, abbiamo investigato l’effetto inibitorio della SPM sull’associazione reversibile o irreversibile della PKC con i liposomi in funzione della concentrazione di Ca++. I risultati ottenuti hanno dimostrato che, a concentrazioni di Ca++ inferiori a 0,1 μM, la SPM inibiva la formazione del complesso fra PKC e membrane. A concentrazioni maggiori la SPM non impediva il processo di associazione, ma faceva diminuire le molecole di enzima inserite nella membrana e insensibile ai chelanti come EGTA, senza alterare l’affinità di legame. L’aumento della concentrazione di PL aumentava i livelli di PKC inserita nei liposomi, confermando che la SPM, complessando i siti di legame sulle membrane sia in presenza che in assenza di Ca++, favorisce le condizioni di legame che ostacolano l’inserimento dell’enzima nella membrana

Effect of Spermine on Membrane-Associated and Membrane-Inserted Forms of Protein Kinase C

Protein kinase C is reported to exist in two membrane-bound states: a reversible one which can be dissociated by calcium chelators (membrane-associated form) and an irreversible one which is chelator stable (membrane-inserted form). In the present work the effects of a naturally occurring polyamine (spermine) on the membrane-associated and membrane-inserted forms of protein kinase C were investigated using a reconstituted system consisting of partially purified protein kinase C from rat brain and phospholipid vesicles of defined composition. The active membrane-bound complex was conveniently determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry. Our experimental data show that, in the absence of calcium ions, the amount of enzyme bound to phospholipids vesicles was dramatically reduced by the presence of spermine whereas the PDBu binding affinity was not significantly affected. The addition of the divalent cation increased the affinity of phorbol ester for the active complex but had no effect on N(max); spermine added in this experimental conditions was no longer able to decrease the total number of enzyme molecules bound to liposomes. Moreover gel filtration experiments of the protein kinase C-phospholipids complex formed in the presence of calcium, indicated that polyamine added during the association process was able to reduce the extent of enzyme insertion into liposomes. Since the increase in phospholipid concentration resulted in a higher level of non-dissociable protein kinase C-liposomes complex we propose that spermine, complexing to membrane binding sites both in the absence and in the presence of Ca++, could promote binding conditions that oppose to the formation of the inserted form of the enzyme. As a consequence the distribution between the reversible and the irreversible membrane-bound forms of protein kinase C is affected