RÉSUMÉ
OBJECTIVE: In the present study, we sought to develop/characterize the pain profile of a rat model of surgically induced osteoarthritis (OA). METHODS: OA was surgically induced in male Lewis rats (200-225 g) by transection of the medial collateral ligament and medial meniscus of the femoro-tibial joint. In order to characterize the pain profile, animals were assessed for a change in hind paw weight distribution (HPWD), development of mechanical allodynia, and the presence of thermal and mechanical hyperalgesia. Rofecoxib and gabapentin were examined for their ability to decrease change in weight distribution and tactile allodynia. RESULTS: Transection of the medial collateral ligament and medial meniscus of male Lewis rats resulted in rapid (<3 days) changes in hind paw weight bearing and the development of tactile allodynia (secondary hyperalgesia). There was, however, no appreciable effect on thermal hyperalgesia or mechanical hyperalgesia. Treatment with a single dose of rofecoxib (10 mg/kg, PO, day 21 post surgery) or gabapentin (100mg/kg, PO, day 21 post surgery) significantly attenuated the change in HPWD, however, only gabapentin significantly decreased tactile allodynia. CONCLUSION: The rat medial meniscal tear (MMT) model mimics both nociceptive and neuropathic OA pain and is responsive to both a selective cylooxygenase-2 (COX-2) inhibitor commonly utilized for OA pain (rofecoxib) and a widely prescribed drug for neuropathic pain (gabapentin). The rat MMT model may therefore represent a predictive tool for the development of pharmacologic interventions for the treatment of the symptoms associated with OA.
Sujet(s)
Arthralgie/anatomopathologie , Hyperalgésie/anatomopathologie , Gonarthrose/anatomopathologie , Amines/usage thérapeutique , Analgésiques/usage thérapeutique , Animaux , Arthralgie/traitement médicamenteux , Arthralgie/étiologie , Acides cyclohexanecarboxyliques/usage thérapeutique , Inhibiteurs de la cyclooxygénase 2/usage thérapeutique , Modèles animaux de maladie humaine , Gabapentine , Membre pelvien , Hyperalgésie/traitement médicamenteux , Hyperalgésie/étiologie , Lactones/usage thérapeutique , Mâle , Gonarthrose/complications , Rats , Sulfones/usage thérapeutique , Mise en charge/physiologie , Acide gamma-amino-butyrique/usage thérapeutiqueRÉSUMÉ
Wee1 protein kinase plays an important regulatory role in cell cycle progression. It inhibits Cdc-2 activity by phosphorylating Tyr15 and arrests cells at G2-M phase. In an attempt to understand Wee1 regulation during cell cycle, yeast two-hybrid screening was used to identify Wee1-binding protein(s). Five of the eight positive clones identified encode 14-3-3beta. In vivo binding assay in 293 cells showed that both full-length and NH2-terminal truncated Wee1 bind with 14-3-3beta. The 14-3-3beta binding site was mapped to a COOH-terminal consensus motif, RSVSLT (codons 639 to 646). Binding with 14-3-3beta increases the protein level of full-length Wee1 but not of the truncated Wee1. Accompanying the protein level increases, the kinase activity of Wee1 also increases when coexpressed with 14-3-3beta. Increased Wee1 protein level/enzymatic activity is accountable, at least in part, to an increased Wee1 protein half-life when coexpressed with 14-3-3beta. The protein half-life of the NH2-terminal truncated Wee1 is much longer than that of the full-length protein and is not affected by 14-3-3beta cotransfection. Biologically, 14-3-3beta/Wee1 coexpression increases the cell population at G2-M phase. Thus, Wee1 binding with 14-3-3beta increases its biochemical activity as well as its biological function. The finding reveals a novel mechanism by which 14-3-3 regulates G2-M arrest and suggests that the NH2-terminal domain of Wee1 contains a negative regulatory sequence that determines Wee1 stability.
Sujet(s)
Protéines du cycle cellulaire , Cycle cellulaire/physiologie , Protéines nucléaires , Protein-tyrosine kinases/métabolisme , Protéines/métabolisme , Tyrosine 3-monooxygenase , Protéines 14-3-3 , Sites de fixation , Lignée cellulaire , ADN recombiné/génétique , Phase G2 , Humains , Mitose , Plasmides , Liaison aux protéines , Protein-tyrosine kinases/composition chimique , Protein-tyrosine kinases/génétique , Protéines/génétique , Saccharomyces cerevisiae/génétique , Protéines de Schizosaccharomyces pombe , Transfection , Techniques de double hybrideRÉSUMÉ
A class of high-affinity inhibitors is disclosed that selectively target and irreversibly inactivate the epidermal growth factor receptor tyrosine kinase through specific, covalent modification of a cysteine residue present in the ATP binding pocket. A series of experiments employing MS, molecular modeling, site-directed mutagenesis, and 14C-labeling studies in viable cells unequivocally demonstrate that these compounds selectively bind to the catalytic domain of the epidermal growth factor receptor with a 1:1 stoichiometry and alkylate Cys-773. While the compounds are essentially nonreactive in solution, they are subject to rapid nucleophilic attack by this particular amino acid when bound in the ATP pocket. The molecular orientation and positioning of the acrylamide group in these inhibitors in relation to Cys-773 entirely support these results as determined from docking experiments in a homology-built molecular model of the ATP site. Evidence is also presented to indicate that the compounds interact in an analogous fashion with erbB2 but have no activity against the other receptor tyrosine kinases or intracellular tyrosine kinases that were tested in this study. Finally, a direct comparison between 6-acrylamido-4-anilinoquinazoline and an equally potent but reversible analog shows that the irreversible inhibitor has far superior in vivo antitumor activity in a human epidermoid carcinoma xenograft model with no overt toxicity at therapeutically active doses. The activity profile for this compound is prototypical of a generation of tyrosine kinase inhibitors with great promise for therapeutic significance in the treatment of proliferative disease.
Sujet(s)
Antienzymes/pharmacologie , Récepteurs ErbB/antagonistes et inhibiteurs , Protein-tyrosine kinases/antagonistes et inhibiteurs , Quinazolines/pharmacologie , Récepteur ErbB-2/antagonistes et inhibiteurs , Animaux , Antinéoplasiques/composition chimique , Antinéoplasiques/pharmacologie , Sites de fixation/génétique , Lignée cellulaire , Cystéine/composition chimique , Antienzymes/composition chimique , Récepteurs ErbB/composition chimique , Récepteurs ErbB/génétique , Humains , Souris , Souris nude , Modèles moléculaires , Mutagenèse dirigée , Transplantation tumorale , Tumeurs expérimentales/traitement médicamenteux , Tumeurs expérimentales/métabolisme , Conformation des protéines , Protein-tyrosine kinases/composition chimique , Protein-tyrosine kinases/génétique , Quinazolines/composition chimique , Récepteur ErbB-2/composition chimique , Récepteur ErbB-2/génétique , Protéines de fusion recombinantes/antagonistes et inhibiteurs , Protéines de fusion recombinantes/génétique , Transplantation hétérologue , Cellules cancéreuses en cultureRÉSUMÉ
PD153035 is a potent (Ki = 6 pm) and specific inhibitor of the epidermal growth factor (EGF) receptor tyrosine kinase that suppresses tyrosine phosphorylation of the EGF receptor in A431 cells at nanomolar concentrations in cell culture. We have examined the pharmacokinetics of this compound and its ability to rapidly suppress phosphorylation of the EGF receptor in A431 human epidermoid tumors grown as xenografts in immunodeficient nude mice. Following a single i.p. dose of 80 mg/kg, the drug levels in the plasma and tumor rose to 50 and 22 microM within 15 minutes. While the plasma levels of PD153035 fell below 1 microM by 3 hours, in the tumors it remained at micromolar concentrations for at least 12 hours. The tyrosine phosphorylation of the EGF receptor was rapidly suppressed by 80-90% in the tumors. However receptor phosphorylation returned to control levels after 3 hours despite the continued presence of the drug at concentrations which, based on previous in vitro results, were predicted to maintain inhibition. EGF-stimulated tyrosine kinase activity in tumor extracts was decreased and recovered in parallel with the effects of PD153035 on receptor phosphorylation though the activity had reached only about half of the control activity after three hours. These results demonstrate the potential for using small molecule inhibitors to inhibit the EGF receptor tyrosine kinase in vivo, though a fair evaluation of their potential anti-cancer activity will have to wait for solutions to problems with sustained delivery which may allow us to maintain suppression of EGF receptor phosphorylation.
Sujet(s)
Carcinome épidermoïde/traitement médicamenteux , Carcinome épidermoïde/enzymologie , Antienzymes/pharmacologie , Récepteurs ErbB/antagonistes et inhibiteurs , Tumeurs/traitement médicamenteux , Tumeurs/enzymologie , Quinazolines/pharmacologie , Animaux , Division cellulaire/effets des médicaments et des substances chimiques , Antienzymes/pharmacocinétique , Humains , Souris , Souris nude , Tumeurs/métabolisme , Quinazolines/pharmacocinétique , Transplantation hétérologue , Cellules cancéreuses en cultureRÉSUMÉ
CI-980, originally synthesized as a potential folate antagonist, is a tubulin-binding mitotic inhibitor currently in pediatric phase I and adult phase II clinical trials. Because of its extensive tissue distribution in animals and its favorable activity against multidrug resistant (MDR)-cells compared with other mitotic inhibitors, such as vincristine, we examined the membrane transport properties of CI-980. CI-980 accumulated rapidly in L1210 and CHO/K1 cells, reaching intracellular levels 40- and 8-fold higher, respectively, than those in the extracellular medium. Efflux was also quite rapid, but a small fraction of drug remained associated with the cells in drug-free medium. The uptake of CI-980 was not temperature or energy dependent, nor was it saturable up to an extracellular concentration of 100 microM. Inhibitors of nucleoside transport had no effect on CI-980 uptake. A cell line deficient in the transport of reduced folate was not resistant to CI-980, nor did it exhibit reduced CI-980 uptake. A 100-fold excess of the R-enantiomer inhibited CI-980 uptake by only 50%. These results are consistent with a model of CI-980 uptake involving passive diffusion followed by significant but largely reversible binding to intracellular or membrane components.
Sujet(s)
Antinéoplasiques/pharmacocinétique , Carbamates/pharmacocinétique , Antifoliques/pharmacocinétique , Pyrazines/pharmacocinétique , Pyridines/pharmacocinétique , Animaux , Transport biologique , Cellules CHO/métabolisme , Protéines de transport/physiologie , Bovins , Cricetinae , Dipyridamole/pharmacologie , Tests de criblage d'agents antitumoraux , Humains , Leucémie L1210/métabolisme , Antiagrégants plaquettaires/pharmacologie , StéréoisomérieRÉSUMÉ
The mechanism of action of the novel anti-cancer compound CI-994 was studied in C26 murine colon tumor and HCT-8 human colon adenocarcinoma cells. Treatment of either cell line resulted in the specific loss of a 16-kDa phosphoprotein in a time- and concentration-dependent manner. Treatment with salicylanilide, CI-940, mimosine, aphidicolin, quercetin or ciclopirolxalamine, which, like CI-994, block cells in the G1-S phase of the cell cycle, did not affect the production of this protein. Loss of the 16-kDa protein preceded the block in cell proliferation induced by CI-994 treatment, and recovery of this protein was evident prior to the resumption of cell growth. Cellular fractionation studies demonstrated that the 16 kDa phosphoprotein is confined to the nuclear compartment. Our data indicate that loss the 16-kDa nuclear phosphoprotein appears to be a direct effect of CI-994 treatment and that the inhibition of this phosphoprotein may play a critical role in the mechanism of action of CI-994.
Sujet(s)
Antinéoplasiques/pharmacologie , Phénylènediamines/pharmacologie , Phosphoprotéines/métabolisme , Adénocarcinome/traitement médicamenteux , Animaux , Benzamides , Cycle cellulaire/effets des médicaments et des substances chimiques , Noyau de la cellule/métabolisme , Tumeurs du côlon/traitement médicamenteux , Humains , Techniques in vitro , Souris , Masse moléculaire , Phosphoprotéines/composition chimique , Salicylanilides/pharmacologie , Cellules cancéreuses en cultureRÉSUMÉ
Carmethizole, a novel bis-carbamate alkylating agent, was evaluated in vitro for potential mechanisms of interaction with DNA and in vivo for spectrum and degree of antitumor activity. In vitro, the concentration of carmethizole required to produce a 50% reduction in clonogenic cell survival was identical in O6-alkylguanine DNA alkyltransferase-positive and -negative human cell lines. The CHO cell line UV4, hypersensitive to mono- and bifunctional alkylating agents, was 37-fold more sensitive to carmethizole than normal cells. The UV5 cell line, which is not hypersensitive to cross-linkers, was 13-fold more sensitive to carmethizole than normal cells. Alkaline elution studies in L1210 cells exposed to carmethizole showed the presence of DNA-protein and DNA-DNA cross-links but not DNA strand breaks. These data suggested that the interaction of carmethizole with DNA produces monoadducts, DNA-protein, and DNA-DNA interstrand cross-links at several sites. In vivo, carmethizole was not cross-resistant with 1,3-bis(2-chloroethyl)-1-nitrosourea or Cytoxan as determined by testing against P388 leukemias resistant to the latter 2 agents. Carmethizole activity was similar to that of melphalan across the murine solid tumor panel, which consisted of B16 melanoma; colon adenocarcinomas 11a, 26, and 36; and the KHT sarcoma. Carmethizole, Cytoxan, and melphalan were all active and had comparable activity against the HCT-8 and MX-1 human tumor xenografts. The in vivo spectrum of activity and efficacy of carmethizole was similar to that of melphalan.
Sujet(s)
Antinéoplasiques/pharmacologie , ADN tumoral/effets des médicaments et des substances chimiques , Imidazoles/pharmacologie , Leucémie L1210/traitement médicamenteux , Leucémie P388/traitement médicamenteux , Animaux , Carmustine/pharmacologie , Réparation de l'ADN , Tests de criblage d'agents antitumoraux , Leucémie L1210/génétique , Leucémie P388/génétique , Melphalan/pharmacologie , Cellules cancéreuses en culture/effets des médicaments et des substances chimiquesRÉSUMÉ
1. APP is activated by adenosine kinase to its 5'-phosphate (APP-MP). 2. APP-MP inhibits PRPP synthetase, and depletes cellular PRPP and purine and pyrimidine nucleotides. 3. APP inhibits synthesis of DNA and RNA, and blocks cells in G1 phase of the cell cycle. 4. APP retains full activity against MDR cells. 5. APP is equally active against quiescent and proliferating CHO cells. 6. APP has only weak activity against L1210 leukemia in vivo, but has substantial activity against mammary carcinoma 16/c. 7. In vitro, APP has a relatively high ratio of solid tumor: leukemia activity.
Sujet(s)
Antimétabolites antinéoplasiques/pharmacologie , Leucémie L1210/traitement médicamenteux , Phosphotransferases/antagonistes et inhibiteurs , Ribose phosphate pyrophosphokinase/antagonistes et inhibiteurs , Cellules cancéreuses en culture/effets des médicaments et des substances chimiques , Animaux , Lignée cellulaire , Survie cellulaire/effets des médicaments et des substances chimiques , Humains , Souris , Purines/antagonistes et inhibiteurs , Purines/pharmacologie , Nucléosides pyrimidiques/pharmacologie , Nucléosides pyrimidiques/usage thérapeutiqueRÉSUMÉ
The cell cycle phase specificity of trimetrexate (CI-898) was examined. CHO cells synchronized by mitotic selection were exposed to 50 microM trimetrexate for 2 h at various time points after release from Colcemid block. Only S phase cells were sensitive to trimetrexate when survival was measured by a cloning assay. Comparison of plateau phase and log phase cultures indicated that plateau phase CHO cells were relatively insensitive to 5 microM trimetrexate. Exponentially growing L1210 cells were continuously exposed to either 30 nM or 3 nM trimetrexate and analyzed by DNA flow cytometry. Incubation with 30 nM trimetrexate produced cell cycle arrest in late G1 or early S phase, while exposure to 3 nM trimetrexate caused only a delay in progression through S phase. In an in vivo schedule dependence study with mice bearing approximately 3 X 10(6) P388 leukemia cells, trimetrexate was most effective with frequent administration. Mice treated on the optimal schedule, every 3 h X 8 on days 1, 5, and 9 after tumor implant, had life-span increases in excess of 100%.
Sujet(s)
Antinéoplasiques/usage thérapeutique , Ovaire/cytologie , Quinazolines/usage thérapeutique , Animaux , Antinéoplasiques/toxicité , Cycle cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , Cellules cultivées , Cricetinae , Démécolcine , Évaluation préclinique de médicament , Femelle , Cytométrie en flux , Injections péritoneales , Interphase , Leucémie L1210/traitement médicamenteux , Méthotrexate/pharmacologie , Souris , Tumeurs expérimentales/traitement médicamenteux , Quinazolines/toxicité , TrimétrexateRÉSUMÉ
CI-920 is a structurally novel, phosphate-containing polyene lactone antitumor agent isolated from a previously undescribed subspecies of Streptomyces pulveraceus cultured from a Brazilian soil sample. CI-920 was active against murine leukemia P388, and highly active and curative against L1210 leukemia in vivo. CI-920 was less active or inactive against the murine solid tumors tested. Daily administration for five to nine days was more effective against L1210 leukemia than a single dose or doses every four days. Given three times daily for five days, CI-920 was more toxic and less active. CI-920 had similar activity intravenously and intraperitoneally. Oral administration was inactive and nontoxic. Subcutaneous treatment was less effective and more toxic. Structure-activity relationship studies showed that the phosphate group was essential for antitumor activity in vivo and in vitro. Hydrolyzing the lactone ring also resulted in loss of antitumor activity, as did acetylation of the 6-hydroxyl group. Hydroxylation at the 5-position of the lactone ring resulted in partial retention of antitumor activity, but in greater toxicity to mice. Removal of the 13-hydroxyl group resulted in retention of high antitumor activity with approximately three-fold improvement in dose-potency. CI-920 is not cytotoxic to prokaryotic cells. CI-920 causes inhibition of biosynthesis of RNA and DNA in intact L1210 cells. Protein synthesis is also inhibited at higher drug concentrations. The inhibition of nucleic acid synthesis is not an antimetabolite effect, since pools of ribonucleoside triphosphates and deoxyribonucleoside triphosphates are not depleted. CI-920 does not cause DNA strand breakage, as measured by alkaline elution, and is not mutagenic in the Ames test at concentrations up to 200 micrograms/ml. CI-920 does not cause direct inhibition of RNA polymerase or DNA polymerase in permeabilized cells. It is possible that CI-920 must be metabolically activated within the target cells; alternatively it may interact with a component of chromatin other than DNA or the polymerases. Flow cytometry studies showed that growth-inhibitory levels of CI-920 caused accumulation of cells in the G2+M region. Higher drug concentrations caused an S-phase block. CI-920 is an inhibitor and irreversible inactivator of reduced folate membrane transport, and appears to enter cells by this receptor. L1210 cells selected for resistance to CI-920 are cross-resistant to methotrexate, and deficient in reduced folate transport.(ABSTRACT TRUNCATED AT 400 WORDS)