velikost textu

Modulation of human telomerase activity by nucleoside and nucleotide analogues

Upozornění: Informace získané z popisných dat či souborů uložených v Repozitáři závěrečných prací nemohou být použity k výdělečným účelům nebo vydávány za studijní, vědeckou nebo jinou tvůrčí činnost jiné osoby než autora.
Název:
Modulation of human telomerase activity by nucleoside and nucleotide analogues
Typ:
Disertační práce
Autor:
Mgr. Miroslav Hájek, Ph.D.
Školitel:
RNDr. Ivan Votruba, CSc.
Oponenti:
prof. RNDr Jiří Fajkus, CSc
RNDr. Jiří Hejnar, CSc.
Id práce:
112528
Fakulta:
Přírodovědecká fakulta (PřF)
Pracoviště:
Katedra biochemie (31-250)
Program studia:
Biochemie (P1406)
Obor studia:
-
Přidělovaný titul:
Ph.D.
Datum obhajoby:
18. 9. 2008
Výsledek obhajoby:
Prospěl/a
Jazyk práce:
Angličtina
Abstrakt:
CONCLUSIONS Considering human telomerase as a promising target of anti-cancer therapy, the thesis deals with the study of inhibitory potency of selected ANP diphosphates towards telomerase, and the capability of nucleoside-type DNA methylation inhibitors to inhibit hTERT expression, knowing that hTERT expression closely correlates with telomerase activity in vitro and in vivo. The results can be summarized as follows: • All the purine ANP diphosphates except for (S)-PMPApp and 6-Me2PMEDAPpp show dose- dependent inhibition of human telomerase in cell-free assay, the adenine derivatives are less effective inhibitors than the guanine derivatives. The only two pyrimidine ANP diphosphates tested (PMECpp and PMETpp) do not show any significant inhibitory potency towards telomerase. • Activity of tested ANPs on telomerase is limited to their diphosphates (ANPpp) only. • (R)-enantiomers are more inhibitory compared to (S)-enantiomers. This indicates that absolute configuration plays an important role in the telomerase inhibition and that the enzyme distinguishes between the (R)- and (S)-enantiomers. • PMEGpp is the most potent human telomerase inhibitor among all ANPs studied with the IC50 value of 12.7 ± 0.5 mol.l-1 at 125 M dNTPs. Its inhibitory potency towards telomerase is comparable to that of ddGTP (IC50 value of 8.1 ± 0.4 mol.l-1 at 125 M dNTPs), which is known to be one of the most effective nucleotide analogue based telomerase inhibitors. • (S)-PMPApp and 6-Me2PMEDAPpp do not inhibit telomerase, on the contrary, they increase the repeat addition processivity of telomerase in a dose-dependent manner in cell-free assay. • Although PMEGpp is a much more potent human telomerase inhibitor than any of the other ANPs tested, only a moderate and reversible telomere shortening can be achieved by exposure to 0.75 M PMEG over a period of 9 weeks of treatment – CCRF-CEM cells lost about 20% of their initial mean telomere length. In contrast, treatment with 20 M PMEDAP caused a progressive and irreversible telomere shortening in CCRF-CEM cell line. Cells lost more than 60% of their initial mean telomere length until the removal of PMEDAP from the growth media in the end of 11th week. • The increase of telomerase processivity in vitro caused by (S)-PMPApp has not been shown to be manifested in telomere elongation in the growing cells - (S)-PMPA does not cause any significant changes in telomere length in CCRF-CEM cells when supplied in the growth medium for 11 weeks at concentration of 100 mol.l-1. • Both -5-azadCyd and -5-azadCyd down-regulate hTERT expression, however, treatment with these compounds induces a distinct pattern of hTERT expression in HL-60 cells. -5-AzadCyd inhibited hTERT expression in the whole range of tested concentrations whereas the beta anomer (decitabine) causes a transient elevation of hTERT mRNA at low micromolar concentrations followed by subsequent hTERT down-regulation at higher concentrations of -5-azadCyd. The increase of hTERT expression correlates with up-regulation of c-myc, however, the subsequent decrease in hTERT expression seems to be independent of c-myc expression. • The reversible SAH-hydrolase inhibitor (S)-DHPA causes up-regulation of hTERT within a broad range of concentrations up to 1000 M. c-Myc expression is significantly elevated at all tested concentrations. • The irreversible SAH-hydrolase inhibitor (R,S)-AHPA-ibu exhibites stronger potency to inhibit hTERT expression when compared to (S)-DHPA. In contrast to (S)-DHPA, we observed a significant decrease in hTERT mRNA levels from concentration corresponding to its GIC50 value (174 M). Again, there is an up-regulation of hTERT expression at lower concentrations of (R,S)- AHPA-ibu and c-myc remains overexpressed within the whole range of tested concentrations. Similar to -5-azadCyd and (S)-DHPA, the down-regulation of hTERT seems to be independent of c-myc expression. • Treatment with (S)-HPMPazaC results in the decrease of hTERT mRNA levels. The effect of (S)-HPMPazaC on expression of hTERT and c-myc is similar to that of -5-azadCyd, however, at considerably higher concentrations. No transient elevation of hTERT mRNA levels and no c-myc overexpression is observed. • From the studied compounds, F-PymRf was shown to have the highest potency to increase c- myc and hTERT mRNA levels.
Abstract v angličtině:
CONCLUSIONS Considering human telomerase as a promising target of anti-cancer therapy, the thesis deals with the study of inhibitory potency of selected ANP diphosphates towards telomerase, and the capability of nucleoside-type DNA methylation inhibitors to inhibit hTERT expression, knowing that hTERT expression closely correlates with telomerase activity in vitro and in vivo. The results can be summarized as follows: • All the purine ANP diphosphates except for (S)-PMPApp and 6-Me2PMEDAPpp show dose- dependent inhibition of human telomerase in cell-free assay, the adenine derivatives are less effective inhibitors than the guanine derivatives. The only two pyrimidine ANP diphosphates tested (PMECpp and PMETpp) do not show any significant inhibitory potency towards telomerase. • Activity of tested ANPs on telomerase is limited to their diphosphates (ANPpp) only. • (R)-enantiomers are more inhibitory compared to (S)-enantiomers. This indicates that absolute configuration plays an important role in the telomerase inhibition and that the enzyme distinguishes between the (R)- and (S)-enantiomers. • PMEGpp is the most potent human telomerase inhibitor among all ANPs studied with the IC50 value of 12.7 ± 0.5 mol.l-1 at 125 M dNTPs. Its inhibitory potency towards telomerase is comparable to that of ddGTP (IC50 value of 8.1 ± 0.4 mol.l-1 at 125 M dNTPs), which is known to be one of the most effective nucleotide analogue based telomerase inhibitors. • (S)-PMPApp and 6-Me2PMEDAPpp do not inhibit telomerase, on the contrary, they increase the repeat addition processivity of telomerase in a dose-dependent manner in cell-free assay. • Although PMEGpp is a much more potent human telomerase inhibitor than any of the other ANPs tested, only a moderate and reversible telomere shortening can be achieved by exposure to 0.75 M PMEG over a period of 9 weeks of treatment – CCRF-CEM cells lost about 20% of their initial mean telomere length. In contrast, treatment with 20 M PMEDAP caused a progressive and irreversible telomere shortening in CCRF-CEM cell line. Cells lost more than 60% of their initial mean telomere length until the removal of PMEDAP from the growth media in the end of 11th week. • The increase of telomerase processivity in vitro caused by (S)-PMPApp has not been shown to be manifested in telomere elongation in the growing cells - (S)-PMPA does not cause any significant changes in telomere length in CCRF-CEM cells when supplied in the growth medium for 11 weeks at concentration of 100 mol.l-1. • Both -5-azadCyd and -5-azadCyd down-regulate hTERT expression, however, treatment with these compounds induces a distinct pattern of hTERT expression in HL-60 cells. -5-AzadCyd inhibited hTERT expression in the whole range of tested concentrations whereas the beta anomer (decitabine) causes a transient elevation of hTERT mRNA at low micromolar concentrations followed by subsequent hTERT down-regulation at higher concentrations of -5-azadCyd. The increase of hTERT expression correlates with up-regulation of c-myc, however, the subsequent decrease in hTERT expression seems to be independent of c-myc expression. • The reversible SAH-hydrolase inhibitor (S)-DHPA causes up-regulation of hTERT within a broad range of concentrations up to 1000 M. c-Myc expression is significantly elevated at all tested concentrations. • The irreversible SAH-hydrolase inhibitor (R,S)-AHPA-ibu exhibites stronger potency to inhibit hTERT expression when compared to (S)-DHPA. In contrast to (S)-DHPA, we observed a significant decrease in hTERT mRNA levels from concentration corresponding to its GIC50 value (174 M). Again, there is an up-regulation of hTERT expression at lower concentrations of (R,S)- AHPA-ibu and c-myc remains overexpressed within the whole range of tested concentrations. Similar to -5-azadCyd and (S)-DHPA, the down-regulation of hTERT seems to be independent of c-myc expression. • Treatment with (S)-HPMPazaC results in the decrease of hTERT mRNA levels. The effect of (S)-HPMPazaC on expression of hTERT and c-myc is similar to that of -5-azadCyd, however, at considerably higher concentrations. No transient elevation of hTERT mRNA levels and no c-myc overexpression is observed. • From the studied compounds, F-PymRf was shown to have the highest potency to increase c- myc and hTERT mRNA levels.
Dokumenty
Stáhnout Dokument Autor Typ Velikost
Stáhnout Text práce Mgr. Miroslav Hájek, Ph.D. 2.22 MB
Stáhnout Abstrakt v českém jazyce Mgr. Miroslav Hájek, Ph.D. 243 kB
Stáhnout Abstrakt anglicky Mgr. Miroslav Hájek, Ph.D. 243 kB
Stáhnout Posudek oponenta prof. RNDr Jiří Fajkus, CSc 101 kB
Stáhnout Posudek oponenta RNDr. Jiří Hejnar, CSc. 259 kB
Stáhnout Záznam o průběhu obhajoby 416 kB