Cyclin E-cdk2 activation is associated with cell cycle arrest and inhibition of DNA replication induced by the thymidylate synthase inhibitor Tomudex.
Tomudex (ZD1694) is a specific antifolate-based thymidylate synthase inhibitor active in a variety of solid tumor malignancies. Studies were carried out in vitro to evaluate downstream molecular alterations induced as a consequence of the potent and sustained inhibition of thymidylate synthase by Tomudex. Twenty-four hours following the initial 2-h treatment with Tomudex, human A253 head and neck squamous carcinoma cells, not expressing p53 and p21(WAF1), were accumulated with DNA content characteristic of early S phase of the cell cycle with a concomitant reduction of cells in G1 and G2/M phases. The changes in cyclin and cdk protein expression and their kinase activities were examined in control and drug-treated A253 cells. Tomudex treatment resulted in the decrease in p27(kip1) expression, with an increase in cyclin E and cdk2 protein expression and kinase activities 24 h after a 2-h exposure. Although cyclin A protein expression was markedly increased, cyclin A kinase activity was only slightly increased. Cyclin D1, cyclin B, cdk4, and cdc2 protein expression and kinase activities remain constant. Lack of activation of cyclin A- and B-cdc2 was associated with a reduced proportion of cells in G2/M phases. Increased cyclin E-cdk2 protein expression was accompanied by the inhibition of DNA synthesis, with a decrease in E2F-1 expression. These results propose that cyclin E-cdk2 kinase can negatively regulate DNA replication. The studies with dThyd rescue from cyclin E-cdk2 protein overexpression and growth inhibition by Tomudex indicate that increased cyclin E-cdk2 protein expression is associated with effective inhibition of thymidylate synthase and resultant dNTP pool imbalance. Provision of dThyd more than 24 h after exposure to Tomudex allowed cells to replicate DNA for a single cycle back to G1, but did not prevent the profound growth-inhibitory effect manifested in the following 5 days. Tomudex treatment resulted in a time-dependent induction of the megabase DNA fragments, followed by secondary 50- to 300-kb DNA fragmentation. The 50- to 300-kb DNA fragmentation may be derived from the inhibition of DNA synthesis associated with cyclin E-cdk2 activation. These results suggest that the megabase DNA fragmentation is induced as a consequence of inhibition of thymidylate synthase by Tomudex and kilobase DNA fragmentation may correlate with the reduction of p27(kip1) expression and the increase in cyclin E and cdk2 kinase activities. Activation of cyclin E and cdk2 kinases allows cells to transit from G1 to S phase accompanied by the inhibition of DNA synthesis. The changes in cell cycle regulatory proteins associated with growth inhibition and DNA damage by Tomudex are not p53 dependent.
Yin
M B
MB
Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York, 14263, USA.
Guo
B
B
Panadero
A
A
Frank
C
C
Wrzosek
C
C
Slocum
H K
HK
Rustum
Y M
YM
D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins; D019925:Cyclin A / Q000737:chemistry; D019927:Cyclin E / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D004247:DNA / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D053938:DNA Fragmentation / Q000187:drug effects; D004273:DNA, Neoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006131:Growth Inhibitors / Q000494:pharmacology; D006801:Humans; D046911:Macromolecular Substances; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D011799:Quinazolines / Q000494:pharmacology*; D056465:Retinoblastoma-Binding Protein 1; D013876:Thiophenes / Q000494:pharmacology*; D013936:Thymidine / Q000502:physiology; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000096:biosynthesis; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D019925:Cyclin A; D019927:Cyclin E; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D004791:Enzyme Inhibitors; D006131:Growth Inhibitors; D046911:Macromolecular Substances; D011799:Quinazolines; D056465:Retinoblastoma-Binding Protein 1; D013876:Thiophenes; D050680:Transcription Factor DP1; D014157:Transcription Factors; D004247:DNA; D013940:Thymidylate Synthase; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; C068874:raltitrexed; D013936:Thymidine