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.
Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York, 14263, USA.
M B
MB
Yin
B
B
Guo
A
A
Panadero
C
C
Frank
C
C
Wrzosek
H K
HK
Slocum
Y M
YM
Rustum
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