p53 and WAF1 are induced and Rb protein is hypophosphorylated during cell growth inhibition by the thymidylate synthase inhibitor ZD1694 (Tomudex).
In a previous study, we found that treatment of HCT-8 cells with ZD1694, a specific antifolate-based thymidylate synthase inhibitor, resulted in DNA fragmentation. In this study, we have demonstrated the dose- and time-dependent induction of DNA fragmentation accompanied by elevation of p53 and WAF1 protein expression by ZD1694. WAF1 mRNA showed a time-dependent increase, whereas p53 mRNA was not found to be significantly overexpressed. The initial increase in WAF1 mRNA was detected at 4 hr, but increased WAF1 protein expression was detected 8-24 hr after a 2-hr exposure. The amount of total and hypophosphorylated pRb seems to be rising greatly after ZD1694 exposure. The effects of ZD1694 on the expression of E2F1 and formation of the E2F1-Rb complex were investigated after a 2-hr drug exposure (IC90). The results showed a time-dependent decrease in E2F1 mRNA and protein expression; an increase in the abundance of the E2F-Rb complex could be demonstrated beginning 4 hr after drug exposure by a gel shift assay. Kinetic analysis showed increased availability of hypophosphorylated pRb for inhibition of E2F, which could indirectly result from WAF1-induced inhibition cyclin-dependent kinase activity. Whereas thymidylate synthase inhibition by ZD1694 was rapid in onset and maintained for at least 24 hr after drug treatment, drug-induced cellular growth inhibition was significant 24 hr after drug exposure. The increased abundance of hypophosphorylated pRb and binding to transcription factor E2F-1 is consistent with ZD1694-induced cell growth inhibition in HCT-8 cells. Therefore, the observed effect on downstream events after effective inhibition of thymidylate synthase may offer the critical determinants of response to ZD1694.
Yin
M B
MB
Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Voigt
W
W
Panadero
A
A
Vanhoefer
U
U
Frank
C
C
Pajovic
S
S
Azizkhan
J
J
Rustum
Y M
YM
D000230:Adenocarcinoma / Q000378:metabolism; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis*; D004273:DNA, Neoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D007078:Ileal Neoplasms / Q000378:metabolism; D010766:Phosphorylation; D011799:Quinazolines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D013876:Thiophenes / Q000494:pharmacology*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis*
C493956:CDKN1A protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D004791:Enzyme Inhibitors; D011799:Quinazolines; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D013876:Thiophenes; D050680:Transcription Factor DP1; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D013940:Thymidylate Synthase; C068874:raltitrexed