tetracycline and raltitrexed

Thymidylate synthase (TS) is responsible for the de novo synthesis of thymidylate, which is required for DNA synthesis and repair and which is an important target for fluoropyrimidines such as 5-fluorouracil (5-FU), and antifolates such as Tomudex (TDX), ZD9331, and multitargeted antifolate (MTA). To study the importance of TS expression in determining resistance to these agents, we have developed an MDA435 breast cancer-derived cell line with tetracycline-regulated expression of TS termed MTS-5. We have demonstrated that inducible expression of TS increased the IC(50) dose of the TS-targeted therapeutic agents 5-FU, TDX, and ZD9331 by 2-, 9- and 24-fold respectively. An IC(50) dose for MTA was unobtainable when TS was overexpressed in these cells, which indicated that MTA toxicity is highly sensitive to increased TS expression levels. The growth inhibitory effects of the chemotherapeutic agents CPT-11, cisplatin, oxaliplatin, and Taxol were unaffected by TS up-regulation. Cell cycle analyses revealed that IC(50) doses of 5-FU, TDX and MTA caused an S-phase arrest in cells that did not overexpress TS, and this arrest was overcome when TS was up-regulated. Furthermore, the S-phase arrest was accompanied by 2- to 4-fold increased expression of the cell cycle regulatory genes cyclin E, cyclin A, and cyclin dependent kinase 2 (cdk2). These results indicate that acute increases in TS expression levels play a key role in determining cellular sensitivity to TS-directed chemotherapeutic drugs by modulating the degree of S-phase arrest caused by these agents. Moreover, CPT-11, cisplatin, oxaliplatin, and Taxol remain highly cytotoxic in cells that overexpress TS.

Topics: Blotting, Northern; Blotting, Western; Camptothecin; CDC2-CDC28 Kinases; Cell Cycle; Cell Division; Cisplatin; Cyclin A; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Fluorouracil; Folic Acid Antagonists; Gene Expression Regulation, Enzymologic; Humans; Irinotecan; Neoplasms; Organoplatinum Compounds; Oxaliplatin; Paclitaxel; Protein Serine-Threonine Kinases; Quinazolines; RNA, Messenger; Tetracycline; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured

The effect of overexpression of p21waf1 on drug sensitivity was studied in an osteosarcoma cell line (SaOs-2) lacking both p53 and functional retinoblastoma protein using a tetracycline (TC)-inducible expression system. p21waf1 expression was barely detectable in SaOS-2 cells incubated in the presence of TC. After TC withdrawal, high levels of p21waf1 were induced in these cells. These p21waf1-induced cells showed increased sensitivity to doxorubicin, tomudex, and methotrexate as compared to uninduced cells; this condition is associated with increased apoptosis. Expression of p21waf1 reduced cyclin A-associated kinase activity and, surprisingly, resulted in inhibition of phosphorylation of E2F-1 and increased E2F-1 binding activity. An S-G2 cell cycle arrest/delay and an increase in expression of E2F-responsive genes (dihydrofolate reductase and thymidylate synthase) was correspondingly observed. Overexpression of p21waf1 in cells lacking functional retinoblastoma protein may mediate sensitivity to anticancer drugs by inhibiting E2F-1 phosphorylation, which may contribute to increased S-G2 cell cycle delay and increased cell susceptibility to apoptosis.

Topics: Anti-Bacterial Agents; Antibiotics, Antineoplastic; Antimetabolites, Antineoplastic; Apoptosis; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; DNA-Binding Proteins; Doxorubicin; E2F Transcription Factors; E2F1 Transcription Factor; Gene Expression Regulation, Neoplastic; Genes, p53; Humans; Methotrexate; Osteosarcoma; Phosphorylation; Quinazolines; Retinoblastoma Protein; Retinoblastoma-Binding Protein 1; Tetracycline; Tetrahydrofolate Dehydrogenase; Thiophenes; Thymidylate Synthase; Transcription Factor DP1; Transcription Factors; Tumor Cells, Cultured