Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 2 de 2
Filter
Add more filters










Database
Language
Publication year range
1.
Cancer Res ; 61(5): 1964-9, 2001 Mar 01.
Article in English | MEDLINE | ID: mdl-11280753

ABSTRACT

In this study, we characterized the structure and function of topoisomerase I (top1) protein in the camptothecin (CPT)-resistant prostate cancer cell lines, DU-145/RC0.1 and DU-145/RC1 (RC0.1 and RC1, respectively). Both of the cell lines were previously selected by continuous exposure to 9-nitro-CPT. The RC0.1 and RC1 cells have high cross-resistance to CPT derivatives including SN-38 and topotecan, but are not cross-resistant to the non-top1 inhibitors etoposide, doxorubicin, and vincristine. Although the top1 protein levels were not decreased in the resistant cells compared with the parental cells, CPT-induced DNA cleavage was markedly reduced in the RC0.1 and RC1 nuclear extracts. The resistant-cell-line nuclear extracts also demonstrated top1 catalytic activity and resistance to CPT, in in vitro assays. Reverse transcription-PCR products from the resistant cell lines were sequenced, and revealed a point mutation resulting in a R364H mutation in the top1 of both RC0.1 and RC1. No wild-type top1 RNA or genomic DNA was detected in the resistant cell lines. Using a purified recombinant R364H top1, we found that the R364H mutant top1 was CPT resistant and fully active. In the published top1 crystal structure, the R364H mutation is close to the catalytic tyrosine and other well-known mutations leading to CPT resistance.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Camptothecin/pharmacology , DNA Topoisomerases, Type I/genetics , Enzyme Inhibitors/pharmacology , Mutation , Prostatic Neoplasms/genetics , Amino Acid Sequence , Base Sequence , Catalysis , DNA Topoisomerases, Type I/metabolism , DNA, Viral/metabolism , Drug Resistance, Neoplasm/genetics , Humans , Male , Molecular Sequence Data , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/enzymology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Simian virus 40/genetics , Topoisomerase I Inhibitors , Tumor Cells, Cultured
2.
Proc Natl Acad Sci U S A ; 97(4): 1885-90, 2000 Feb 15.
Article in English | MEDLINE | ID: mdl-10677551

ABSTRACT

1-beta-d-Arabinofuranosylcytosine (Ara-C) is a nucleoside analog commonly used in the treatment of leukemias. Ara-C inhibits DNA polymerases and can be incorporated into DNA. Its mechanism of cytotoxicity is not fully understood. Using oligonucleotides and purified human topoisomerase I (top1), we found a 4- to 6-fold enhancement of top1 cleavage complexes when ara-C was incorporated at the +1 position (immediately 3') relative to a unique top1 cleavage site. This enhancement was primarily due to a reversible inhibition of top1-mediated DNA religation. Because ara-C incorporation is known to alter base stacking and sugar puckering at the misincorporation site and at the neighboring base pairs, the observed inhibition of religation at the ara-C site suggests the importance of the alignment of the 5'-hydroxyl end for religation with the phosphate group of the top1 phosphotyrosine bond. This study also demonstrates that ara-C treatment and DNA incorporation trap top1 cleavage complexes in human leukemia cells. Finally, we report that camptothecin-resistant mouse P388/CPT45 cells with no detectable top1 are crossresistant to ara-C, which suggests that top1 poisoning is a potential mechanism for ara-C cytotoxicity.


Subject(s)
Cytarabine/pharmacology , DNA Topoisomerases, Type I/metabolism , DNA/chemistry , Enzyme Induction/drug effects , Animals , Antineoplastic Agents/pharmacology , Camptothecin/pharmacology , Drug Resistance , Humans , Mice , Molecular Structure , Tumor Cells, Cultured
SELECTION OF CITATIONS
SEARCH DETAIL
...