Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites.

Camptothecin (CPT), a plant alkaloid with antitumor activity, is a specific inhibitor of eukaryotic DNA topoisomerase I. We have previously isolated and characterized a CPT-resistant topoisomerase I isolated from a CPT-resistant human leukemia cell line, CPT-K5. cDNA clones of topoisomerase I were isolated from the CPT-resistant and the parental CPT-sensitive cell lines, respectively. Sequencing of the clones identified two mutations in the cDNA isolated from the resistant cells, which cause amino acid changes from aspartic acid to glycine at residues 533 and 583 of the parental topoisomerase I. When the CPT-K5 topoisomerase I was expressed in E. coli as a fusion protein with Staphylococcal Protein A fragment, the activity was resistant to CPT at a dose level up to 125 microM, whereas the parental fusion protein was sensitive to CPT as low as 1 microM. The resistance index (greater than 125) of the CPT-K5 fusion topoisomerase I is similar to that of the native CPT-K5 topoisomerase I. These results indicate that either or both of the two amino acid changes identified in the mutant enzyme is responsible for the resistance to CPT.     

Differential expression of DNA topoisomerase I gene between CPT-11 acquired- and native-resistant human pancreatic tumor cell lines: detected by RNA/PCR-based quantitation assay.

RNA/PCR quantitation method was developed to determine DNA Topoisomerase I(Topo I)-specific mRNA in order to study its gene expression in CPT-11 sensitive, acquired- or native-resistant human pancreatic tumor cell lines. The results were supported by Northern blotting and Western blotting analyses. Acquired-resistant cells have shown decreased levels of Topo I mRNA, compared with their parental cells. On the contrary, in the wild type cells no correlation was shown between sensitivity and gene expression. On the other, specific Topo I activity of the native resistant cell lines was fairly lower than that of sensitive cell lines, suggesting that immunoreactive Topo I protein contains low levels of active form enzyme which could be targets of CPT-11 in these native-resistant ones. Finally, the different mechanisms might be operative between acquired- and native-resistant tumor cells.     

Synthesis and biological evaluation of 7-alkenyl homocamptothecins as potent topoisomerase I inhibitors

Homocamptothecin (hCPT) is a camptothecin (CPT) derivative with a seven-membered β-hydroxylactone E ring, which shows higher lactone stability and improves topoisomerase I (Topo I) inhibition activity. In an attempt to improve the antitumor activity of homocamptothecins, a series of 7-alkenyl-homocamptothecin derivatives was designed and synthesized based on a semisynthetic route starting from CPT. Most of the synthesized compounds exhibit higher cytotoxic activities on the A-549 tumor cell line than topotecan (TPT). Some compounds such as 2a and 2o show a broad in vitro antitumor spectrum and exhibit superior Topo I-inhibition activity.     

Fanconi anemia cells have a normal gene structure for topoisomerase I

Cells from Fanconi anemia (FA) patients have defective DNA repair and are hypersensitive to DNA crosslinking agents such as mitomycin C (MMC). We examined the possibility that topoisomerase I is involved in the DNA crosslink repair system and is deficient in FA group A cells. FA cells and control cells were exposed to MMC with or without camptothecin (CPT), a topoisomerase I inhibitor. The cells did not show any increased sensitivity to killing by MMC with CPT, suggesting that the topoisomerase I is not involved in MMC-damaged DNA repair. However, FA cells showed increased sensitivity to CPT in comparison to control cells, raising the possibility of altered topoisomerase I in FA cells. Therefore, a mutation analysis was performed on topoisomerase I cDNA from FA cells by using chemical cleavage mismatch scanning and nucleotide sequencing. No mutation was detected from GM1309, a group A FA cell line. A base transition (C to T) at position 241, causing an amino acid change (His to Tyr), was found in GM2061, a FA cell line of unknown complementation group. However, allele-specific oligonucleotide hybridization analysis showed that this is a gene polymorphism. We conclude that FA cells have normal gene structure for topoisomerase I.     

Synthesis and Biological Evaluation of 7‐Alkenyl Homocamptothecins as Potent Topoisomerase I Inhibitors

Homocamptothecin (hCPT) is a camptothecin (CPT) derivative with a seven‐membered β‐hydroxylactone E ring, which shows higher lactone stability and improves topoisomerase I (Topo I) inhibition activity. In an attempt to improve the antitumor activity of homocamptothecins, a series of 7‐alkenyl‐homocamptothecin derivatives was designed and synthesized based on a semisynthetic route starting from CPT. Most of the synthesized compounds exhibit higher cytotoxic activities on the A‐549 tumor cell line than topotecan (TPT). Some compounds such as 2a and 2o show a broad in vitro antitumor spectrum and exhibit superior Topo I‐inhibition activity.     

Synthesis of new camptothecin analogs with improved antitumor activities

Novel hexacyclic camptothecin analogs containing cyclic amidine, urea, or thiourea moiety were designed and synthesized based on the proposed 3D-structure of the topoisomerase I (Topo I)/DNA/camptothecin ternary complex. The analogs were prepared from 9-nitrocamptothecin via 7,9-diaminocamptothecin derivatives as a key intermediate. Among them, 7c exhibited in vivo antitumor activities superior to CPT-11 in human cancer xenograft models in mice at their maximum tolerated doses though its in vitro antiproliferative activity was comparable to SN-38 against corresponding cell lines.     

The role of topoisomerase I inhibitor in cisplatin-resistant ovarian cancer.

We discussed the role of DNA topoisomerase I (topo I) inhibitor, which is now widely used in clinical practice, in cisplatin-resistant ovarian cancer. Our study showed the synergistic actions between cisplatin and 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of 7-ethyl-10-[4-(1-pyperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11), in two cisplatin-resistant cancer cell lines, HeLa/CDDP and KFr cells, but not in each parent cell line, HeLa and KF cells. Furthermore, HeLa/CDDP cells had a collateral sensitivity to SN-38. The levels of topo I protein in the cisplatin-resistant cells did not differ from those of their parent cell lines and were unaffected by exposure to cisplatin. In contrast, topo I enzymatic activity was 2-4 fold higher in the cisplatin-resistant cell lines compared with their respective parent cell lines. A significant correlation between the sensitivity for SN-38 and topo I activity human clear cell carcinoma cell lines, which are known as intrinsically ciasplatin-resistant cancer, was observed. Next, we examined the relationship between topo I activity and sensitivity to second-line chemotherapy consisting of cisplatin and CPT-11. A total of 30 patients with ovarian cancer who had initially undergone chemotherapy consisting of cisplatin, doxorubicin, and cyclophosphamide (CAP) and exhibited measurable lesions were entered in the study. Tumor samples were obtained in the period between the initial and the second-line chemotherapy. Of those 30 patients, 18 responded to second-line chemotherapy and 12 did not. Topo I activity in tumor samples of responder was significantly greater than that of in nonresponders. In 8 cases whose samples could be obtained before and after CAP, topo I activity significantly increased after CAP therapy. Consequently, the combination therapy with cisplatin and CPT-11 may be effective for patients with cisplatin-resistant ovarian cancer. In addition, topo I enzymatic activity may be a predictor of the sensitivity for topo I inhibitor.     

Spinocerebellar ataxia with axonal neuropathy: consequence of a Tdp1 recessive neomorphic mutation?

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) cleaves the phosphodiester bond between a covalently stalled topoisomerase I (Topo I) and the 3' end of DNA. Stalling of Topo I at DNA strand breaks is induced by endogenous DNA damage and the Topo I-specific anticancer drug camptothecin (CPT). The H493R mutation of Tdp1 causes the neurodegenerative disorder spinocerebellar ataxia with axonal neuropathy (SCAN1). Contrary to the hypothesis that SCAN1 arises from catalytically inactive Tdp1, Tdp1-/- mice are indistinguishable from wild-type mice, physically, histologically, behaviorally, and electrophysiologically. However, compared to wild-type mice, Tdp1-/- mice are hypersensitive to CPT and bleomycin but not to etoposide. Consistent with earlier in vitro studies, we show that the H493R Tdp1 mutant protein retains residual activity and becomes covalently trapped on the DNA after CPT treatment of SCAN1 cells. This result provides a direct demonstration that Tdp1 repairs Topo I covalent lesions in vivo and suggests that SCAN1 arises from the recessive neomorphic mutation H493R. This is a novel mechanism for disease since neomorphic mutations are generally dominant.     

Camptothecin analog (CPT-11)-sensitive human pancreatic tumor cell line QGP-1N shows resistance to SN-38, an active metabolite of CPT-11.

In the course of our study to determine the cross-sensitivity between CPT-11 and its active metabolite, SN-38, we found a SN-38-resistant human pancreatic tumor cell line, QGP-1N, which shows sensitivity to CPT-11. The IC50 of SN-38 was 152 times greater for QGP-1N than for SUIT-2, also a human pancreatic tumor cell line, whose IC50 of CPT-11 was similar to that for QGP-1N. The uptakes of CPT-11 and SN-38 and the intracellular conversion of CPT-11 to SN-38 could not explain the difference in sensitivity. DNA synthesis of QGP-1N cells was inhibited by CPT-11 which did not affect that of SUIT-2, while SN-38 inhibited the DNA synthesis of SUIT-2 at lower concentrations than that of QGP-1N. The inhibition test of topoisomerase I catalytic activity by CPT-11 or SN-38 revealed no difference in the biochemical properties of the topoisomerase I enzymes to the compounds between these two cell lines. These results indicate that CPT-11 should have its own inhibitory effect on DNA synthesis through a yet unknown mechanism in QGP-1N cells, although SN-38 plays an essential role in the antitumor activity of CPT-11 in SUIT-2 cells. In some cases, the antitumor effect of CPT-11 might be consequent not only on SN-38 but also on CPT-11 itself.     

The C-terminal domain but not the tyrosine 723 of human DNA topoisomerase I active site contributes to kinase activity.

Human DNA topoisomerase I not only has DNA relaxing activity, but also splicing factors phosphorylating activity. Topo I shows strong preference for ATP as the phosphate donor. We used photoaffinity labeling with the ATP analogue [alpha-32P] 8-azidoadenosine-5'-triphosphate combined with limited proteolysis to characterize Topo I domains involved in ATP binding. The majority of incorporated analogue was associated with two fragments derived from N-terminal and C-terminal regions of Topo I, respectively. However, mutational analysis showed that deletion of the first 138 N-terminal residues, known to be dispensable for topoisomerase activity, did not change the binding of ATP or the kinase activity. In contrast, deletion of 162 residues from the C-terminal domain was deleterious for ATP binding, kinase and topoisomerase activities. Furthermore, a C-terminal tyrosine 723 mutant lacking topoisomerase activity is still able to bind ATP and to phosphorylate SF2/ASF, suggesting that the two functions of Topo I can be separated. These findings argue in favor of the fact that Topo I is a complex enzyme with a number of potential intra-cellular functions.     

Suppression of pea nuclear topoisomerase I enzyme activity by pea PCNA

Proliferating cell nuclear antigen (PCNA), a highly conserved DNA polymerase accessory protein of eukary- otic kingdom, has not been studied thoroughly in bio- chemical terms in plants. We describe the isolation of the cDNA encoding PCNA from the pea cDNA library using the PCR approach. The cDNA was used for expression of pea PCNA in bacteria as a fusion protein (GST.PCNA) with the GST tag at the amino terminal end. The GST.PCNA stimulated the partially purified pea DNA polymerases approximately 30-fold. The stimulation was due to the oligomeric form of GST.PCNA. The pea PCNA interacted with the recombinant type I pea topoiso- merase as well as the native pea nuclear topoisomerase I and repressed the DNA relaxation activities. However, the DNA binding activity of Topo I remained undisturbed in the presence of high amounts of PCNA, thereby signify- ing that the catalysis of Topo I was probably affected by PCNA.     

Evolutionary trace analysis of eukaryotic DNA topoisomerase I superfamily: Identification of novel antitumor drug binding site

During protein evolutionary processes, protein fam-ily members undergo extensive random mutations and a long period of natural selections, and thus induce the functional evolution and the emergence of subfamily. The evolutionary variation events were recorded in the sequences of protein family members. Therefore, identification of functionally important residues can be achieved by studying residue conservation in protein sequence families. Generally, the residues conserved across the family of…     

Characterization of DNA Topoisomerase-1 in Spodoptera exigua for Toxicity Evaluation of Camptothecin and Hydoxy-Camptothecin

Camptothecin (CPT), a plant alkaloid originally isolated from the native Chinese tree, Camptotheca acuminate, exerts the toxic effect by targeting eukaryotic DNA topoisomerase 1 (DNA Topo1). Besides as potent anti-cancer agents, CPT and its derivatives are now being explored as potential pesticides for insect control. In this study, we assessed their toxicity to an insect homolog, the Topo1 protein from beet armyworms (Spodoptera exigua Hübner), a worldwide pest of many important crops. The S. exigua Topo1 gene contains an ORF of 2790 base pairs that is predicted to encode a polypeptide of 930 amino acids. The deduced polypeptide exhibits polymorphism at residue sites V420, L530, A653 and T729 (numbered according to human Topo1) among insect species, which are predicted to confer sensitivity to CPT. The DNA relaxation activity of this protein was subsequently examined using a truncated form that contained the residues 337–930 and was expressed in bacteria BL21 cells. The purified protein retained the ability to relax double-stranded DNA and was susceptible to CPT and its derivative hydroxy-camptothecin (HCPT) in a dose-dependent manner. The same inhibitory effect was also found on the native Topo1 extracted from IOZCAS-Spex-II cells, a cell line established from beet armyworms. Additionally, CPT and HCPT treatment reduced the steady accumulation of Topo1 protein despite the increased mRNA expression in response to the treatment. Our studies provide information of the S. exigua Topo1 gene and its amino acid polymorphism in insects and uncover some clues about potential mechanisms of CPT toxicity against insect pests. These results also are useful for development of more effective Topo1-targeted CPT insecticides in the future.     

Evolutionary trace analysis of eukaryotic DNA topoisomerase I superfamily: Identification of novel antitumor drug binding site

The studies of novel inhibitors of DNA topoisomerase I (Topo I) have already become very promising in cancer chemotherapy. Identifying the new drug-binding residues is playing an important role in the design and optimization of Topo I inhibitors. The designed compounds may have novel scaffolds, thus will be helpful to overcome the toxicities of current camptothecin (CPT) drugs and may provide a solution to cross resistance with these drugs. Multiple sequence alignments were performed on eukaryotic DNA topoisomerase I superfamily and thus the evolutionary tree was constructed. The Evolutionary Trace method was applied to identify functionally important residues of human Topo I. It has been demonstrated that class-specific hydrophobic residues Ala351, Met428, Pro431 are located around the 7,9-position of CPT, indicating suitable substitution of hydrophobic group on CPT will increase antitumor activity. The conservative residue Lys436 in the superfamily is of particular interest and new CPT derivatives designed based on this residue may greatly increase water solubility of such drugs. It has also been demonstrated that the residues Asn352 and Arg364 were conservative in the superfamily, whose mutation will render CPT resistance. As our molecular docking studies demonstrated they did not make any direct interaction with CPT, they are important drug-binding site residues for future design of novel non-camptothecin lead compounds. This work provided a strong basis for the design and synthesis of novel highly potent CPT derivatives and virtual screening for novel lead compounds.     

Altered catalytic activity of and DNA cleavage by DNA topoisomerase II from human leukemic cells selected for resistance to VM-26

The simultaneous development of resistance to the cytotoxic effects of several classes of natural product anticancer drugs, after exposure to only one of these agents, is referred to as multiple drug resistance (MDR). At least two distinct mechanisms for MDR have been postulated: that associated with P-glycoprotein and that thought to be due to an alteration in DNA topoisomerase II activity (at-MDR). We describe studies with two sublines of human leukemic CCRF-CEM cells approximately 50-fold resistant (CEM/VM-1) and approximately 140-fold resistant (CEM/VM-1-5) to VM-26, a drug known to interfere with DNA topoisomerase II activity. Each of these lines is cross-resistant to other drugs known to affect topoisomerase II but not cross-resistant to vinblastine, an inhibitor of mitotic spindle formation. We found little difference in the amount of immunoreactive DNA topoisomerase II in 1.0 M NaCl nuclear extracts of the two resistant and parental cell lines. However, topoisomerase II in nuclear extracts of the resistant sublines is altered in both catalytic activity (unknotting) of and DNA cleavage by this enzyme. Also, the rate at which catenation occurs is 20-30-fold slower with the CEM/VM-1-5 preparations. The effect of VM-26 on both strand passing and DNA cleavage is inversely related to the degree of primary resistance of each cell line. Our data support the hypothesis that at-MDR is due to an alteration in topoisomerase II or in a factor modulating its activity.     

Enhanced killing of cancer cells by poly(ADP-ribose) polymerase inhibitors and topoisomerase I inhibitors reflects poisoning of both enzymes

Poly(ADP-ribose) polymerase-1 (PARP1) plays critical roles in the regulation of DNA repair. Accordingly, small molecule inhibitors of PARP are being developed as agents that could modulate the activity of genotoxic chemotherapy, such as topoisomerase I poisons. In this study we evaluated the ability of the PARP inhibitor veliparib to enhance the cytotoxicity of the topoisomerase I poisons topotecan and camptothecin (CPT). Veliparib increased the cell cycle and cytotoxic effects of topotecan in multiple cell line models. Importantly, this sensitization occurred at veliparib concentrations far below those required to substantially inhibit poly(ADP-ribose) polymer synthesis and at least an order of magnitude lower than those involved in selective killing of homologous recombination-deficient cells. Further studies demonstrated that veliparib enhanced the effects of CPT in wild-type mouse embryonic fibroblasts (MEFs) but not Parp1(-/-) MEFs, confirming that PARP1 is the critical target for this sensitization. Importantly, parental and Parp1(-/-) MEFs had indistinguishable CPT sensitivities, ruling out models in which PARP1 catalytic activity plays a role in protecting cells from topoisomerase I poisons. To the contrary, cells were sensitized to CPT in a veliparib-independent manner upon transfection with PARP1 E988K, which lacks catalytic activity, or the isolated PARP1 DNA binding domain. These results are consistent with a model in which small molecule inhibitors convert PARP1 into a protein that potentiates the effects of topoisomerase I poisons by binding to damaged DNA and preventing its normal repair.     

Indolocarbazole poisons of human topoisomerase I: regioisomeric analogues of ED-110

All four "symmetrical" regioisomers of ED-110, an indolocarbazole derivative having potent activity against human topoisomerase I (Topo I) were synthesized. The isomer containing hydroxyl groups in the 3- and 9-positions was approximately ten-fold more active against Topo I, and 5- to 35-fold more active against human solid tumor cell lines in vitro, relative to ED-110.