Isolation of genetic suppressor elements, inducing resistance to topoisomerase II-interactive cytotoxic drugs, from human topoisomerase II cDNA.

Gudkov, Andrei V.; Zelnick, Carolyn R.; Kazarov, Alexander R.; Thimmapaya, Rama; Suttle, D P; Beck, William T.; Roninson, Igor B.

doi:10.1073/pnas.90.8.3231

Cited by 126 publications

(86 citation statements)

References 22 publications

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“…Drug-resistant tumour cells usually incur multiple mutations, however, and the resistance is an accumulative effect of mutations in MDR], topoisomerase IIc and perhaps other unknown targets of mutation. Our degree of enhanced resistance is comparable to the levels observed in transfected HeLa cells, in which small subfragments of human Top20t gene were randomly expressed (Gudkov et al, 1993). In this HeLa cell study, it was not determined whether the cloned fragment made a stable polypeptide product, nor was the subcellular localisation of the products examined.…”

Section: Resultssupporting

confidence: 53%

Ectopic expression of inactive forms of yeast DNA topoisomerase II confers resistance to the anti-tumour drug, etoposide

Vassetzky¹,

Alghisi²,

Roberts³

et al. 1996

Br J Cancer

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Section: Resultssupporting

confidence: 53%

Ectopic expression of inactive forms of yeast DNA topoisomerase II confers resistance to the anti-tumour drug, etoposide

Vassetzky¹,

Alghisi²,

Roberts³

et al. 1996

Br J Cancer

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“…Although previous work has suggested a relationship between Top2A levels and doxorubicin sensitivity (26), the effect has not been studied extensively or validated in vivo. The effects of Top2A knockdown were specific to topoisomerase 2 poisons: shTop2A caused resistance to another, structurally unrelated TOP2A poison, etoposide, but not to the alkylating agent maphosphamide (an active metabolite of cyclophosphamide) nor the topoisomerase 1 poison camptothecin ( Fig.…”

Section: Top2a Shrnas Cause Resistance Specifically To Topoisomerasementioning

confidence: 99%

Topoisomerase levels determine chemotherapy response in vitro and in vivo

Burgess

Doles²,

Zender

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

280

236

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Topoisomerase poisons are chemotherapeutic agents that are used extensively for treating human malignancies. These drugs can be highly effective, yet tumors are frequently refractory to treatment or become resistant upon tumor relapse. Using a pool-based RNAi screening approach and a well characterized mouse model of lymphoma, we explored the genetic basis for heterogeneous responses to topoisomerase poisons in vitro and in vivo. These experiments identified Top2A expression levels as major determinants of response to the topoisomerase 2 poison doxorubicin and showed that suppression of Top2A produces resistance to doxorubicin in vitro and in vivo. Analogously, using a targeted RNAi approach, we demonstrated that suppression of Top1 produces resistance to the topoisomerase 1 poison camptothecin yet hypersensitizes cancer cells to doxorubicin. Importantly, lymphomas relapsing after treatment display spontaneous changes in topoisomerase levels as predicted by in vitro gene knockdown studies. These results highlight the utility of pooled shRNA screens for identifying genetic determinants of chemotherapy response and suggest strategies for improving the effectiveness of topoisomerase poisons in the clinic.A myriad of genetic factors influence the efficacy of cancer chemotherapy, including both somatic changes in the tumor itself as well as genetic polymorphisms present in the patient. These factors include increased expression of detoxification pumps that prevent access of the drug to its target (1), point mutations that disrupt the drug-target interaction (2, 3), and mutations in stress response pathways [e.g., p53 loss (4)]. To tailor treatment successfully to the individual patient, a more complete understanding of the genetic determinants of therapy response is necessary.RNA interference (RNAi) exploits a mechanism of gene regulation whereby double-stranded RNAs are processed by a conserved cellular machinery to suppress the expression of genes containing homologous sequences (5). Importantly, libraries of DNA-based vectors encoding short hairpin RNAs (shRNAs) capable of targeting most genes in the human and mouse genomes have been produced and enable forward genetic screens to be performed in mammalian cells. Indeed, by using human tumor-derived cell lines treated in vitro, RNAi has been used to evaluate potential drug targets (6) or to investigate mechanisms of drug action and drug resistance by screening for new molecules that modulate the response of tumor-derived cell lines to a given chemotherapeutic agent (7-10).Here, we evaluate the suitability of combining mouse models and RNAi to identify genetic modifiers of drug action in tumors in their natural site. Initially, we chose to investigate resistance to doxorubicin in the E -Myc mouse lymphoma system. Doxorubicin (Adriamycin) is an anthracycline DNA-damaging agent that exerts its effects primarily by targeting of the topoisomerase 2 activity and DNA intercalation (11). Along with etoposide and the camptothecin derivatives, doxorubicin is one of several t...

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“…Biologically active cDNA fragments are then isolated from cells expressing the library by a positive functional selection for a specific phenotype. This concept has been used previously to identify functional regions in several genes including topoisomerase II (9), kinesin heavy chain (10,11), and p53 (12,13). Here we adjusted the screen to the conditions and principles of the TKO selection procedure (1).…”

mentioning

confidence: 99%

A functional genetic screen identifies regions at the C-terminal tail and death-domain of death-associated protein kinase that are critical for its proapoptotic activity

Raveh

Berissi

Eisenstein

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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Death-associated protein kinase (DAP-kinase) is a Ca ؉2 ͞calmodulin-regulated serine͞threonine kinase with a multidomain structure that participates in apoptosis induced by a variety of signals. To identify regions in this protein that are critical for its proapoptotic activity, we performed a genetic screen on the basis of functional selection of short DAP-kinase-derived fragments that could protect cells from apoptosis by acting in a dominant-negative manner. We expressed a library of randomly fragmented DAP-kinase cDNA in HeLa cells and treated these cells with IFN-␥ to induce apoptosis. Functional cDNA fragments were recovered from cells that survived the selection, and those in the sense orientation were examined further in a secondary screen for their ability to protect cells from DAP-kinase-dependent tumor necrosis factor-␣-induced apoptosis. We isolated four biologically active peptides that mapped to the ankyrin repeats, the ''linker'' region, the death domain, and the C-terminal tail of DAP-kinase. Molecular modeling of the complete death domain provided a structural basis for the function of the death-domain-derived fragment by suggesting that the protective fragment constitutes a distinct substructure. The last fragment, spanning the C-terminal serine-rich tail, defined a new regulatory region. Ectopic expression of the tail peptide (17 amino acids) inhibited the function of DAP-kinase, whereas removal of this region from the complete protein caused enhancement of the killing activity, indicating that the C-terminal tail normally plays a negative regulatory role. Altogether, this unbiased screen highlighted functionally important regions in the protein and revealed an additional level of regulation of DAP-kinase apoptotic function that does not affect the catalytic activity. D eath-associated protein kinase (DAP-kinase) is a positive mediator of apoptosis. It was isolated by a function-based gene-cloning methodology named technical knockout (TKO) selection, which involved expression of an anti-sense cDNA library in cells, followed by selection of clones that survived in the continuous presence of an apoptotic stimulus (1). In this system, specific inhibition of DAP-kinase protein expression by anti-sense mRNA protected HeLa cells from apoptosis induced by IFN-␥ (2). Furthermore, DAP-kinase was shown to modulate cell death triggered by Fas, tumor necrosis factor ␣ (TNF-␣) (3), and detachment from extracellular matrix (4), indicating its general relevance to apoptosis.DAP-kinase is a Ca ϩ2 ͞calmodulin (CaM)-regulated serine͞ threonine kinase that is localized to the cytoskeleton, where it associates with the actin microfilament system (5). In addition to the kinase domain, which is essential for the death-promoting effects, the protein carries eight ankyrin repeats, a cytoskeleton-binding region, and a death domain. The multidomain structure of DAPkinase and its participation in a wide range of apoptotic systems imply that this protein may interact with various intracellular components to exert its act...

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Isolation of genetic suppressor elements, inducing resistance to topoisomerase II-interactive cytotoxic drugs, from human topoisomerase II cDNA.

Abstract: Many cytotoxic anticancer drugs act at topoisomerase 11 (topo 11) by

Cited by 126 publications

References 22 publications

Ectopic expression of inactive forms of yeast DNA topoisomerase II confers resistance to the anti-tumour drug, etoposide

Ectopic expression of inactive forms of yeast DNA topoisomerase II confers resistance to the anti-tumour drug, etoposide

Topoisomerase levels determine chemotherapy response in vitro and in vivo

A functional genetic screen identifies regions at the C-terminal tail and death-domain of death-associated protein kinase that are critical for its proapoptotic activity

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