What is topoisomerase

DNA topoisomerases are essential enzymes that control the topological state of DNA replication during mitosis ¹. DNA topoisomerases catalyze the interconversion of different topological forms of DNA, such as the knotting and unknotting of DNA and catenation and decatenation of DNA rings ². All organisms contain at least two classes of DNA topoisomerases, namely type 1 and type 2 DNA topoisomerases in which the enzymes are classified based on their mechanisms and physical properties. Type 1 topoisomerases are able to unknot or decatenate single strand of the DNA duplex and catenanes, but are unable to carry out these reactions on intact double-stranded DNA ³, ⁴, ⁵, ⁶, ⁵. Type 2 topoisomerases are able to catalize the knotting/unknotting of intact double-stranded DNA and the catenation/decatenation of intact double-stranded DNA ³, ⁴, ⁵, ⁶, ⁵. Both type 1 and type 2 topoisomerases can relax supercoiled DNA ³, ⁴, ⁵, ⁶, ⁵. Because type 1 topoisomerases cleave only a single strand DNA, they can modulate DNA supercoiling, but cannot remove knots or tangles from intact duplex DNA ⁷, ⁸, ⁹. In contrast, because type 2 topoisomerases cleave both strands of the double helix DNA, they can regulate DNA under- and overwinding and can also remove tangles and knots from the genome ⁷, ⁸, ⁹, ¹⁰, ¹¹.

The DNA topoisomerase subtypes (A, B or C) are differentiated by amino acid sequences or structures ¹². For example, enzymes that cleave only one strand of DNA are defined as type 1 with a further classification of type 1-A subtypes for proteins linked to a 5‟-phosphate and type 1-B subtypes for proteins attached to a 3‟-phosphate during the relaxation of the cleavable DNA. The enzymes are further divided into subfamilies based on the structural changes induced by gene duplication, such as DNA topoisomerase IIα and IIβ ¹³.

DNA topoisomerase enzymes are classified based on their mechanisms and physical properties. During mitosis, superhelical DNA must be unwound or relaxed by DNA topoisomerases prior to a decoding step by DNA processing enzymes, such as DNA polymerase and RNA polymerase. By blocking the reaction of resealing the breaks in the DNA ultimately can result in cellular death. Compounds that inhibit the catalytic function of DNA topoisomerase enzymes represent highly relevant targets for the treatment of various types of cancer and DNA topoisomerases are under continuous investigation as targets for novel classes of inhibitory agents. While nearly all clinically marketed topoisomerase inhibitors used to treat cancer fall within the topoisomerase poison mechanistic category, several very promising catalytic inhibitors have been disclosed recently and offer promise as potentially novel anti-cancer agents with significantly reduced toxicity profiles. Of note are the novel chemical classes with activity against type I topoisomerases, as there is currently only one marketed class of inhibitors in clinical use.

DNA topoisomerases are essential enzymes that control the topological state of DNA during cellular processes in both prokaryotic and eukaryotic cells. The cellular processes include DNA replication, transcription, recombination and chromatin segregation, to control the synthesis of proteins and to facilitate DNA replication during mitosis ¹⁴.

Studies in eukaryotes have shown that type 1 DNA topoisomerase is associated with actively transcribed genes, whereas type 2 DNA topoisomerase is required for DNA replication and the successful traverse of mitosis ¹⁵. Type 1 and type 2α DNA topoisomerases are well known targets for chemotherapy of advanced or recurrent human cancers.

DNA topoisomerase types

The DNA topoisomerases can be categorized into two general subfamilies, type I and type II topoisomerases. Further sub-divisions of the topoisomerases, types IA, IB, and IIA are categorized based upon the polarity of the DNA-protein bond (i.e., tyrosine attachment to the 5′- or 3′-phosphate), the mechanism (strand passage or rotation), and the number of overhanging DNA bases in the staggered double strand cleavage of the type II topoisomerases ¹⁶. Structurally, eukaryotic type I topoisomerases are monomeric protein, single-gene products, while eukaryotic type II topoisomerases are homodimers, composed of the products of a distinct gene. Functionally, type I topoisomerases are not dependent on ATP hydrolysis to power the energy required for their reaction, instead they derive the energy required from the intrinsic strain energy of the supercoiled DNA itself ¹⁷. Type II topoisomerases are typically ATP-dependent and possess an ATP-binding domain separate from the DNA-binding domain. Additionally, the DNA topoisomerases can also be distinguished by their dependence, or lack of dependence, on Mg2+ as a requirement for catalytic activity. The type IA and all type II topoisomerases appear to require Mg2+, while the type IB topoisomerases are catalytically active in the absence of Mg2+ ¹⁸. The targets of the currently marketed cancer chemotherapeutic agents are topoisomerase I, IIα, and IIβ ¹⁶.

Topoisomerase 1

DNA topoisomerase 1 or type 1 DNA topoisomerases are monomeric proteins and ATP-independent enzymes that induce a single strand DNA break via a phosphodiester bond between the tyrosine group of the enzyme and the phosphate group of the DNA (Figure 1). DNA topoisomerase III is an example of a type I-A enzyme that is more active as a decatenating enzyme than as a DNA-relaxing enzyme ¹⁹.

Figure 1. DNA topoisomerase type 1

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Topoisomerase 2

Type 2 DNA topoisomerases are tetrameric proteins formed by two different subunits, GyrA2GyrB2 for gyrase and ParC2ParE2 for DNA topoisomerase 4 ²⁰. Requiring ATP, these enzymes act by making a transient break on the double stranded DNA, passing through an intact duplex DNA via the broken strand followed by a resealing of the transient break ²¹.

Figure 2. Human DNA topoisomerases type 1 and type 2 structure

Footnote: Structures of human topoisomerases. Shown are the structures of the full length human topoisomerase 1 (left, PDB ID 1k4t) and topoisomerase 2α (right, PDB ID 5qwk) enzymes with bound DNA, representative of the overall structure and domains of the two sub-family types (DNA ribbon is colored yellow; chain A of topoisomerase 1 is blue; chain A of topoisomerase IIα is green; chain B of topoisomerase 2α is red).

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Topoisomerase function

The various topoisomerases enzymes deal with the topological issues related to DNA transcription and replication and are capable of relaxing positive or negatively supercoiled DNA, introducing negative or positive supercoils into DNA, and catenating or decatenating (disentangling) circular and linear DNA ²².

Table 1. Summary of eukaryotic DNA topoisomerase enzyme function and mechanism

Type	Subtype	Protein	Gene	Function	Mechanism	Multimericity	Metal dependence	Cleavage polarity
1	A	Topoisomerase 3α	top3A	(—) Supercoil relaxation	Strand passage	Monomer	Yes (Mg2+)	5′
		Topoisomerase 3β	top3B	Unknown
	B	Topoisomerase 1	top1	(—) and (+) supercoil relaxation	Strand rotation	Monomer	No	3′
2	A	Topoisomerase 2α	top2A	Decatenation during replication	Strand passage (ATPase)	Homodimer	Yes (Mg2+)	5′
		Topoisomerase 2β	top2B	Various, neuronal transcription

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The type IA topoisomerases represented by eukaryotic topoisomerase IIIα and IIIβ consist of four domains that coordinate DNA binding, cleavage, and strand passage ²³. Domain I contains the so-called TOPRIM fold, a Rossman-fold like structure that can bind magnesium ions ²³. Domain II consists primarily of β-strands that forms the central core and links domain III, which contains the catalytic tyrosine residue, to domain IV. The type IB topoisomerases, represented by eukaryotic topoisomerase I, is also composed of four domains including an N-terminal domain, a linker domain, a core domain, and a C-terminal domain ²⁴. The core domain is responsible for DNA binding and appears to be highly conserved. The catalytic tyrosine is in the C-terminal domain and operates as part of a catalytic pentad with four residues located in the core domain. Type IIA topoisomerases, represented by eukaryotic topoisomerase IIα and IIβ, consist of a three-domain structure spanning the A and B subunits that form the homodimer (or heterotetramer in prokaryotes). The N-terminal domain contains the ATB-binding region (ATPase domain), a core domain that contains a TOPRIM fold and DNA-binding region, and a C-terminal domain with unclear function ²⁵.

The sub-types of topoisomerases are mechanistically distinct with respect to how they act on their DNA substrates. Type IA topoisomerases operate using a “strand-passage” mechanism, whereby a single strand of DNA is passed through a break in a second single DNA stand. In this mechanism, the first DNA single strand binds to domain I and III of the topoisomerase and is cleaved by the catalytic tyrosine residue located on domain III, creating a 5′-phosphodiester bond between the enzyme and the DNA. Domain II is then believed to act as a hinge, separating the cleaved DNA strand and permitting a second strand to pass through, after which domains I and III come back together and the cleaved DNA strand is re-joined. The type IB topoisomerases operate using a “hindered rotation mechanism”, whereby the enzyme binds to DNA and cleaves a single strand via a 3′-phosphodiester bond using an active site tyrosine residue. The 5′-end then rotates about the second DNA strand, relaxing the DNA. As discussed above, both type IA and type IB topoisomerases use the DNA torsional strain (torque) to drive the uncoiling process rather than ATP hydrolysis. Type IA topoisomerases relax negatively supercoiled DNA and perform DNA decatenation, while type IB topoisomerases can relax both negatively and positively supercoiled DNA. Like the type IA topoisomerases, the type IIA topoisomerases exert their effect through a strand passage mechanism, in this case using what is known as a “two-gate” mechanism24. Double-stranded DNA, called the “G-segment”, binds to the topoisomerase in a central DNA-binding region called the “DNA-gate”. A second double-stranded DNA, the “T-segment”, binds to the N-terminal ATPase domain facilitated by ATP binding and domain dimerization. ATP hydrolysis and the release of inorganic phosphate causes a double-stranded break with 4-base overhang in the G-segment DNA facilitated by the formation of 5′-phosphodiester bonds between two tyrosine residues and individual strands of the G-segment DNA. The DNA-binding gate separates, and the T-segment is passed through the G-segment into the C-terminal region, or “C-gate”. The C-gate opens and releases the T-segment DNA, and ADP product is released, resetting the enzyme for another catalytic cycle.

Topoisomerase in DNA replication

In general, DNA is wound around a basic protein called histone in a tightly coiled or supercoiled formation ¹⁴. Sequences of DNA must be unwound or relaxed prior to being read by DNA processing enzymes, such as DNA polymerase and RNA polymerase. Type II DNA topoisomerases or DNA topoisomerase II temporarily break the DNA strand, allowing both ends to rotate within the enzyme freely. This mechanism allows the DNA to unwind. The enzyme then reconnects the two ends, leaving a part of the relaxed DNA ready for processing. DNA topoisomerase II inhibitors, such as etoposide, stabilize the enzyme with the DNA strand cut in the enzyme-DNA complex, leaving a permanent break in the double strand of the DNA. This DNA strand break is capped by the remnants of the enzyme and is difficult to repair.

The expressions of DNA topoisomerases in cancer tissues and cells

DNA topoisomerases are over-expressed at different levels in both normal and cancerous tissues. The expression of DNA topoisomerase I in colorectal cancer, as detected by immunohistochemical methods, was a biological marker for the chemosensitivity of tumour against DNA topoisomerase I inhibitors ²⁶. Using the investigation with reverse transcription PCR, differential expression studies in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) demonstrated that the DNA topoisomerase IIα gene in tumors was expressed at significantly higher level than in normal lung tissues, and the relative expression in small cell lung cancer was significantly higher than in non-small cell lung cancer ²⁷. Meanwhile, immunostaining for topoisomerase IIIβ was found in 33.9% of breast carcinoma and the immunopositivity was correlated with distant metastasis and death ²⁸. Decreased expression of topoisomerase IIIβ was correlated with low expression of Ki67, positivity to BRCA1, but negativity for HER2 and p53 expressions in the study.

The highly invasive human cancer cell lines HT-29 and MDA-MB-231 expressed higher levels of DNA topoisomerases than the levels measured in HeLa and MCF-7. Both DNA topoisomerases I and II were not detected in HeLa and MCF-7 by conventional PCR. These results indicated that both HT-29 and MDA-MB-231 may be sensitive to DNA topoisomerase inhibitors whereas HeLa and MCF-7 may not be sensitive to these compounds. Expression levels of DNA topoisomerase II were observed to be higher than DNA topoisomerase I in both HT-29 and MDA-MB-231, indicating that DNA topoisomerase II plays a more important role than DNA topoisomerase I in these highly invasive human cancer cells. In addition, these cells might be more sensitive to DNA topoisomerase II inhibitors than DNA topoisomerase inhibitor I. Decreased expression of DNA topoisomerase I and II had been demonstrated to indicate a resistance against certain anticancer agents ²⁹, suggesting that these enzymes are promising targets for anticancer agents.

Topoisomerase inhibitors

The first known class of topoisomerase inhibitors used for the treatment of cancer was the anthracycline agents. Table 2 summarizes the currently marketed topoisomerase active agents used for cancer chemotherapy. The anthracyclines were first extracted from bacterial Streptomyces species and discovered to possess antibiotic and antitumor activity ³⁰. The clinically marketed anthracycline derivatives include doxorubicin, epirubicin, valrubicin, daunorubicin, and idarubicin. Their structures are shown in Figure 3. Doxorubicin has several current indications including treatment of breast cancer, various types of leukemia, lymphoma, sarcomas, carcinomas, and other tumors. Other anthracyclines which have indications for treatment of leukemia include daunorubicin and idarubicin. Epirubicin is indicated in breast cancer following resection, while valrubicin is indicated in urinary bladder carcinoma. The anthracycline agents affect their cytotoxic activity by acting as topoisomerase “poisons”, as discussed in the previous section. They primarily affect type IIa topoisomerases, topoisomerase IIα and topoisomerase IIβ indiscriminately, by intercalation into the bound and cleaved DNA, stabilizing the DNA and topoisomerase complex and preventing DNA re-ligation by the topoisomerase. Interestingly, this appears to be one of the mechanisms that the anthracyclines induce cell death ³¹. The production of free radical species in an iron-dependent manner appears to be another mechanism behind the anthracyclines׳ cytotoxic effects in cancer cells. Unfortunately, this mechanism also appear to be responsible for additional toxicities associated with this class of agents ³². Anthracyclines are well known for their cardiotoxic properties, causing both acute and chronic cardiac complications in a dose-dependent manner. Menna and colleagues ³³ discuss the mechanisms behind this toxicity, including anthracyclines׳ production of superoxide anions and hydrogen peroxide in cardiac muscle cells, which contribute to oxidative stress and eventually apoptosis. Anthracyclines also disrupt calcium and iron levels in cardiac cells, which leads to even more free-radical generation ³³. Some recent studies have implied another potential mechanism of the cardiotoxicity ³⁴. These studies suggest that inhibition of topoisomerase IIβ, which is overexpressed in the heart, by the anthracycline agents may result cardiotoxicity from apoptosis and ROS. Dexrazoxane, discussed above, is an FDA-approved agent indicated to reduce adverse cardiac affects in women who have received a total dose of 300 mg/m² of doxorubicin and are continuing to receive doxorubicin ³⁵. Interestingly, the mechanism of cardiotoxicity mitigation appears to be related, at least partially, to the ability of the drug to chelate iron. The drug is also known to catalytically inhibit topoisomerase IIβ, as discussed above, which may reduce cardiotoxicity related to generation of the topoisomerase IIβ poisons formed by anthracyclines. Other investigational measures to prevent cardiotoxicity include the use of the beta blocker carvedilol. Spallarossa and colleagues found a decrease in free radical production and apoptosis in heart muscle cells when treating cells with carvedilol prior to treatment with doxorubicin, due to carvedilol׳s unique antioxidant effect ³⁶.

The anthracenedione agents include the drugs mitoxantrone and pixantrone. The anthracenedione (anthraquinone) agents are synthetic agents, designed to act similarly to the anthracycline drugs, with fewer adverse effects and toxicities. Mitoxantrone was approved in 1987 in the U.S. and pixantrone, a newer agent, was approved in 2012 in Europe. The structures of these agents are shown in Figure 4. Mitoxantrone can cross the blood brain barrier and is indicated for the reduction of the frequency and intensity of multiple sclerosis relapses in addition to its indication as a chemotherapeutic agent in leukemia and prostate cancer ³⁷. Similar to the anthracyclines, the anthracenedione agents are topoisomerase poisons, primarily affecting type II topoisomerases. Like the previously discussed agents, mitoxantrone intercalates topoisomerase-bound DNA, preventing DNA re-ligations, and ultimately resulting in DNA strand breakage and disruption of DNA repair. Pixantrone, an aza-anthracenedione approved in treatment of non-hodgkin B-cell lymphoma, exhibits its cytotoxic effects by intercalating into DNA like the anthracyclines, but also causes long term cell damage and eventual death by causing errors in mitosis and segregation of chromosomes ³⁸. Pixantrone has shown less toxicity than doxorubicin in cardiac muscle cells because of its inability to bind iron and contribute to free radical production in the heart; as a result, animal models showed decreased heart weight in animals treated with doxorubicin as compared to pixantrone ³⁹.

Clinically marketed camptothecin derivatives include topotecan, irinotecan, and belotecan. Their structures are shown in Figure 5. The camptothecin alkaloid was first derived from the Chinese tree, Camptotheca acuminata. The camptothecin-derived agents are topoisomerase poisons which primarily affect type I topoisomerases. In vitro studies showed camptothecin, a cytotoxic alkaloid, was capable of inhibiting topoisomerase I and causing DNA strand breaks, thus preventing DNA replication ⁴⁰. The water-soluble forms of camptothecin include the clinically marketed irinotecan and topotecan, which reversibly bind and form a ternary complex with topoisomerase I and DNA ⁴¹. Topotecan is approved as second-line small cell lung cancer and, in combination with cisplatin, for patients with stage IV-B cervical carcinoma not treated by surgery or radiation. Irinotecan is approved following failure or progression following treatment with fluorouracil or in combination with 5-fluorouracil and leucovorin for patients with metastatic colon or rectal carcinoma. Belotecan is a relatively new camptothecin derivative agent, approved in South Korea for treatment of non-small-cell lung cancer and ovarian cancer in 2003 ⁴². The mechanism of action is the same as other agents in this class. Compared with older camptothecin agents, belotecan is reported to have a similar efficacy profile, with reduced toxicities ⁴³.

The drugs etoposide and teniposide are epipodophyllotoxin-derived agents. Epipodophyllotoxins are natural substances derived from the Mayapple plant (wild mandrake), Podophyllum peltatum ⁴⁴. The drugs have been available in the U.S. and other countries since the early 1980s. Their structures are shown in Figure 6. Both agents act as topoisomerase poisons and cause DNA strand breaks by binding to type II topoisomerases, similar to the agents described above ⁴⁵. Etoposide is indicated as part of a multi-drug chemotherapy regimen for refractory testicular tumors and in combination with cisplatin to treat small-cell lung cancer. Teniposide is approved in patients with refractory childhood acute lymphoblastic leukemia in combination with other chemotherapy drugs.

The sole marketed agent it its chemical class, amsacrine (m-AMSA, Figure 4), is a synthetic agent composed of a planar, acridine ring system. Like the agents discussed above, amsacrine is a topoisomerase poison targeting the type II topoisomerases. Interestingly, amsacrine was the first drug proven to poison eukaryotic topoisomerase II ⁴⁶. The acridine ring system is the component of the drug that intercalates DNA and contributes to the activity of the drug, while the non-intercalative 4′-amino-methane-sulfon-m-anisidide (m-AMSA) headgroup imparts specificity for the DNA-topoisomerase cleavage complex ⁴⁷. Amsacrine is approved in Canada to induce remission in adults with acute leukemia resistant to conventional therapy ⁴⁸.

Table 2. Summary of marketed topoisomerase inhibitors and indications

Drug	Class	Approval date	Mechanism/Target	Indication
Doxorubicin	Anthracycline	8/7/1974 (U.S.)	Type IIA poison	In combination with other chemotherapy agents to treat women after surgical resection of breast cancer with axillary lymph node involvement; acute lymphoblastic and myeloblastic leukemias; Hodgkin and non-Hodgkin lymphomas; metastatic neuroblastoma, Wilms′ tumor, cancers of the breast, soft tissue sarcoma, and bone sarcomas; metastatic ovarian, transitional cell bladder, thyroid, gastric, and bronchogenic carcinomas
Epirubicin	Anthracycline	9/15/1999 (U.S.)	Type IIA poison	Combination with other chemotherapy agents to treat women after surgical resection of breast cancer with axillary lymph node involvement
Valrubicin	Anthracycline	9/25/1998 (U.S.)	Type IIA poison	Intravesical administration for urinary bladder carcinoma refractory to BCG therapy in patients who are not candidates for cystectomy
Daunorubicin	Anthracycline	12/19/1979 (U.S.)	Type IIA poison	In combination with other approved chemotherapy agents to induce remission in acute myelogenous, monocytic, and erythroid lymphocytic leukemias in adults and in acute lymphocytic leukemia in children
Idarubicin	Anthracycline	9/27/1990 (U.S.)	Type IIA poison	In combination with other approved agents to treat adults with acute myeloid leukemia, including French-American-British M1—M7 classifications
Mitoxantrone	Anthracenedione	12/23/1987 (U.S.)	Type IIA poison	For patients with secondary (chronic) multiple sclerosis with significantly abnormal neurologic status between relapses to reduce neurologic disability and or/the frequency of relapses; in combination with corticosteroids as initial chemotherapy to treat pain related to advanced hormone-refractory prostate cancer; in combination with other approved agents to as initial therapy of acute nonlymphocytic anemia in adults
Pixantrone	Anthracenedione	2/16/2012 (E.U.)	Type IIA poison	Monotherapy for the treatment of adult patients with multiply relapsed or refractory aggressive Non-Hodgkin B-cell lymphomas
Etoposide	Epipodophyllotoxin	11/10/1983 (U.S.)	Type IIA poison	Refractory testicular tumors in combination with other chemotherapy agents; first-line treatment in combination with cisplatin for small-cell lung cancer
Teniposide	Epipodophyllotoxin	7/14/1992 (U.S.)	Type IIA poison	Induction of remission in patients with refractory childhood acute lymphoblastic leukemia
Amsacrine	Miscellaneous	12/31/1983 (Canada)	Type IIA poison	Induction of remission in acute adult leukemia refractory to conventional therapy
Topotecan	Camptothecin	5/28/1996 (U.S.)	Type IB poison	Small-cell lung cancer after failure of first-line chemotherapy; combination with cisplatin for persistent or recurrent stage IV-B carcinoma of the cervix not cured by surgery and/or radiation
Irinotecan	Camptothecin	6/14/1996 (U.S.)	Type IB poison	First-line chemotherapy in combination with 5-fluorouracil and leucovorin in patients with metastatic carcinoma of the colon or rectum; recurrent or progressive metastatic carcinoma of the colon or rectum following initial fluorouracil-based therapy
Belotecan	Camptothecin	12/10/2003 (S. Korea)	Type IB poison	Non-small-cell lung cancer; ovarian cancer

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Figure 3. Anthracycline topoisomerase inhibitors

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Figure 4. Anthracenedione and acridine-derived topoisomerase inhibitors

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Figure 5. Camptothecin-derived topoisomerase inhibitors

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Figure 6. Epipodophyllotoxin-derived topoisomerase inhibitors

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