Doxorubicin is among the most effective and widely used anticancer drugs in the clinic. However, cardiotoxicity is one of the life-threatening side effects of doxorubicin-based therapy. Dexrazoxane (Zinecard, also known as ICRF-187) has been used in the clinic as a cardioprotectant against doxorubicin cardiotoxicity. The molecular basis for doxorubicin cardiotoxicity and the cardioprotective effect of dexrazoxane, however, is not fully understood. In the present study, we showed that dexrazoxane specifically abolished the DNA damage signal ;-H2AX induced by doxorubicin, but not camptothecin or hydrogen peroxide, in H9C2 cardiomyocytes. Doxorubicin-induced DNA damage was also specifically abolished by the proteasome inhibitors bortezomib and MG132 and much reduced in top2B À/À mouse embryonic fibroblasts (MEF) compared with TOP2B +/+ MEFs, suggesting the involvement of proteasome and DNA topoisomerase IIB (Top2B). Furthermore, in addition to antagonizing Top2 cleavage complex formation, dexrazoxane also induced rapid degradation of Top2B, which paralleled the reduction of doxorubicin-induced DNA damage. Together, our results suggest that dexrazoxane antagonizes doxorubicin-induced DNA damage through its interference with Top2B, which could implicate Top2B in doxorubicin cardiotoxicity. The specific involvement of proteasome and Top2B in doxorubicininduced DNA damage is consistent with a model in which proteasomal processing of doxorubicin-induced Top2B-DNA covalent complexes exposes the Top2B-concealed DNA doublestrand breaks.

Drugs that target DNA topoisomerase II (Top2), including etoposide (VP-16), doxorubicin, and mitoxantrone, are among the most effective anticancer drugs in clinical use. However, Top2-based chemotherapy has been associated with higher incidences of secondary malignancies, notably the development of acute myeloid leukemia in VP-16-treated patients. This association is suggestive of a link between carcinogenesis and Top2-mediated DNA damage. We show here that VP-16-induced carcinogenesis involves mainly the ␤ rather than the ␣ isozyme of Top2. In a mouse skin carcinogenesis model, the incidence of VP-16-induced melanomas in the skin of 7,12-dimethylbenz[a]anthracene-treated mice is found to be significantly higher in TOP2␤ ؉ than in skin-specific top2␤-knockout mice. Furthermore, VP-16-induced DNA sequence rearrangements and double-strand breaks (DSBs) are found to be Top2␤-dependent and preventable by cotreatment with a proteasome inhibitor, suggesting the importance of proteasomal degradation of the Top2␤-DNA cleavage complexes in VP-16-induced DNA sequence rearrangements. VP-16 cytotoxicity in transformed cells expressing both Top2 isozymes is, however, found to be primarily Top2␣-dependent. These results point to the importance of developing Top2␣-specific anticancer drugs for effective chemotherapy without the development of treatment-related secondary malignancies.DNA rearrangements ͉ melanoma ͉ skin-specific topoisomerase II␤-knockout ͉ tumor cell killing ͉ carcinogenesis A nticancer drugs that target DNA topoisomerase II (Top2), including etoposide (VP-16), doxorubicin, and mitoxantrone, are often referred to as Top2 poisons and are among the most effective and widely used anticancer drugs in the clinic. However, life-threatening toxic side effects, including drug-induced secondary malignancies, have been noted in patients receiving Top2-based chemotherapy. An association between infant leukemia and in utero exposure to Top2 poisons has also been reported (reviewed in refs. 1-3). In all cases, the molecular basis underlying carcinogenesis in Top2-based chemotherapy is unclear.Clinical evidence for a direct link between VP-16 treatment and treatment-related acute myeloid leukemia (t-AML) is particularly strong (1-3). VP-16-induced t-AML is frequently associated with balanced translocations between the mixed lineage leukemia (MLL) gene on chromosome 11q23 and Ͼ50 partner genes (the MLL gene is also known as ALL-1, hTRX, or HRX) (4-7). These rearrangements, as well as those found in infant leukemia, cluster within a well characterized 8.3-kb breakpoint cluster region (bcr) (8-16). The bcr of MLL is AT-rich and contains Alu sequences, putative recognition sites of Top2-mediated DNA cleavage, and chromosome scaffold/matrix attachment regions (SAR/MAR) (5,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). There is substantial evidence that chromosome 11q23 translocations in t-AML and infant leukemia are a consequence of drug-induced formation of double-strand breaks (DSBs) (6-9). VP-16 is known to induce DSBs by the format...

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Mice lacking topoisomerase II␤ (TopII␤) are known to exhibit a perinatal death phenotype. In the current study, transcription profiles of the brains of wild-type and top2␤ knockout mouse embryos were generated. Surprisingly, only a small number (1 to 4%) of genes were affected in top2␤ knockout embryos. However, the expression of nearly 30% of developmentally regulated genes was either up-or down-regulated. By contrast, the expression of genes encoding general cell growth functions and early differentiation markers was not affected, suggesting that TopII␤ is not required for early differentiation programming but is specifically required for the expression of developmentally regulated genes at later stages of differentiation. Consistent with this notion, immunohistochemical analysis of brain sections showed that TopII␤ and histone deacetylase 2, a known TopII␤-interacting protein, were preferentially expressed in neurons which are in their later stages of differentiation. Chromatin immunoprecipitation analysis of the developing brains revealed TopII␤ binding to the 5 region of a number of TopII␤-sensitive genes. Further studies of a TopII␤-sensitive gene, Kcnd2, revealed the presence of TopII␤ in the transcription unit with major binding near the promoter region. Together, these results support a role of TopII␤ in activation/repression of developmentally regulated genes at late stages of neuronal differentiation.

Since the original descriptions of gain-of function mutations in anaplastic lymphoma kinase (ALK), interest in the role of this receptor tyrosine kinase in neuroblastoma development and as a potential therapeutic target has escalated. As a group, the activating point mutations in full-length ALK, found in approximately 8% of all neuroblastoma tumors, are distributed evenly across different clinical stages. However, the most frequent somatic mutation, F1174L, is associated with amplification of the MYCN oncogene. This combination of features appears to confer a worse prognosis than MYCN amplification alone, suggesting a cooperative effect on neuroblastoma formation by these two proteins. Indeed, F1174L has shown more potent transforming activity in vivo than the second most common activating mutation, R1275Q, and is responsible for innate and acquired resistance to crizotinib, a clinically relevant ALK inhibitor that will soon be commercially available. These advances cast ALK as a bona fide oncoprotein in neuroblastoma and emphasize the need to understand ALK-mediated signaling in this tumor. This review addresses many of the current issues surrounding the role of ALK in normal development and neuroblastoma pathogenesis, and discusses the prospects for clinically effective targeted treatments based on ALK inhibition.