Topo IIIalpha and BLM act within the Fanconi anemia pathway in response to DNA-crosslinking agents.

The Bloom protein (BLM) and Topoisomerase IIIalpha are found in association with proteins of the Fanconi anemia (FA) pathway, a disorder manifesting increased cellular sensitivity to DNA crosslinking agents. In order to determine if the association reflects a functional interaction for the maintenance of genome stability, we have analyzed the effects of siRNA-mediated depletion of the proteins in human cells. Depletion of Topoisomerase IIIalpha or BLM leads to increased radial formation, as is seen in FA. BLM and Topoisomerase IIIalpha are epistatic to the FA pathway for suppression of radial formation in response to DNA interstrand crosslinks since depletion of either of them in FA cells does not increase radial formation. Depletion of Topoisomerase IIIalpha or BLM also causes an increase in sister chromatid exchanges, as is seen in Bloom syndrome cells. Human Fanconi anemia cells, however, do not demonstrate increased sister chromatid exchanges, separating this response from radial formation. Primary cell lines from mice defective in both Blm and Fancd2 have the same interstrand crosslink-induced genome instability as cells from mice deficient in the Fancd2 protein alone. These observations demonstrate that the association of BLM and Topoisomerase IIIalpha with Fanconi proteins is a functional one, delineating a BLM-Topoisomerase IIIalpha-Fanconi pathway that is critical for suppression of chromosome radial formation.

Role of the Bloom's syndrome helicase in maintenance of genome stability.

The RecQ family of DNA helicases has members in all organisms analysed. In humans, defects in three family members are associated with disease conditions: BLM is defective in Bloom's syndrome, WRN in Werner's syndrome and RTS in Rothmund-Thomson syndrome. In each case, cells from affected individuals show inherent genomic instability. The focus of our work is the Bloom's syndrome gene and its product, BLM. Here, we review the latest information concerning the roles of BLM in the maintenance of genome integrity.

The Bloom's syndrome gene product interacts with topoisomerase III.

Bloom's syndrome is a rare genetic disorder associated with loss of genomic integrity and a large increase in the incidence of many types of cancer at an early age. The Bloom's syndrome gene product, BLM, belongs to the RecQ family of DNA helicases, which also includes the human Werner's and Rothmund-Thomson syndrome gene products and the Sgs1 protein of Saccharomyces cerevisiae. This family shows strong evolutionary conservation of protein structure and function. Previous studies have shown that Sgs1p interacts both physically and genetically with topoisomerase III. Here, we have investigated whether this interaction has been conserved in human cells. We show that BLM and hTOPO IIIalpha, one of two human topoisomerase III homologues, co-localize in the nucleus of human cells and can be co-immunoprecipitated from human cell extracts. Moreover, the purified BLM and hTOPO IIIalpha proteins are able to bind specifically to each other in vitro, indicating that the interaction is direct. We have mapped two independent domains on BLM that are important for mediating the interaction with hTOPO IIIalpha. Furthermore, through characterizing a genetic interaction between BLM and TOP3 in S. cerevisiae, we have identified a functional role for the hTOPO IIIalpha interaction domains in BLM.

Overexpression of the RI alpha subunit of protein kinase A confers hypersensitivity to topoisomerase II inhibitors and 8-chloro-cyclic adenosine 3'5'-monophosphate in Chinese hamster ovary cells.

We have shown that a mutant derivative of Chinese hamster ovary CHO-K1 cells, ADR-5, which shows hypersensitivity to topoisomerase II (topo II)-inhibitory drugs, is cross-sensitive to the site-selective cyclic AMP analogue 8-chloro-cyclic AMP. We tested the hypothesis that overexpression of the type I alpha regulatory subunit of protein kinase A may represent a common element conferring hypersensitivity to both topo II inhibitors and 8-chloro-cyclic AMP in ADR-5 cells. We have demonstrated that ADR-5 cells overexpress RI alpha protein, compared to parental CHO-K1 cells. Moreover, retroviral vector-mediated transfer of the RI alpha gene into CHO-K1 cells was able to confer a drug-hypersensitive phenotype similar to that exhibited by ADR-5 cells. Analysis of topo II protein levels and activity revealed no differences between parental and infected cells, suggesting that protein kinase A may be involved in the downstream processing of topo II-mediated events.

Evidence for the involvement of DNA topoisomerase II in neutrophil-granulocyte differentiation.

Agents that slow cellular proliferation usually stimulate myeloid differentiation. The demonstration in this report of an anomalous inhibitory behavior of the epipodophyllotoxin VP16-213, an agent known to inhibit the enzyme DNA topoisomerase II, prompted us to investigate the role of this enzyme in both changes in DNA supercoiling and in DNA strand breakage and reunion events occurring during the induction of neutrophil-granulocyte differentiation. We recently reported that retinoic acid, an inducer of granulocytic differentiation, stimulates transient relaxation of DNA supercoiling. We now show that this is associated with the formation of small numbers of protein-linked DNA breaks (a characteristic of topoisomerase reactions). Both events are perturbed by VP16-213, and since this agent inhibits subsequent differentiation, these observations raise the possibility of a role for DNA topoisomerase II in granulocytic differentiation. The possible relevance of these findings to mechanisms of leukemogenesis is discussed.