Transcriptional regulation of topoisomerase II at confluence and pharmacological modulation of expression by bis-benzimidazole drugs

ABSTRACT Topoisomerase II␣ is a critical gene involved in DNA replication and maintenance of genomic stability. Several chemotherapeutic agents target topoisomerase II and levels of expression are an important factor in chemosensitivity. Transcriptional regulation has been demonstrated to regulate topoisomerase II␣ levels under several circumstances, including cellular confluence, heat shock, and expression of oncogenes including ras and myb. Expression of topoisomerase II␣ is regulated by cellular proliferation; transcriptional down-regulation in confluent cells is modulated through sequences within the promoter. In this study, we examined DNA-protein interactions within the topoisomerase II␣ promoter in exponential and confluent phase NIH3T3 cells. Using electrophoretic mobility shift assay and in vitro DNase I footprint experiments, the involvement of NF-Y in transcriptional regulation was established. Incubation of the DNA minor groove-binding agents Hoechst 33342 and Hoechst 33258 with nuclear extracts revealed drug binding to regions surrounding the inverted CCAAT boxes within the topoisomerase II␣ promoter and displacement of proteins binding to these elements. Addition of both Hoechst 33342 and Hoechst 33258 to NIH3T3 cells at confluence resulted in increased expression of topoisomerase II␣. In addition, MTT cytotoxicity assays in confluent cells showed an additive effect of incubation with Hoechst 33342 and the topoisomerase II␣ poison etoposide. Therefore, DNA binding drugs which block transcription factor activation of the promoter may deregulate topoisomerase II␣ and this strategy may be of value in modifying gene expression and modulating chemosensitivity

Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus

A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli EI, HPr, and IIAMtl. Degenerate primers based on regions of high amino acid similarity in the E. coli and Staphylococcus carnosus EIIMtl were used to develop a digoxigenin-labeled probe by PCR. Using this probe, we isolated three overlapping DNA fragments totaling 7.2 kb which contain the genesmtlA,mtlR,mtlF, andmtlD, encoding the mannitol IICB, a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, respectively. The mtlA gene consists of 1,413 bp coding for a 471-amino-acid protein with a calculated mass of 50.1 kDa. The amino acid sequence shows high similarity with the sequence of IICBMtl of S. carnosus and the IICB part of the IICBAMtls of E. coli and B. subtilis. The enzyme could be functionally expressed in E. coli by placing it behind the strong tac promoter. The rate of thermal inactivation at 60&C of B. stearothermophilus IICBMtl expressed in E. coli was two times lower than that of E. coli IICBMtl. IICBMtl in B. stearothermophilus is maximally active at 85&C and thus very thermostable. The enzyme was purified on Ni-nitrilotriacetic acid resin to greater than 95 % purity after six histidines were fused to the C-terminal part of the transporter. Many bacteria transport D-mannitol and other carbohy-drates via a phosphoenolpyruvate (PEP)-dependent phospho