Identification of a monopartite sequence in PU.1 essential for nuclear import, DNA-binding and transcription of myeloid-specific genes.

The Ets transcription factor PU.1 is an essential regulator of normal hematopoiesis, especially within the myeloid lineage. As such, endogenous PU.1 predominantly localizes to the nucleus of mammalian cells to facilitate gene regulation. However, to date, little is known regarding the mechanisms of PU.1 nuclear transport. We found, using HeLa and RAW 264.7 macrophage cells, that PU.1 enters the nucleus via passive diffusion and active transport. The latter can be facilitated by: (i) the classical pathway requiring importin alpha and beta; (ii) the non-classical pathway requiring only importin beta; or (iii) direct interaction with nucleoporins. A group of six positively charged lysine or arginine residues within the Ets DNA-binding domain was determined to be crucial in active nuclear import. These residues directly interact with importin beta to facilitate a predominantly non-classical import pathway. Furthermore, luciferase reporter assays demonstrated that these same six amino acids are crucial for PU.1-mediated transcriptional activation of myeloid-specific genes. Indeed, these residues may represent a consensus sequence vital for nuclear import, DNA-binding and transcriptional activity of Ets family members. By identifying and characterizing the mechanisms of PU.1 nuclear import and the specific amino acids involved, this report may provide insights into the molecular basis of diseases.

Identification of nuclear import and export signals within Fli-1: roles of the nuclear import signals in Fli-1-dependent activation of megakaryocyte-specific promoters.

The Ets factor Friend leukemia integration 1 (Fli-1) is an important regulator of megakaryocytic (Mk) differentiation. Here, we demonstrate two novel nuclear localization signals (NLSs) within Fli-1: one (NLS1) is located at the N terminus, and another (NLS2) is within the Ets domain. Nuclear accumulation of Fli-1 reflected the combined functional effects of the two discrete NLSs. Each NLS can independently direct nuclear transport of a carrier protein, with mutations within the NLSs affecting nuclear accumulation. NLS1 has a bipartite motif, whereas the NLS2 region contains a nonclassical NLS. Both NLSs bind importin alpha (IMPalpha) and IMPbeta, with NLS1 and NLS2 being predominantly recognized by IMPalpha and IMPbeta, respectively. Fli-1 also contains one nuclear export signal. Leptomycin B abolished its cytoplasmic accumulation, showing CRM1 dependency. We demonstrate that Ets domain binding to specific target DNA effectively blocks IMP binding, indicating that the targeted DNA binding plays a role in localizing Fli-1 to its destination and releasing IMPs for recycling back to the cytoplasm. Finally, by analyzing full-length Fli-1 carrying NLS1, NLS2, and combined NLS1-NLS2 mutations, we conclude that two functional NLSs exist in Fli-1 and that each NLS is sufficient to target Fli-1 to the nucleus for activation of Mk-specific genes.

Unexpected anthracycline-mediated alterations in iron-regulatory protein-RNA-binding activity: the iron and copper complexes of anthracyclines decrease RNA-binding activity.

Anthracyclines are effective antineoplastic agents. However, the interaction of these drugs with iron (Fe) is an important cause of myocardial toxicity, limiting their therapeutic use (J Lab Clin Med 122:245-251, 1993). To overcome this limitation, it is crucial to understand how anthracyclines interact with the Fe metabolism of myocardial and neoplastic cells. Iron-regulatory proteins (IRPs) play vital roles in regulating cellular Fe metabolism via their mRNA-binding activity. We showed that doxorubicin (DOX) and its analogs interfere with tumor and myocardial cell Fe metabolism by affecting the RNA-binding activity of IRPs. Unexpectedly, experiments with the free radical scavengers, catalase, superoxide dismutase, ebselen, and Mn(III) tetrakis (4-benzoic acid) porphyrin complex, suggested that the effects of DOX on IRP-RNA-binding activity were not due to anthracycline-mediated free radical production. In contrast to previous studies, we showed that the DOX metabolite, doxorubicinol, had no effect on IRP-RNA-binding activity. Rather, the anthracycline-Fe and -copper (Cu) complexes decreased IRP-RNA-binding activity, indicating that formation of anthracycline-metal complexes may affect cellular Fe metabolism. In addition, anthracyclines prevented the response of IRPs to the depletion of intracellular Fe by chelators. This information may be useful in designing novel therapeutic strategies against tumor cells by combining chelators and anthracyclines. Interestingly, the effect of DOX on primary cultures of cardiomyocytes was similar to that observed using neoplastic cells, and particularly notable was the decrease in IRP2-RNA-binding activity. Our results add significant new information regarding the effects of anthracyclines on Fe metabolism that may lead to the design of more effective treatments.

The iron metabolism of neoplastic cells: alterations that facilitate proliferation?

For many years it has been known that neoplastic cells express high levels of the transferrin receptor 1 (TfR1) and internalize iron (Fe) from transferrin (Tf) at a tremendous rate. Considering the high requirement of neoplastic cells for Fe, understanding its metabolism is vital in terms of devising potential new therapies. Apart from TfR1, a number of molecules have been identified that may have roles in Fe metabolism and cellular proliferation. These molecules include transferrin (Tf), the oestrogen-inducible transferrin receptor-like protein, transferrin receptor 2 (TfR2), melanotransferrin (MTf), ceruloplasmin, and ferritin. In the present review these latter molecules are discussed in terms of their potential functions in tumour cell Fe metabolism and proliferation. Further studies are essential to determine the specific roles of these proteins in the pathogenesis of cancer.