Iron is an essential nutrient that is severely limited in the mammalian host. Candida albicans encodes a family of 15 putative ferric reductases, which are required for iron acquisition and utilization. Despite the central role of ferric reductases in iron acquisition and mobilization, relatively little is known about the regulatory networks that govern ferric reductase gene expression in C. albicans. Here we have demonstrated the differential regulation of two ferric reductases, FRE2 and FRP1, in response to distinct iron-limited environments. FRE2 and FRP1 are both induced in alkaline-pH environments directly by the Rim101 transcription factor. However, FRP1 but not FRE2 is also induced by iron chelation. We have identified a CCAAT motif as the critical regulatory sequence for chelatormediated induction and have found that the CCAAT binding factor (CBF) is essential for FRP1 expression in iron-limited environments. We found that a hap5⌬/hap5⌬ mutant, which disrupts the core DNA binding activity of CBF, is unable to grow under iron-limited conditions. C. albicans encodes three CBF-dependent transcription factors, and we identified the Hap43 protein as the CBF-dependent transcription factor required for iron-limited responses. These studies provide key insights into the regulation of ferric reductase gene expression in the fungal pathogen C. albicans.

Two signaling pathways are activated by antineoplastic therapies that damage DNA and stall replication. In one pathway, double-strand breaks activate ataxia-telangiectasia mutated kinase (ATM) and checkpoint kinase 2 (Chk2), two protein kinases that regulate apoptosis, cell-cycle arrest, and DNA repair. In the second pathway, other types of DNA lesions and replication stress activate the Rad9-Hus1-Rad1 complex and the protein kinases ataxia-telangiectasia mutated and Rad3-related kinase (ATR) and checkpoint kinase 1 (Chk1), leading to changes that block cell-cycle progression, stabilize stalled replication forks, and influence DNA repair. Gemcitabine and cytarabine are two highly active chemotherapeutic agents that disrupt DNA replication. Here, we examine the roles these pathways play in tumor cell survival after treatment with these agents. Cells lacking Rad9, Chk1, or ATR were more sensitive to gemcitabine and cytarabine, consistent with the fact that these agents stall replication forks, and this sensitization was independent of p53 status. Interestingly, ATM depletion sensitized cells to gemcitabine and ionizing radiation but not cytarabine. Together, these results demonstrate that 1) gemcitabine triggers both checkpoint signaling pathways, 2) both pathways contribute to cell survival after gemcitabine-induced replication stress, and 3) although gemcitabine and cytarabine both stall replication forks, ATM plays differential roles in cell survival after treatment with these agents.Gemcitabine (2Ј,2Ј-difluoro 2Ј-deoxycytidine), a pyrimidine-based antimetabolite, is currently licensed for the treatment of pancreatic cancer. Recent clinical studies have also demonstrated extensive activity of this agent against a variety of additional neoplasms, including carcinomas of the ovary, lung, and breast, acute leukemias, and refractory lymphomas (Carmichael, 1998;Nabhan et al., 2001). Because of its widespread use, there is considerable interest in understanding factors that affect sensitivity and resistance to this agent. Earlier studies demonstrated that gemcitabine is taken into cells on concentrative nucleoside transporter 1 and phosphorylated to gemcitabine 5Ј-monophosphate by deoxycytidine kinase (Plunkett et al., 1996). Subsequent addition of 5Ј-phosphates results in the formation of gemcitabine diphosphate and gemcitabine triphosphate, both of which contribute to the antiproliferative effects of gemcitabine (Plunkett et al., 1996). Gemcitabine diphosphate inhibits ribonucleotide reductase, thereby depleting deoxyribonucleotide levels. Gemcitabine triphosphate is a substrate for replicative DNA polymerases and causes chain termination one base pair beyond the site of incorporation.Because gemcitabine inhibits replication, this drug is predicted to activate the S-phase checkpoint, a series of reactions that inhibit DNA synthesis and enhance survival when cells experience replication stress. According to current understanding, the kinases ATR and Chk1 play critical roles in this checkpoint. When replicati...

Candida albicans is a commensal fungus of mucosal surfaces that can cause disease in susceptible hosts. One aspect of the success of C. albicans as both a commensal and a pathogen is its ability to adapt to diverse environmental conditions, including dramatic variations in environmental pH. The response to a neutral-toalkaline pH change is controlled by the Rim101 signal transduction pathway. In neutral-to-alkaline environments, the zinc finger transcription factor Rim101 is activated by the proteolytic removal of an inhibitory C-terminal domain. Upon activation, Rim101 acts to induce alkaline response gene expression and repress acidic response gene expression. Previously, recombinant Rim101 was shown to directly bind to the alkalinepH-induced gene PHR1. Here, we demonstrate that endogenous Rim101 also directly binds to the alkalinepH-repressed gene PHR2. Furthermore, we find that of the three putative binding sites, only the ؊124 site and, to a lesser extent, the ؊51 site play a role in vivo. In C. albicans, the predicted Rim101 binding site was thought to be CCAAGAA, divergent from the GCCAAG site defined in Aspergillus nidulans and Saccharomyces cerevisiae. Our results suggest that the Rim101 binding site in C. albicans is GCCAAGAA, but slight variations are tolerated in a context-dependent fashion. Finally, our data suggest that Rim101 activity is governed not only by proteolytic processing but also by an additional mechanism not previously described.

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