Cdc7p is a protein kinase that is required for G 1 ͞S transition and initiation of DNA replication in Saccharomyces cerevisiae. The mechanisms whereby Cdc7p and its substrates exerts their effects are unknown. We report here the characterization in S. cerevisiae of a recessive mutation in a member of the MCM family, MCM5͞CDC46, which bypasses the requirement for Cdc7p and its interacting factor Dbf4p. Because the MCM family of evolutionarily conserved proteins have been implicated in restricting DNA replication to once per cell cycle, our studies suggest that Cdc7p is required late in G 1 because in its absence the Mcm5p͞Cdc46p blocks the initiation of DNA replication. Moreover, Mcm5p͞Cdc46p may have both positive and negative effects on the ability of cell to initiate replication.

Iron is an essential micronutrient for the growth and function of all cells. It is, therefore, an attractive target for chemotherapeutic compounds. Numerous studies in vitro and in vivo provide evidence that iron chelators may be effective antitumor agents. Lipophilic iron chelators that are readily cell permeable and can bind intracellular iron stores may selectively kill cancer cells without damaging normal cells. In this review we discuss the role of iron in cellular processes and how these processes differ between normal and neoplastic cells. We also review the effects on normal and cancer cell growth of several lipophilic iron chelators.

Proliferation of vascular smooth muscle cells (VSMC)is characteristic of restenosis following balloon angioplasty. We show here that a low concentration of a novel iron chelator, desferri-exochelin 772SM, reversibly arrests the growth of human VSMC in vitro, specifically in G 0 /G 1 and S phases. The lipophilic desferri-exochelin is effective more rapidly and at a 10-fold lower concentration than the nonlipophilic iron chelator deferoxamine. Treatment of growth-synchronized VSMC with the desferri-exochelin results in down-regulation of cyclin E/ Cdk2 and cyclin A/Cdk2 activity but does not affect the cyclin D/Cdk4/retinoblastoma phosphorylation pathway. Both DNA replication and RNA transcription are inhibited in exochelin-treated cells, but protein synthesis is not. The ability of desferri-exochelin 772SM to reversibly block the growth of VSMC in vitro with no apparent cytotoxicity suggests that the exochelin may be useful as a therapeutic agent to limit restenosis in injured vessels.Although vascular smooth muscle cells (VSMC) 1 are normally quiescent, they enter the cell cycle when exposed to growth factors in vitro or following vascular injury in vivo. In animal models, proliferation and migration of VSMC begin soon after vascular injury occurs and culminate in formation of a neointima that encroaches on the interior space of the vessel (1). Such neointima formation is seen in a substantial number of patients following balloon angioplasty and may result in a narrowing or blockage (restenosis) of the vessel that requires further intervention (2). By preventing the immediate proliferation of VSMC following vascular injury, it may be possible to avert neointima formation and restenosis. In this report, we describe the cell cycle-specific growth inhibitory effects of a novel iron chelator, desferri-exochelin 772SM, on serum-and growth factor-stimulated human VSMC in vitro.Iron is required for a variety of cellular functions, including respiration, energy metabolism, and DNA synthesis. In addition, iron participates in redox reactions that generate free radicals, which may activate signaling pathways for cell proliferation. Treatment with iron chelators has previously been shown to cause growth arrest in several types of cells (3-5). However, the effect of chelation varied depending upon both the cell type and the chelator used (5-7).The iron chelator deferoxamine (DFO) inhibited DNA synthesis in cultured rat VSMC (8) and blocked neointimal growth in a rat model of vascular injury (8). However, DFO enters cells only very slowly by pinocytosis (9) and causes hypotension when given in large doses in vivo (10). Therefore, DFO is of limited usefulness for determining mechanisms by which iron deprivation prevents cell cycle progression or for the clinical treatment of restenosis.Exochelins, the secreted siderophores of Mycobacterium tuberculosis, are a family of high affinity iron chelators that are both water-and lipid-soluble, a property that allows them to enter cells rapidly and chelate specific intracellular iro...

A major goal of cancer chemotherapy is the identification of cytotoxic compounds that are highly selective for cancer cells. We describe here one such compound - a novel iron chelator, desferri-exochelin 772SM. This desferri-exochelin has unique chemical and pharmacological properties, including extremely high iron binding affinity, the capacity to block iron-mediated redox reactions, and lipid solubility which enables it to enter cells rapidly. At low concentrations, this desferri-exochelin kills T47D-YB and MCF-7 human breast cancer cells by inducing apoptosis, but only reversibly arrests the growth of normal human mammary epithelial cells without cytotoxicity. Since iron-loaded exochelin is ineffective, iron chelation accounts for the efficacy of desferri-exochelin. For both the killing of breast cancer cells and the growth arrest of normal breast epithelial cells, desferri-exochelin was effective at much lower concentrations than the lipid-insoluble iron chelator deferoxamine, which has shown only limited potential as an anti-cancer agent. Growth arrest of progesterone receptor positive T47D-YB cells with the progestin R5020 transiently protects them from the cytotoxic effects of desferri-exochelin, but the cells are killed after cell growth resumes. Similarly, MCF-7 cells arrested with the estrogen antagonist ICI182780 are transiently resistant to killing by desferri-exochelin. Thus the desferri-exochelin is cytotoxic only to actively growing tumor cells. Since desferri-exochelin 772SM can selectively and efficiently destroy proliferating cancer cells without damaging normal cells, it may prove useful for the treatment of cancer.

Peroxiredoxin (Prx) is an important antioxidant defense enzyme that reduces hydrogen peroxide to molecular oxygen by using reducing equivalents from thioredoxin. We report that lung Prx I messenger RNA (mRNA) is specifically upregulated by oxygen. Throughout the third trimester, mRNA for Prx I was expressed constitutively at low levels in fetal baboon lung. However, after premature birth (125 or 140 d gestation), lung Prx I mRNA increased rapidly with the onset of oxygen exposure. Premature animals (140 d) breathing 100% O(2) developed chronic lung disease within 7 to 14 d. These animals had greater lung Prx I mRNA after 1, 6, or 10 d of life than did fetal controls. In 140-d animals given lesser O(2) concentrations (as needed) that did not develop chronic lung disease, lung Prx I mRNA also was increased on Days 1 and 6, but not Day 10. In fetal distal lung explant culture, Prx I mRNA was elevated in 95% O(2), relative to 1% oxygen, and remained elevated at 24 h. Prx protein activity increased in 140-d premature baboons exposed to as-needed oxygen. By contrast, there was a decrease in Prx activity in 140-d premature baboons exposed to 100% oxygen. In the lung explants from prematures (140 d), there was no significant increase in Prx activity in response to 24 h exposure to hyperoxia, whereas exposure of explants to 48 h hyperoxia caused a nonsignificant decrease in Prx activity. Treatment of lung explants with actinomycin D inhibited Prx mRNA increases in 95% oxygen, indicating transcriptional regulation. In cellular signaling studies we demonstrated that protein kinase (PK) C activity increased when A549 cells were exposed to 95% oxygen, compared with 21% oxygen exposure. In lung explant cultures, specific PKC inhibitors calphostin C or GF109203X inhibited the increase in Prx I mRNA with 95% oxygen exposure, indicating PKC-mediated signaling. The acute increase in gene expression of Prx I in response to oxygen suggests an important role for this protein during the transition from relatively anaerobic fetal life to oxygen-breathing at birth.