The Rb/E2F pathway regulates the expression of genes essential for cell proliferation but that also trigger apoptosis. During normal proliferation, PI3K/Akt signaling blocks E2F1-induced apoptosis, thus serving to balance proliferation and death. We now identify a subset of E2F1 target genes that are specifically repressed by PI3K/Akt signaling, thus distinguishing the E2F1 proliferative or apoptotic function. RNAi-mediated inhibition of several of these PI3K-repressed E2F1 target genes, including AMPK alpha 2, impairs apoptotic induction by E2F1. Activation of AMPK alpha 2 with an AMP analog further stimulates E2F1-induced apoptosis. We also show that the presence of the E2F1 apoptotic expression program in breast and ovarian tumors coincides with good prognosis, emphasizing the importance of the balance in the E2F1 proliferation/apoptotic program.

Multiple or pleiotropic drug resistance most often occurs in Saccharomyces cerevisiae due to substitution mutations within the Cys 6 -Zn(II) transcription factors Pdr1p and Pdr3p. These dominant transcriptional regulatory proteins cause elevated drug resistance and overexpression of the ATP-binding cassette transporterencoding gene, PDR5. We have carried out a genetic screen to identify negative regulators of PDR5 expression and found that loss of the mitochondrial genome ( o cells) causes up-regulation of Pdr3p but not Pdr1p function. Additionally, loss of the mitochondrial inner membrane protein Oxa1p generates a signal that results in increased Pdr3p activity. Both of these mitochondrial defects lead to increased expression of the PDR3 structural gene. Importantly, the signaling pathway used to enhance Pdr3p function in o cells is not the same as in oxa1 cells. Loss of previously described nuclear-mitochondrial signaling genes like RTG1 reduce the level of PDR5 expression and drug resistance seen in o cells but has no effect on oxa1-induced phenotypes. These data uncover a new regulatory pathway connecting expression of multidrug resistance genes with mitochondrial function.

Semidominant mutations in the PDR1 or PDR3 gene lead to elevated resistance to cycloheximide and oligomycin. PDR1 and PDR3 have been demonstrated to encode zinc cluster transcription factors. Cycloheximide resistance mediated by PDR1 and PDR3 requires the presence of the PDR5 membrane transporterencoding gene. However, PDR5 is not required for oligomycin resistance. Here, we isolated a gene that is necessary for PDR1-and PDR3-mediated oligomycin resistance. This locus, designated YOR1, causes a dramatic elevation in oligomycin resistance when present in multiple copies. A yor1 strain exhibits oligomycin hypersensitivity relative to an isogenic wild-type strain. In addition, loss of the YOR1 gene blocks the elevation in oligomycin resistance normally conferred by mutant forms of PDR1 or PDR3. The YOR1 gene product is predicted to be a member of the ATP-binding cassette transporter family of membrane proteins. Computer alignment indicates that Yor1p shows striking sequence similarity with multidrug resistance-associated protein, Saccharomyces cerevisiae Ycf1p, and the cystic fibrosis transmembrane conductance regulator. Use of a YOR1-lacZ fusion gene indicates that YOR1 expression is responsive to PDR1 and PDR3. While PDR5 expression is strictly dependent on the presence of PDR1 or PDR3, control of YOR1 expression has a significant PDR1/PDR3-independent component. Taken together, these data indicate that YOR1 provides the link between transcriptional regulation by PDR1 and PDR3 and oligomycin resistance of yeast cells.

Saccharomyces cerevisiae has large number of genes that can be genetically altered to produce a multiple or pleiotropic drug resistance phenotype.

Previous work has demonstrated a role for the E2F1 gene product in signaling apoptosis, both as a result of the deregulation of the Rb͞E2F pathway as well as in response to DNA damage. We now show that the ability of cells to suppress the apoptotic potential of E2F1, as might occur during the course of normal cellular proliferation, requires the action of the Ras-phosphoinositide 3-kinaseAkt signaling pathway. In addition, we also identify a domain within the E2F1 protein, previously termed the marked-box domain, that is essential for the apoptotic activity of E2F1 and that distinguishes the E2F1 protein from E2F3. We also show that the E2F1-marked-box domain is essential for the induction of both p53 and p73 accumulation. Importantly, a role for the marked-box domain in the specificity of E2F1-mediated apoptosis coincides with recent work demonstrating a role for this domain in achieving specificity in the activation of transcription. We conclude that the unique capacity of E2F1 to trigger apoptosis reflects a specificity of transcriptional activation potential, and that this role for E2F1 is regulated through the action of the Akt protein kinase. T he role of the Rb͞E2F pathway in the regulation of cell cycle progression, particularly the G 1 ͞S transition, is now well established (1, 2). A variety of experiments have suggested distinct roles for individual members of the E2F family in the control of cell proliferation and cell fate (1, 2). For example, various experiments have suggested a particularly important role for E2F3 in the control of cell proliferation, consistent with a role in the activation of genes encoding DNA replication proteins (3, 4). In contrast, E2F4 may not be so critical for proliferation, but rather it may foster the ability of various cell types to exit the cell cycle and differentiate (5, 6). E2F1 would appear to play dual roles in the control of cell proliferation and cell fate. Overexpression of E2F1 can drive quiescent cells into S phase (7), and studies of cells deleted for multiple E2F proteins provide evidence of a role for E2F1 in the ability of cells to proliferate (8). But, in addition to this role, E2F1 appears to have the unique ability to induce apoptosis when expressed in the absence of proliferative signals (9)(10)(11)(12)(13)(14).A physiological role for the E2F1-induced apoptosis has been seen in several contexts. E2F1 Ϫ/Ϫ mice develop thymic hyperplasia caused by a defect in thymocyte apoptosis (15), and the absence of E2F1 activity has been associated with decreased negative selection of T cells (16). In addition, mice lacking Rb function exhibit excessive apoptosis in the developing neurons, lens, and myocytes (17, 18). At least part of the Rb Ϫ/Ϫ phenotype is caused by E2F1 deregulation (19-21), but it is also true that other studies have provided evidence of a role for E2F3 in the Rb-dependent apoptosis pathway (22). Additional studies have shown that E2F1 is uniquely induced in response to DNA damage (23). The specificity of the response reflects the fact that E2...