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The importance of the mitochondria in UV-induced apoptosis has become increasingly apparent. Following DNA damage cytochrome c and other pro-apoptotic factors are released from the mitochondria, allowing for formation of the apoptosome and subsequent cleavage and activation of caspase-9. Active caspase-9 then activates downstream caspases-3 and/or -7, which in turn cleave poly(ADP)-ribose polymerase (PARP) and other downstream targets, resulting in apoptosis. In an effort to understand the mechanisms of Akt-mediated cell survival in breast cancer, we studied the effects of insulin-like growth factor (IGF)-I treatment on UV-treated MCF-7 human breast cancer cells. Apoptosis was induced in MCF-7 cells after UV treatment, as measured by caspase-7 and PARP cleavage, and IGF-I co-treatment protected against this response. Surprisingly caspase-9 cleavage was unchanged with UV and/or IGF-I treatment. Using MCF-7 cells overexpressing caspase-3 we have shown that resistance of caspase-9 to cleavage was not altered by the expression of caspase-3. Furthermore, overexpression of caspase-9 did not enhance PARP or caspase-7 cleavage after UV treatment. Because caspase-8 was activated with UV treatment alone, we believe that UV-induced apoptosis in MCF-7 cells occurs independently of cytochrome c and caspase-9, supporting the existence of a cytoplasmic inhibitor of cytochrome c in MCF-7 cells. We anticipate that such inhibitors may be overexpressed in cancer cells, allowing for treatment resistance.

Pyrrole-imidazole (Py-Im) polyamides are synthetic ligands that can be designed to bind predetermined DNA sequences (40,44). These minor-groove DNA-binding molecules block eukaryotic transcription factors from binding to their cognate DNA sequences and inhibit transcription, both in vitro and, in a few cases, in cell culture experiments (reviewed in reference 13). Polyamides are effective inhibitors of tissue-specific and general transcription factors (7, 8) as well as viral repressors (9) and transactivators (29). Recently, activation of gene expression has been achieved in vitro by tethering a small peptide activation domain to a sequence-specific Py-Im polyamide (31). Remarkably, activation and repression of selected genes have been achieved in Drosophila melanogaster by targeting polyamides to highly repeated satellite DNA sequences (17,18).Since batteries of genes utilize common general and tissuespecific transcription factors, polyamides have been synthesized to bind sequences adjacent to the binding sites for required transcription factors (7). A polyamide targeted to sequences adjacent to the human immunodeficiency virus type 1 (HIV-1) TATA box effectively inhibits TATA box binding protein (TBP) binding and basal transcription by RNA polymerase II (7). The binding of the TBP subunit of TFIID in the minor groove nucleates assembly of the polymerase II transcription machinery for TATA-containing genes (24,25). Since TFIID and the other general transcription factors TFIIA, -B, -E, -F, and -H (28, 33) occupy at least 40 bp of promoter DNA upstream from the transcription start site of mRNA-coding genes, this raises the question of whether sites nonoverlapping and distant from the TATA box might also serve as effective polyamide targets for inhibition of transcription. To address this issue, we generated a series of DNA constructs in which a common polyamide-binding site was scanned through a promoter and determined the effect of binding site position on inhibition of TBP binding and basal RNA polymerase II transcription. Our results show that essential protein-DNA contacts on the HIV-1 core promoter are not simply restricted to the TATA box and initiator element (20, 45) but rather extend both upstream and downstream of the TATA box. Some of these contacts are likely due to TFIID, the multiprotein complex containing TBP. Importantly, transcription inhibition can be achieved by targeting polyamides to promoter sequences distant from the TATA element that are gene specific. Py-Im polyamides thus provide simple and convenient chemical probes for discovery of functionally important protein-DNA contacts within specific gene promoters. MATERIALS AND METHODSPolyamide synthesis and characterization. Three Py-Im polyamides (1-3), whose structures are shown in Fig. 1A, were synthesized by solid-phase methods (1). Polyamide-EDTA conjugates were also prepared (40). The purity and identity of each compound were verified by analytical high-pressure liquid chromatography, 1 H nuclear magnetic resonance, and matrix-assisted ...

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Significant discoveries have recently contributed to our knowledge of intracellular growth factor and nutrient signaling via mTOR (mammalian target of rapamycin). This signaling pathway is essential in cellular metabolism and cell survival by enhancing protein translation through phosphorylation of 4EBP-1 and p70S6K. Growth factors like insulin-like growth factor-I induce mTOR to prevent cell death during cellular stress. Agents targeting mTOR are of major interest as anticancer agents. We show here, using human breast cancer cells, that certain types of stress activate mTOR leading to 4E-BP1 and p70S6K phosphorylation. UV treatment increased phosphorylation of the translation inhibitor eIF2␣, suggesting a potential mechanism for UV activation of Akt and mTOR. c-Myc, a survival protein regulated by cap-dependent protein translation, increased with IGF-I treatment, but this response was not inhibited by rapamycin. Additionally, UV treatment potently increased c-Myc degradation, which was reduced by co-treatment with the proteasomal inhibitor, MG-132. Together, these data suggest that protein translation does not strongly mediate cell survival in these models. In contrast, the phosphorylation status of retinoblastoma protein (pRB) was mediated by mTOR through its inhibitory effects on phosphatase activity. This effect was most notable during DNA damage and rapamycin treatment. Hypophosphorylated pRB was susceptible to inactivation by caspase-mediated cleavage, resulting in cell death. Reduction of pRB expression inhibited IGF-I survival effects. Our data support an important role of phosphatases and pRB in IGF-I/mTOR-mediated cell survival. These studies provide new directions in optimizing anticancer efficacy of mTOR inhibitors when used in combination with DNA-damaging agents.Overexpression of tyrosine kinase receptors (TKRs) has long been appreciated to contribute to tumorigenesis and resistance to treatment. Receptor activation of insulin-like growth factor-I (IGF-I) 2 , insulin, PDGF (platelet-derived growth factor), and some ErbB receptors induce Akt activity via PI3K (phosphatidylinositol 3-kinase). The p110 catalytic subunit of PI3K stimulates the phosphorylation of PI(4,5)P 2 to PI(3,4,5)P 3 activating PDK1 (3-phosphoinositol-dependent kinase-1). PDK1 then enhances the activity of several kinases including Akt, PKC isoforms, SGK (serum and glucocorticoid-induced protein kinase), mTOR (mammalian target of rapamycin), and p70S6K. Loss of PTEN function in cancer cells leads to similar signaling events as activation of TKRs. Moreover, many of IGF-I-mediated functions in breast cancer cells, such as proliferation and survival, are thought to be conveyed through PI3K and Akt.Given the Akt potency as a survival mediator, much attention has focused on how it conveys this response. Increased protein translation occurs via Akt and its downstream mTOR. mTOR contains an Akt phosphorylation site, but current evidence indicates that Akt induces mTOR activity indirectly by phosphorylating tuberous sclerosis 2 (TSC2) (1)...