Characterization of the unimolecular water elimination reaction from 1-propanol, 3,3,3-propan-1-ol-d3, 3,3,3-trifluoropropan-1-ol, and 3-chloropropan-1-ol.

The unimolecular reactions of 1-propanol, 3,3,3-propan-1-ol-d3, 3,3,3-trifluoropropan-1-ol, and 3-chloropropan-1-ol have been studied by the chemical activation technique. The recombination of CH3, CD3, CF3, and CH2Cl radicals with CH2CH2OH radicals at room temperature was used to generate vibrationally excited CH3CH2CH2OH, CD3CH2CH2OH, CF3CH2CH2OH, and CH2ClCH2CH2OH molecules. The principal unimolecular reaction for propanol and propanol-d3 with 90 kcal mol(-1) of vibrational energy is 1,2-H2O elimination with rate constants of 3.4 x 10(5) and 1.4 x 10(5) s(-1), respectively. For CH2ClCH2CH2OH also with 90 kcal mol(-1) of energy, 2,3-HCl elimination with a rate constant of 3.0 x 10(7) s(-1) is more important than 1,2-H2O elimination; the branching fractions are 0.95 and 0.05. For CF3CH2CH2OH with an energy of 102 kcal mol(-1), 1,2-H2O elimination has a rate constant of 7.9 x 10(5) and 2,3-HF elimination has a rate constant of 2.6 x 10(5) s(-1). Density functional theory was used to obtain models for the molecules and their transition states. The frequencies and moments of inertia from these models were used to calculate RRKM rate constants, which were used to assign threshold energies by comparing calculated and experimental rate constants. This comparison gives the threshold energy for H2O elimination from 1-propanol as 64 kcal mol(-1). The threshold energies for 1,2-H2O and 2,3-HCl elimination from CH2ClCH2CH2OH were 59 and 54 kcal mol(-1), respectively. The threshold energies for H2O and HF elimination from CF3CH2CH2OH are 62 and 70 kcal mol(-1), respectively. The structures of the transition states for elimination of HF, HCl, and H2O are compared.

Stress and IGF-I differentially control cell fate through mammalian target of rapamycin (mTOR) and retinoblastoma protein (pRB).

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 eIF2alpha, 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.

PKCdelta and mTOR interact to regulate stress and IGF-I induced IRS-1 Ser312 phosphorylation in breast cancer cells.

IRS-1 (Insulin Receptor Substrate-1) is an adaptor protein important for insulin and IGF-I receptor (Insulin-like Growth Factor-IR) transduction to downstream targets. One mechanism recently identified to downregulate IGF-I or insulin receptor signaling in diabetic models is IRS-1 Ser(312) phosphorylation. To date, the importance of this residue in cancer is unknown. This paper identifies mechanisms leading to Ser(312) regulation in MCF-7 breast cancer cells. Whereas IGF-I phosphorylation of IRS(312) is PI (phosphatidylinositol) 3-kinase dependent, anisomycin stress treatment requires JNK activation to induce phosphorylation of IRS(312). We show that both IGF-I and anisomycin stress treatment converge downstream onto mTOR (Mammalian Target of Rapamycin) and PKCdelta (Protein Kinase C-delta) to induce IRS-1 Ser(312) phosphorylation. mTOR associates with IRS-1 and is primarily required for Ser(312) phosphorylation in response to stress or IGF-I treatment. PKCdelta binds to mTOR and its activity is also important for stress or IGF-I mediated Ser(312) phosphorylation. Thus, mTOR and PKCdelta convey diverse signals to regulate IRS-1 function.

UV-induced apoptosis is mediated independent of caspase-9 in MCF-7 cells: a model for cytochrome c resistance.

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 down-stream 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.

Promoter scanning for transcription inhibition with DNA-binding polyamides.

When targeted to sequences adjacent to a TATA element, pyrrole-imidazole (Py-Im) polyamides inhibit the DNA binding activity of TATA box binding protein (TBP) and basal transcription by RNA polymerase II. In the present study, we scanned the human immunodeficiency virus type 1 promoter for polyamide inhibition of TBP binding and transcription using a series of DNA constructs in which a polyamide binding site was placed at various distances from the TATA box. Polyamide interference with either TBP-DNA or TFIID-TFIIA-DNA contacts both upstream and downstream of the TATA element resulted in inhibition of transcription. Our results define important protein-DNA interactions outside of the TATA element and suggest that transcription inhibition of selected gene promoters can be achieved with polyamides that target unique sequences within these promoters at a distance from the TATA element. Our studies also demonstrate the utility of the Py-Im polyamides for discovery of functionally important protein-DNA contacts involved in transcription.