Electron-Mediated Aminyl and Iminyl Radicals from C5 Azido-Modified Pyrimidine Nucleosides Augment Radiation Damage to Cancer Cells.

Two classes of azido-modified pyrimidine nucleosides were synthesized as potential radiosensitizers; one class is 5-azidomethyl-2'-deoxyuridine (AmdU) and cytidine (AmdC), while the second class is 5-(1-azidovinyl)-2'-deoxyuridine (AvdU) and cytidine (AvdC). The addition of radiation-produced electrons to C5-azido nucleosides leads to the formation of π-aminyl radicals followed by facile conversion to σ-iminyl radicals either via a bimolecular reaction involving intermediate α-azidoalkyl radicals in AmdU/AmdC or by tautomerization in AvdU/AvdC. AmdU demonstrates effective radiosensitization in EMT6 tumor cells.

Prehydrated One-Electron Attachment to Azido-Modified Pentofuranoses: Aminyl Radical Formation, Rapid H-Atom Transfer, and Subsequent Ring Opening.

Methyl 2-azido-2-deoxy-α-d-lyxofuranoside (1a) and methyl 2-azido-2-deoxy-ß-d-ribofuranoside (2) were prepared from d-xylose or d-arabinose, respectively. Employing ESR and DFT/B3LYP/6-31G* calculations, we investigated (i) aminyl radical (RNH·) formation and (ii) reaction pathways of RNH·. Prehydrated electron attachment to 1a and 2 at 77 K produced transient azide anion radical (RN3·-) which reacts via rapid N2 loss at 77 K, forming nitrene anion radical (RN·-). Rapid protonation of RN·- at 77 K formed RNH· and -OH. 15N-labeled-1a confirmed this mechanism. Investigations employing in-house synthesized site-specifically deuterated derivatives of 1a (e.g., CH3 (1b), C4 (1c), and C5 (1d)) established that (a) a facile intramolecular H atom transfer from C5 to RNH· generated C5· and RNH2. C5· formation had a small deuterium kinetic isotope effect suggesting that this reaction does not occur via direct H atom abstraction. (b) Subsequently, C5· underwent a facile unimolecular conversion to ring-opened C4·. Identification of ring-opened C4· intermediate confirms the mechanism of C5'· mediated unaltered base release associated with DNA-strand break. However, for 2, ESR studies established thermally activated intermolecular H atom abstraction by RNH· from the methyl group at C1. Thus, sugar ring configuration strongly influences the site and pathway of RNH· mediated reactions in pentofuranoses.

UV-Induced Adenine Radicals Induced in DNA A-Tracts: Spectral and Dynamical Characterization.

Adenyl radicals generated in DNA single and double strands, (dA)20 and (dA)20·(dT)20, by one- and two-photon ionization by 266 nm laser pulses decay at 600 nm with half-times of 1.0 ± 0.1 and 4 ± 1 ms, respectively. Though ionization initially forms the cation radical, the radicals detected for (dA)20 are quantitatively identified as N6-deprotonated adenyl radicals by their absorption spectrum, which is computed quantum mechanically employing TD-DFT. Theoretical calculations show that deprotonation of the cation radical induces only weak spectral changes, in line with the spectra of the adenyl radical cation and the deprotonated radical trapped in low temperature glasses.

Cell cycle and apoptosis regulatory protein (CARP)-1 is a novel, adriamycin-inducible, diffuse large B-cell lymphoma (DLBL) growth suppressor.

UNLABELLED: Diffuse large B-cell lymphoma (DLCL) accounts for 30-40% of adult non-Hodgkin's Lymphoma (NHL). Current anti-NHL therapies often target cellular growth suppression pathways and include R-CHOP (cyclophosphamide, adriamycin, vincristine, and prednisone plus monoclonal anti-CD20 antibody rituximab). However, since many patients relapse, resistant cells to these therapies remain a significant problem and necessitate development of new intervention strategies. Cell cycle and apoptosis regulatory protein (CARP)-1 functions in a biphasic manner to regulate growth factor as well as chemotherapy (adriamycin, etoposide, or iressa)-dependent signaling. PURPOSE: To determine whether CARP-1 is a novel suppressor of lymphoma growth. METHODS: Flow cytometric analyses coupled with Western immunoblotting, cell growth, apoptosis, and immunocytochemistry methodologies were utilized to determine CARP-1-dependent lymphoma growth inhibition in vitro and in vivo. RESULTS: CARP-1 expression correlated with activated caspase-3 and inversely correlated with activated Akt in DLCL. Exposure to adriamycin stimulated CARP-1 expression and inhibited growth of Raji cells, but not CHOP-resistant WSU-DLCL2 cells. Expression of wild-type CARP-1 or its apoptosis-inducing mutants inhibited growth of Raji as well as CHOP-resistant WSU-DLCL2 cells, in part by activating caspase-9 and apoptosis. Since CARP-1 harbors multiple, apoptosis-promoting subdomains, we investigated whether epigenetic compensation of CARP-1 function by intracellular delivery of trans-activator of transcription (TAT) domain-tagged CARP-1 peptide(s) will inhibit lymphoma growth. Treatments with TAT-tagged CARP-1 peptides suppressed growth of the Raji and WSU-DLCL2 cells by stimulating apoptosis. TAT-CARP-1 (1-198) as well as (896-1150) peptides also suppressed growth of WSU-DLCL2 cell-derived tumor xenografts in SCID mice, while administration of TAT-CARP-1 (1-198) also inhibited growth of WSU-FSCCL cell-derived ascites and prolonged host survival. CONCLUSION: CARP-1 is a suppressor of NHL growth and could be exploited for targeting the resistant DLCL.