A Platinum(IV) Anticancer Prodrug Targeting Nucleotide Excision Repair To Overcome Cisplatin Resistance

DNA damage response plays a key role not only in maintaining genome integrity but also in mediating the antitumor efficacy of DNA‐damaging antineoplastic drugs. Herein, we report the rational design and evaluation of a Pt^IV anticancer prodrug inhibiting nucleotide excision repair (NER), one of the most pivotal processes after the formation of cisplatin‐induced DNA damage that deactivates the drug and leads to drug resistance in the clinic. This dual‐action prodrug enters cells efficiently and causes DNA damage while simultaneously inhibiting NER to promote apoptotic response. The prodrug is strongly active against the proliferation of cisplatin‐resistant human cancer cells with an up to 88‐fold increase in growth inhibition compared with cisplatin, and the prodrug is much more active than a mixture of cisplatin and an NER inhibitor. Our study highlights the importance of targeting downstream pathways after the formation of Pt‐induced DNA damage as a novel strategy to conquer cisplatin resistance.     

DNA Ligases I and III Support Nucleotide Excision Repair in DT40 Cells with Similar Efficiency

In eukaryotic cells helix‐distorting DNA lesions like cyclobutane pyrimidine dimers (CPDs) and 6–4 pyrimidine‐pyrimidone photoproducts (6–4 PPs) are efficiently removed by nucleotide excision repair (NER). NER is a multistep process where in the end, subsequent to replication over the gap, the remaining nick is sealed by a DNA ligase. Lig1 has been implicated as the major DNA ligase in NER. Recently, Lig3 has been implicated as a component of a NER subpathway that operates in dividing cells, but which becomes particularly important in nondividing cells. Here, we use DT40 cells and powerful gene targeting approaches for generating DNA ligase mutants to examine the involvement and contribution of Lig1 and Lig3 in NER using cell survival measured by colony formation, and repair kinetics of CPD by immunofluorescence microscopy and immuno‐slot‐blotting. Our results demonstrate an impressive and previously undocumented potential of Lig3 to substitute for Lig1 in removing helix‐distorting DNA lesions by NER in proliferating cells. We show for the first time in a clean genetic background a functional redundancy in NER between Lig1 and Lig3, which appears to be cell cycle independent and which is likely to contribute to the stability of vertebrate genomes.     

Detection of the Excised,Damage‐containing Oligonucleotide Products of Nucleotide Excision Repair in Human Cells

The human nucleotide excision repair system targets a wide variety of DNA adducts for removal from DNA, including photoproducts induced by UV wavelengths of sunlight. A key feature of nucleotide excision repair is its dual incision mechanism, which results in generation of a small, damage‐containing oligonucleotide approximately 24 to 32 nt in length. Detection of these excised oligonucleotides using cell‐free extracts and purified proteins with defined DNA substrates has provided a robust biochemical assay for excision repair activity in vitro. However, the relevance of a number of in vitro findings to excision repair in living cells in vivo has remained unresolved. Over the past few years, novel methods for detecting and isolating the excised oligonucleotide products of repair in vivo have therefore been developed. Here we provide a basic outline of a sensitive and versatile in vivo excision assay and discuss how the assay both confirms previous in vitro findings and offers a number of advantages over existing cell‐based DNA repair assays. Thus, the in vivo excision assay offers a powerful tool for readily monitoring the repair of DNA lesions induced by a large number of environmental carcinogens and anticancer compounds.     

An Experimental Population Study of Nucleotide Excision Repair as a Risk Factor for UVB‐induced Melanoma

Nucleotide excision repair (NER) is the primary defense against the DNA damage implicit in skin cancer formation and is negatively affected by chronic exposure to UVB radiation. However, in situ and in vitro studies consistently yield equivocal results when addressing individual DNA repair capacity and melanoma susceptibility. The primary objective of this study was to determine if individual global NER capacity is a risk factor for melanoma formation in a prominent UVB‐inducible melanoma model, hybrid Xiphophorus fishes. After neonatal UVB irradiation, adult tumor‐bearing and tumor‐free fish were given a challenge UVB dose and (6–4) photoproduct repair was quantified in individual fish at 24 h using radioimmunoassay. Despite considerable inter‐individual variation in repair capacity, ranging from 13% to 91%, we found no difference in mean NER capacity between fish with and without melanomas, thus detaching global NER from melanomagenesis. Furthermore, despite epidemiological data indicating that sex and age are important risk factors underlying melanoma susceptibility, we found no difference in mean NER rates among the sexes or as a function of age. We conclude with a discussion of the apparent paradox of how inter‐individual variation in NER is not a risk factor given the clear evidence that DNA damage underlies melanoma susceptibility.     

Bringing It All Together: Coupling Excision Repair to the DNA Damage Checkpoint

Nucleotide excision repair and the ATR‐mediated DNA damage checkpoint are two critical cellular responses to the genotoxic stress induced by ultraviolet (UV) light and are important for cancer prevention. In vivo genetic data indicate that these global responses are coupled. Aziz Sancar et al. developed an in vitro coupled repair‐checkpoint system to analyze the basic steps of these DNA damage stress responses in a biochemically defined system. The minimum set of factors essential for repair‐checkpoint coupling include damaged DNA, the excision repair factors (XPA, XPC, XPF‐ERCC1, XPG, TFIIH, RPA), the 5′‐3′ exonuclease EXO1, and the damage checkpoint proteins ATR‐ATRIP and TopBP1. This coupled repair‐checkpoint system was used to demonstrate that the ~30 nucleotide single‐stranded DNA (ssDNA) gap generated by nucleotide excision repair is enlarged by EXO1 and bound by RPA to generate the signal that activates ATR.     

Ribose‐Protonated DNA Base Excision Repair: A Combined Theoretical and Experimental Study

Living organisms protect the genome against external influences by recognizing and repairing damaged DNA. A common source of gene mutation is the oxidized guanine, which undergoes base excision repair through cleavage of the glycosidic bond between the ribose and the nucleobase of the lesion. We unravel the repair mechanism utilized by bacterial glycosylase, MutM, using quantum‐chemical calculations involving more than 1000 atoms of the catalytic site. In contrast to the base‐protonated pathway currently favored in the literature, we show that the initial protonation of the lesion’s ribose paves the way for an almost barrier‐free glycosidic cleavage. The combination of theoretical and experimental data provides further insight into the selectivity and discrimination of MutM’s binding site toward various substrates.     

f-block MOFs: A Pathway to Heterometallic Transuranics

A novel series of heterometallic f-block-frameworks including the first examples of transuranic heterometallic ²³⁸U/²³⁹Pu-metal–organic frameworks (MOFs) and a novel monometallic ²³⁹Pu-analog are reported. In combination with theoretical calculations, we probed the kinetics and thermodynamics of heterometallic actinide(An)-MOF formation and reported the first value of a U-to-Th transmetallation rate. We concluded that formation of uranyl species could be a driving force for solid-state metathesis. Density of states near the Fermi edge, enthalpy of formation, band gap, proton affinity, and thermal/chemical stability were probed as a function of metal ratios. Furthermore, we achieved 97 % of the theoretical maximum capacity for An-integration. These studies shed light on fundamental aspects of actinide chemistry and also foreshadow avenues for the development of emerging classes of An-containing materials, including radioisotope thermoelectric generators or metalloradiopharmaceuticals.     

Selenium‐Mediated Dehalogenation of Halogenated Nucleosides and its Relevance to the DNA Repair Pathway

Halogenated nucleosides can be incorporated into the newly synthesized DNA of replicating cells and therefore are commonly used in the detection of proliferating cells in living tissues. Dehalogenation of these modified nucleosides is one of the key pathways involved in DNA repair mediated by the uracil‐DNA glycosylase. Herein, we report the first example of a selenium‐mediated dehalogenation of halogenated nucleosides. We also show that the mechanism for the debromination is remarkably different from that of deiodination and that the presence of a ribose or deoxyribose moiety in the nucleosides facilitates the deiodination. The results described herein should help in understanding the metabolism of halogenated nucleosides in DNA and RNA.     

Intramolecular Baylis-Hillman Reaction: A Pathway to Substituted Coumarins

The acrylate ester of salicylaldehyde, in the presence of DABCO, affords a crystalline coumarin salt. Formation of this derivative confirms a vital intermediate in the mechanism of the reaction. Salicylaldehyde, suitably protected, reacts with methyl acrylate to afford a novel coumarin not unlike the vasodilator chromonar.     

Mutants of Arabidopsis as Tools to Understand the Regulation of Phenylpropanoid Pathway and UVB Protection Mechanisms

Abstract— Plants accumulate certain phenylpropanoid compounds in the vacuoles of their epidermal and subepidermal cell layers thereby protecting the underlying tissue against UVB-induced damage. However, a number of mutants of Arabidopsis thaliana are known that fail to synthesize these protective pigments, thereby allowing harmful UVB radiation to penetrate into their dermal layers. Study of several of these nonlethal mutants, defective in various aspects of flavonoid and lignin biosynthesis, has led to a better understanding of the coordinate regulation and expression of important genes as well as of mechanisms involved in plant defense against UVB radiation. The characteristics of the various phenylpropanoid mutants of Arabidopsis, viz. flavonoid mutants (banyuls [ baity , increased chalcone synthase expression 1 [ icx1 ]; transparent testa [ tt ] and ultraviolet sensitive [ uvs ]) and hydroxycinnamic acid ester mutants (ferulic acid hydroxylase 1 [ fahl ] and sinapoylglucose accumulator 1 [ sng1 ]) are discussed in detail. We have briefly touched upon, wherever relevant, the unique aspects in other plant species too.     

Proteome Analysis: A Pathway to the Functional Analysis of Proteins

The protein equivalent of genomes , proteomes are quantitative protein patterns of an organism, a cell, or a body fluid, and are determined by the development state and environmental parameters. Changes in protein expression and their consequences can be investigated at the molecular level and provide biologically relevant information not obtainable from experiments with mRNA.