an-vitro-enzymatic-assay-to-measure-transcription-inhibition-gallium

An <em>In Vitro</em> Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H<sub>3</sub> 5,10,15-tris(pentafluorophenyl)corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

Beckman Research Institute of the City of Hope

Endogenous cellular oxidation of omega6-polyunsaturated fatty acids (PUFAs) has long been recognized as a contributing factor in the development of various cancers. The accrual of DNA damage as a result of reaction with free radical and electrophilic aldehyde products of lipid peroxidation is believed to be involved; however, the genotoxic and mutation-inducing potential of specific membrane PUFAs remains poorly defined. In the present study we have examined the ability of peroxidizing arachidonic acid (AA, 20:4omega6) to induce DNA strand breaks, base modifications, and mutations. The time-dependent induction of single-strand breaks and oxidative base modifications by AA in genomic DNA was quantified using denaturing glyoxal gel electrophoresis. Mutation spectra were determined in XP-G fibroblasts and a repair-proficient line corrected for this defect by c-DNA complementation (XP-G(+)). Mutation frequencies were elevated from approximately 5- to 30-fold over the background following reaction of DNA with AA for various times. The XPG gene product was found to be involved in the suppression of mutations after extended reaction of DNA with AA. Arachidonic acid-induced base substitutions were consistent with the presence of both oxidized and aldehyde base adducts in DNA. The frequency of multiple-base substitutions induced by AA was significantly reduced upon correction for the XPG defect (14% vs 2%, P = 0.0015). Evidence is also presented which suggests that the induced frequency of multiple mutations is lesion dependent. These results are compared to published data for mutations stimulated by alpha,beta-unsaturated aldehydes identified as products of lipid peroxidation.

DNA damage and mutations induced by arachidonic acid peroxidation.

Endogenous DNA damage induced by lipid peroxidation is believed to play a critical role in carcinogenesis. Lipid peroxidation generates free radical intermediates (primarily peroxyl radicals, ROO(*)) and electrophilic aldehydes as the principal genotoxicants. Although detailed information is available on the role of aldehyde base adducts in mutagenesis and carcinogenesis, the contribution of peroxyl radical mediated DNA base damage is less well understood. In the present study we have mapped oxidative base damage induced by peroxyl radicals in the supF tRNA gene and correlated this information with peroxidation-induced mutations in several human fibroblast cell lines. Nearly identical patterns of oxidative base damage were obtained from reaction of DNA with either peroxidizing arachidonic acid (20:4omega6) or peroxyl radicals generated by thermolysis of ABIP in the presence of oxygen. Oxidative base damage primarily occurred at G and C. Transversions at GC base pairs in the supF gene were the major base substitution detected in all cell lines. Peroxyl radical induced tandem mutations were also observed. Many mutation hot spots coincided with sites of mapped oxidative lesions, although in some cases hot spots occurred adjacent to the damaged base. Evidence is presented for the involvement of 8-oxodG in the oxidation of DNA by ROO(*). These results are used to interpret some key features of previously published mutation spectra induced by lipid peroxidation in human cells.

Peroxyl radical mediated oxidative DNA base damage: implications for lipid peroxidation induced mutagenesis.

Glycation of biopolymers by glucose-derived α-oxo-aldehydes such as methylglyoxal (MG) is believed to play a major role in the complex pathologies associated with diabetes and metabolic disease. In contrast to the extensive literature detailing the formation and physiological consequences of protein glycation, there is little information about the corresponding phenomenon for DNA. To assess the potential contribution of DNA glycation to genetic instability, we prepared shuttle vectors containing defined levels of the DNA glycation adduct N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) and transfected them into isogenic human fibroblasts that differed solely in the capacity to conduct nucleotide excision repair (NER). In the NER-compromised fibroblasts, the induced mutation frequencies increased up to 18-fold relative to background over a range of ∼10-1400 CEdG adducts/10(5) dG, whereas the same substrates transfected into NER-competent cells induced a response that was 5-fold over background at the highest adduct density. The positive linear correlation (R(2) = 0.998) of mutation frequency with increasing CEdG level in NER-defective cells suggested that NER was the primary if not exclusive mechanism for repair of this adduct in human fibroblasts. Consistent with predictions from biochemical studies using CEdG-substituted oligonucleotides, guanine transversions were the predominant mutation resulting from replication of MG-modified plasmids. At high CEdG levels, significant increases in the number of AT → GC transitions were observed exclusively in NER-competent cells (P < 0.0001). This suggested the involvement of an NER-dependent mutagenic process in response to critical levels of DNA damage, possibly mediated by error-prone Y-family polymerases.

Mutagenesis and repair induced by the DNA advanced glycation end product N2-1-(carboxyethyl)-2'-deoxyguanosine in human cells.

A gallium(III)-substituted amphiphilic corrole noncovalently associated with a targeting protein was previously found by us to confer promising cytotoxic and antitumor activities against a breast cancer cell line and a mouse xenograft breast cancer model. To further explore potential anticancer applications, the cytostatic and cytotoxic properties of six nontargeted metallocorroles were evaluated against seven human cancer cell lines. Results indicated that toxicity toward human cancer cells depended on the metal ion as well as corrole functional group substitution. Ga(III)-substituted metallocorrole 1-Ga inhibited proliferation of breast (MDA-MB-231), melanoma (SK-MEL-28), and ovarian (OVCAR-3) cancer cells primarily by arrest of DNA replication, whereas 2-Mn displayed both cytostatic and cytotoxic properties. Confocal microscopy revealed extensive uptake of 1-Ga into the cytoplasm of melanoma and ovarian cancer cells, while prostate cancer cells (DU-145) displayed extensive nuclear localization. The localization of 1-Ga to the nucleus in DU-145 cells was exploited to achieve a 3-fold enhancement in the IC(50) of doxorubicin upon coadministration. Time-course studies showed that over 90% of melanoma cells incubated with 30 μM 1-Ga internalized metallocorrole after 15 min. Cellular uptake of 1-Ga and 1-Al was fastest and most efficient in melanoma, followed by prostate and ovarian cancer cells. Cell cycle analyses revealed that bis-sulfonated corroles containing Al(III), Ga(III), and Mn(III) induced late M phase arrest in several different cancer cell lines, a feature that could be developed for potential therapeutic benefit.

Differential cytostatic and cytotoxic action of Metallocorroles against human cancer cells: potential platforms for anticancer drug development.

We have synthesized and characterized a water-soluble gold(III) corrole (1-Au) that is highly toxic to cisplatin-resistant cancer cells. Relative to its 1-Ga analogue, axial ligands bind only weakly to 1-Au, which likely accounts for its lower affinity for human serum albumin (HSA). We suggest that the cytotoxicity of 1-Au may be related to this lower HSA affinity.

Punnajit Lim

Department of Molecular Medicine

Beckman Research Institute of the City of Hope

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H₃ 5,10,15-tris(pentafluorophenyl)corroles

Punnajit Lim

Department of Molecular Medicine

Beckman Research Institute of the City of Hope

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H₃ 5,10,15-tris(pentafluorophenyl)corroles