LEF-1 recognition of platinated GG sequences within double-stranded DNA. Influence of flanking bases.

The lymphoid enhancer-binding factor 1 (LEF-1) recognizes a double-stranded 9 base-pairs (bp) long motif in DNA which is significantly bent upon binding. This bend is centered at two destacked adenines whose geometry closely resembles that of two adjacent guanines crosslinked by the antitumor drug cisplatin. It has been proposed that cisplatin-GG crosslinks could hijack high mobility group (HMG) box containing transcription factors such as LEF-1. In order to examine such a possibility, we used electrophoretic mobility shift assays to determine the affinity of the HMG box of LEF-1 for a series of 25 oligonucleotides containing a central GG sequence, free or site-specifically modified by cisplatin. The binding affinity of the GG-platinated oligonucleotides was 3-6-fold higher than that determined for the corresponding unplatinated oligonucleotides, however, the binding to all cisplatin-modified oligonucleotides was at least 1 order of magnitude weaker than that to the 25 bp oligonucleotide containing the recognition 9 bp motif. The binding affinity was dependent on the nature of bases flanking the cisplatin-crosslinked G(*)G(*) dinucleotide, the AG(*)G(*)T sequence displaying the strongest affinity and CG(*)G(*)T showing the strongest binding enhancement upon platination. In contrast, modification of the AGGT sequence with the third-generation platinum antitumor drug oxaliplatin did not enhance the affinity significantly. These results suggest that the cisplatin-caused bending of DNA does produce a target for LEF-1 binding, however, the cisplatinated DNA does not appear to be a strong competitor for the LEF-1 recognition sequence.

Mechanism of the formation of DNA-protein cross-links by antitumor cisplatin.

DNA-protein cross-links are formed by various DNA-damaging agents including antitumor platinum drugs. The natures of these ternary DNA-Pt-protein complexes (DPCLs) can be inferred, yet much remains to be learned about their structures and mechanisms of formation. We investigated the origin of these DPCLs and their cellular processing on molecular level using gel electrophoresis shift assay. We show that in cell-free media cisplatin [cis-diamminedichloridoplatinum(II)] forms DPCLs more effectively than ineffective transplatin [trans-diamminedichloridoplatinum(II)]. Mechanisms of transformation of individual types of plain DNA adducts of the platinum complexes into the DPCLs in the presence of several DNA-binding proteins have been also investigated. The DPCLs are formed by the transformation of DNA monofunctional and intrastrand cross-links of cisplatin. In contrast, interstrand cross-links of cisplatin and monofunctional adducts of transplatin are stable in presence of the proteins. The DPCLs formed by cisplatin inhibit DNA polymerization or removal of these ternary lesions from DNA by nucleotide excision repair system more effectively than plain DNA intrastrand or monofunctional adducts. Thus, the bulky DNA-protein cross-links formed by cisplatin represent a more distinct and persisting structural motif recognized by the components of downstream cellular systems processing DNA damage considerably differently than the plain DNA adducts of this metallodrug.

Deoxyribonuclease I footprinting reveals different DNA binding modes of bifunctional platinum complexes.

Deoxyribonuclease I (DNase I) footprinting methodology was used to analyze oligodeoxyribonucleotide duplexes containing unique and single, site-specific adducts of trinuclear bifunctional platinum compound, [{trans-PtCl(NH3)2}2 mu-trans-Pt(NH3)2{H2N(CH2)6NH2}2]4+ (BBR3464) and the results were compared with DNase I footprints of some adducts of conventional mononuclear cis-diamminedichloroplatinum(II) (cisplatin). These examinations took into account the fact that the local conformation of the DNA at the sites of the contacts of DNase I with DNA phosphates, such as the minor groove width and depth, sequence-dependent flexibility and bendability of the double helix, are important determinants of sequence-dependent binding to and cutting of DNA by DNase I. It was shown that various conformational perturbations induced by platinum binding in the major groove translated into the minor groove, allowing their detection by DNase I probing. The results also demonstrate the very high sensitivity of DNase I to DNA conformational alterations induced by platinum complexes so that the platinum adducts which induce specific local conformational alterations in DNA are differently recognized by DNase I.

Azidothymidine and cisplatin increase p14ARF expression in OVCAR-3 ovarian cancer cell line.

p14(ARF) tumor suppressor protein regulates p53 by interfering with mdm2-p53 interaction. p14(ARF) is activated in response to oncogenic stimuli but little is known of the responses of endogenous p14(ARF) to different types of cellular stress or DNA damage. Azidothymidine (AZT) is being tested in several clinical trials as an enhancer of anticancer chemotherapy. However, the knowledge of the relationship between AZT and cellular pathways, e.g. p53 pathway, is very limited. In this study, we show that AZT, cisplatin (CDDP) and docetaxel (DTX) all induce unique molecular responses in OVCAR-3 ovarian carcinoma cells carrying a mutated p53, while in A2780, ovarian carcinoma and MCF-7 breast carcinoma cells with wild type p53, all of these drugs cause similar p53 responses. We found that endogenous p14(ARF) protein in OVCAR-3 cells is down-regulated by DTX but induced by AZT and a short CDDP pulse treatment. In HT-29 colon carcinoma cells with a mutated p53, all treatments down-regulated p14(ARF) protein. Both CDDP and AZT increased the expression of p14ARF mRNA in OVCAR-3 cells. Differences in cell death induced by these drugs did not explain the differences in protein and mRNA expressions. No increase in the level of either c-Myc or H-ras oncoproteins was seen in OVCAR-3 cells after AZT or CDDP-treatment. These results suggest that p14(ARF) can respond to DNA damage without oncogene activation in cell lines without functional p53.

Pb2+-induced toxicity is associated with p53-independent apoptosis and enhanced by glutamate in GT1-7 neurons.

Recent studies indicate that the glutamatergic neurotransmitter system is involved in neurotoxicity caused by inorganic lead (Pb2+). We studied the role of apoptosis in the effects induced by Pb2+ (0.01-100 microM) and glutamate (0.1 and 1 mM) in mouse hypothalamic GT1-7 neurons. Although glutamate alone had no effect on cell viability, it enhanced neuronal cell death induced by Pb2+ (1-100 microM) within 72 h. Glutamate alone neither induced caspase-3-like protease activity nor promoted internucleosomal DNA fragmentation, both biochemical hallmarks of apoptosis. However, concurrent exposure to Pb2+ (10 or 100 microM) and glutamate (1 mM) resulted in more prominent cleavage of the fluorogenic caspase-3 substrate (Ac-DEVD-AMC) than caused by the same Pb2+ concentrations alone at 24-72 h. The highest caspase-3-like protease activities were measured at 48 h. Internucleosomal DNA fragmentation caused by Pb2+ (10 or 100 microM) alone or together with glutamate (1 mM) was evident at 96 h, less clear at 72 h and absent at 48 h. Immunoblotting did not reveal any changes in p53 protein levels in cells exposed to Pb2+, glutamate or their combination at any studied time point (3-72 h). Our results suggest that Pb2+-induced neurotoxicity may partially be mediated through p53-independent apoptosis and enhanced by glutamate.