Intrastrand DNA crosslinking

The trans isomer of a dinuclear platinum complex can form intrastrand DNA crosslinks, which do not distort the molecule s shape but disrupt its function. Here the two chloride ligands of the complex ion are replaced by the N atoms in the five-membered ring of the guanines on two different strands. The rodlike structure represents the bases held together by hydrogen bonds. 

The DNA crosslinks formed by cis-DDP interfere with DNA replication and eventually cause cell death. The activity of DNA polymerases is impaired by DNA-platinum adducts (54-56). Furthermore, although the cell is capable of repairing DNA-platinum adducts (50,57-59), the intrastrand DNA crosslinks produced by cis-DDP do not cause large distortions of the double helix and so may not be easily recognized and repaired (51,60,61). Despite cellular repair mechanisms, a sufficient concentration of cis-DDP will inhibit replication and prevent cell division (20-23,62-64). In some cells, cis-DDP appears to also impair the cell s ability to transcribe genes needed for mitosis (63,64), and even cells which manage to divide initially after cis-DDP treatment often do not display long-term survival (22,23). 

Fig. 7. Schematic representation of cisplatin bonding to DNA. (1) monofunctional binding (X = Cr, OH", OH2) (2) interstrand crosslinking (3) protein-DNA crosslinking (4) intrastrand crosslinking between adjacent guanines (5) intrastrand crosslinking between two guanines separated by a third base (6) intrastrand crosslinking at a-AG-unit...

Very few structural studies have been concentrated on models of ApG adducts of cisplatin. Conformational analysis of two rotameric forms of the complex m-[Pt(NH3)2(9-MeA-A(7))(9-EtGH-A(7))]2+ has recently been described [40], One of the forms, crystallized as a PI 6 salt, can be characterized as a right-handed helicoidal model for the intrastrand ApG crosslink in double-stranded DNA. The bases in this compound assume a head-to-head orientation (Fig. 6) with the interbase dihedral angles of 81.8° and 87.5°. There are two independent complex cations in the unit cell. The left-... 

Figure 2.12 DNA crosslinking. The deleterious properties of nitrogen mustards are explained through the illustrated interstrand linkage mechanism that makes DNA impossible to duplicate or transcribe. Intrastrand crosslinking is the basis of action for anti-cancer drugs such as c/s-platin and carbo-platin. This is intended to prevent DNA duplication and hence cancer cell division. DNA crosslinking to proteins (such as histones) uses a non-covalent DNA intercalator with two azide functional groups. Both azides are activated for covalent coupling under photo-chemical conditions so that DNA subsequently becomes covalently linked to protein.

The different possible adducts formed between mitomycin C and DNA have been isolated by degradation of DNA after in vitro alkylation/crosslinking reactions and structurally characterized. Monoadduct 21 (Scheme 11.3), derived from alkylation at C-l only [53], and monoadducts 22 [54] and 23 [55, 56] (derived from C-10 alkylation by 16 at N-7 or N-2 of guanine, respectively) have been isolated, together with bisadducts 24 [57] and 25 [58], derived from interstrand and intrastrand crosslinks, respectively, and adduct 26 [59], formed by addition of a molecule of water to C-10 instead of the second guanine. All of these adducts have also been isolated from DNA after in vivo crosslinking [60, 61]. 

Binding of cisplatin to the neighbouring bases in the DNA disrupts the orderly stacking of the purine bases when it forms a 1,2-intrastrand crosslink, it bends the DNA helix by some 34° towards the major groove and unwinds the helix by 13°. These cross-links are believed to block DNA replication. 

While the critical role of intrastrand 1,2-crosslinks in the mechanism of action of the anti-tumor drug cis- [PtCl2(NH3)2] is well established, much less is known about the few frans-analogs that exhibit similar efficacy. A series of 1,3-adducts has been characterized with 3-mers generally involving purine N7 coordination (32-34), and either monofunctional or interstrand adducts are formed with duplex DNA (35). However, one of the most active compounds, frans-[PtCl2 ( )-HN = C(OMe)Me 2], (2), has been shown to form a 1,2-adduct with 2-mer ribonucleotide sequence r(AG) (36). Though the formation of the... 

If a DNA adduct involves the nitrogen or oxygen atoms involved in base-pairing, and the adducted DNA is not repaired, base substitution can result. Adducts can be small, such as the simple addition of methyl or ethyl groups, or they can be very bulky, owing to reaction with multiringed structures. The most vulnerable base is guanine, which can form adducts at several of its atoms (e.g., N7, C8, O6 and exocyclic N2) (Venitt and Parry, 1984). Adducts can form links between adjacent bases on the same strand (intrastrand cross-links) and can form interstrand crosslinks between each strand of double-stranded DNA. 

Perez C, Leng M, Malinge JM. Rearrangement of interstrand crosslinks into intrastrand crosslinks in cis-diamminedichloroplatinum(II)-modified DNA. Nucleic Acids Res 1997 25 896-903. 

Data taken from N. Poklar, D. S. Pilch, S. J. Lippard, E. A. Redding, S. U. Dunham, and K. J. Breslauer, Influence of Cisplatin Intrastrand Crosslinking on the Conformation, Thermal Stability and Energetics of a 20-mer DNA Duplex, Proc. Natl. Acad. Sci. USA, 93, 7606-76U (1996). 

The formation of intrastrand crosslinks on GG and AG base sequences in DNA could be deduced from the presence of the adducts ds-Pt(NH3)2(d(pGG)) and cis-Pt(NH3)2(d(pAG)) in digests of platinated DNA. However, no indications for the presence of GA adducts could be found, and therefore crosslinks with a GA sequence are unlikely. 

Recently, it could be established that in living cells the same types of Pt-adducts are formed as in DNA treated with cisplatin in vitro120,121. Also the strong preference of cisplatin to form intrastrand crosslinks on GG base sequences is found upon interaction with DNA in vivo120. ... 

The question which of the induced Pt-DNA adducts is (are) responsible for the antitumor activity is still unanswered. Many investigators have tried to correlate interstrand DNA and DNA-protein crosslink formation with the cytotoxic action of cisplatin. However, conflicting results were obtained18,50 Until now a biological role inside the cell has been indicated for only one type of Pt-DNA adduct. Brouwer et al.48 showed that cisplatin can induce base-pair substitutions in E. coli bacteria at GAG and GCG base sequences. This strongly suggests that the intrastrand crosslink of cisplatin on GBG is responsible for this effect. 

Patients, suffering apparently from the same type of tumor, often respond differently to Pt-chemotherapy. Therefore, a research program has been started on the Pt-adduct formation and repair in the DNA of the nucleated peripheral blood cells of these patients. Figure 22 shows the number of intrastrand crosslinks on pGG sequences per 109... 

Fig. 22. Numbers of intrastrand crosslinks to GG-units in DNA isolated from white blood cells of two different patients at various time intervals after the onset of the cisplatin infusion (indicated by arrows).

Cyclophosphamide (6.31) is a nitrogen mustard used for cancer treatment (Scheme 6.9). In the body, cyclophosphamide is oxidized to aminal 6.32. Compound 6.32 opens and loses acrolein to form phosphoramide mustard (6.33). Structure 6.33 is a strong bis-electrophile and reacts readily with nucleophiles. In DNA, the nucleophile tends to be N7 of guanine, which is oriented outward into the major groove (Figure 6.6). By reacting twice, 6.33 crosslinks DNA either within the same strand (intrastrand) or across the double helix (interstrand).16... 

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