BURs as Targets for Anticancer Therapy

SIDD profiles (i.e. plots of G versus map position) are potent predictive tools for localizing "base-unpairing regions" (BURs), which are the hallmarks of S/MARs. BURs either consist of one dominant G(x) minimum exceeding a threshold extension of 200-300 bp (see inserts to Figs. 1 and 2) or of a succession of multiple, evenly spaced, but moderately destabilized "unpairing elements" (UEs). In the latter case, if spacing between restricted UEs exceeds «500 bp, individual elements lose their capacity to communicate with each other, concomitant with a loss of scaffold-binding activity (Bode et al. 2006). In any event, part or all of the BUR would become single stranded at sufficient superhelicity. The relevance of these features is underlined by the success of BUR-affinity chromatographic separation procedures, which served to isolate and identify a considerable number of S/MAR-associated proteins (review: Galande et al. 2002), among these ubiquitous representatives such as PARP-1, Ku autoantigen, HMG-I(Y), nucleolin, mutant p53 and cell-type specific factors such as SATB1 (T-cells) and BRIGHT (B-cells). The observation that prototype BUR binders are early targets for a caspase-mediated apoptotic cleavage is of particular present interest. Among these, PARP-1 and SAF-A/hnRNP-U are cleaved simultaneously by caspase 3, whereas SATB1 is uniquely cleaved by caspase 6.

BURs have recently emerged as general targets for cancer therapy, most likely since distinct BUR-binding proteins are up-regulated in carcinomas (Galande et al. 2002). The ability of small molecules to target regions with BUR potential may therefore provide a general approach for counteracting deleterious effects in these regions. First of all, the naturally occurring oligopeptide distamycin A prefers A/T-rich DNA sequences for binding to the minor groove of double-stranded DNA, whereby strand unpairing and the association of BUR-specific factors is prevented. This principle can be fortified by using distamycin derivatives to carry cytotoxic alkylating moieties into these regions. On the other hand, there are a number of small molecules available to target BUR-type sequences directly. This group comprises two DNA-specific antitumour agents of the cyclopropylpyrro-leindole family, bizelesin and adozelesin (Schwartz et al. 2003), which share a high specificity for AT-patches/BURs. Bizelesin has the highest potential for conferring regiospecific alkylation and is one of the most cytotoxic compounds ever identified: it produces a significantly higher number of lesions in several prominent AT-rich islands within cancer cells than in the bulk of genomic DNA. Strikingly, the SIDD-derived S/MAR potential (Sect. 3 and Winkelmann et al. 2006) of the affected regions correlates well with the total number of bizelesin sites (Woynarowski et al. 2001). Presently the design, chemical synthesis and in vitro testing of BUR-specific polyamides and other cyclopropylpyrrolein-dole-derived small molecules are underway in order to exploit their highly effective anti-tumor potential.

So far, we have more or less considered the global properties of BUR-binding proteins. If we add, as another criterion, the class of S/MARs they interact with, the following subdivision emerges:

• Constitutive contacts: mostly established by extended S/MARs at the borders of a chromatin domain by association with ubiquitous scaffold proteins, such as scaffold attachment factor A (SAF-A, otherwise known as hnRNP-U), the lam-ins and possibly NuMA/actin. These "bordering elements" are accompanied by DNAse hypersensitive sites in all cell types

° Constitutive matrix proteins bind according to the mass-binding phenomenon in a cooperative process. Series of binding sub-sites (i.e. of multiple UEs, which together form a BUR) may correspond to "AT patches," "AT hooks," "SAF boxes" or their equivalents (see Sec. 5.2 and Fackelmayer 2004). ° Constitutive S/MARs are platforms for the assembly of chromatin-modulat-ing proteins such as topo II and related enzymes, which at these sites initiate apoptotic degradation, histone-acetyltransferases/-deacetylases and methyl-binding proteins such as MeCP2/ARBP (Straetling and Yu 1999).

• Facultative S/MARs: more restricted, domain-internal sites, which in the extreme case consist of a single UE (insert to Fig. 1). These elements preferentially associate with tissue-specific proteins

° These proteins may be rare transcription factors, which are concentrated at the scaffold owing to a nuclear matrix-targeting sequence (NMTS; Zeng et al. 1997) or due to their association with a central player such as PARP, which exhibits many activity-dependent modes of binding (Sect. 5.1). ° Cell-type specific factors (such as SATB1 in T-cells, SATB2 and BRIGHT in B-cells) that establish a dynamic equilibrium within the domain (see Sect. 5.2 and the dynamic model depicted in Fig. 1).

The dynamic properties of nuclear matrix functions may explain why this scheme must not be considered a rigid one and why we have to expect factors commuting between both groups.

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