D

FIGURE 7.7 Fluorescent images of MCF7 cells exposed to the thioredoxin-targeted device. The cells were observed at 400 x under fluorescent light with a FITC filter at the City of Hope Cytogenetics Core Laboratory using high-quality fluorescent photomicroscopes and computerized imaging system. MCF7 cells exposed to the Y-junction linked to M-EcoRII-Trx (A) had a high overall fluorescence and localized fluorescence around the cell surface while cells exposed to Y-junction linked to M-EcoRII (B) and Y-junction DNA (C) had a similar level of fluorescence to cells exposed to PBS (D).

FIGURE 7.7 Fluorescent images of MCF7 cells exposed to the thioredoxin-targeted device. The cells were observed at 400 x under fluorescent light with a FITC filter at the City of Hope Cytogenetics Core Laboratory using high-quality fluorescent photomicroscopes and computerized imaging system. MCF7 cells exposed to the Y-junction linked to M-EcoRII-Trx (A) had a high overall fluorescence and localized fluorescence around the cell surface while cells exposed to Y-junction linked to M-EcoRII (B) and Y-junction DNA (C) had a similar level of fluorescence to cells exposed to PBS (D).

7.3.4.1 Biology of DNA Methylation

Selective gene activation and repression in normal cells is not fully understood, however these processes appear to center on promoter activation or repression mediated by protein-DNA interaction combinatorics. These patterns are tissue specific and stably maintained in a given cell lineage. Once a transcription state is established, it appears to be stably maintained by a self-reinforcing network of protein and DNA modifications involving histone methylation, histone acetylation, and cytosine methylation in DNA [42].

In general, gene expression patterns are randomized during tumorigenesis by genetic damage and natural selection during tumor progression. Hallmarks of this process are the establishment of patterns of ectopic gene expression and ectopic gene silencing that adapt them for their role as invasive tumors. This has led to the development of drugs directed at the disruption of stable patterns of gene expression, in the hope that selective delivery to tumor cells will inhibit growth or induce cell death [43]. Among the drugs that have been discovered are a variety of histone deacetylase inhibitors and DNA (cytosine-5) methyltransferase inhibitors [44] that tend to act synergistically [45,46] to disrupt these gene silencing systems. These drugs can also be effective alone. In principle, DNA methyltransferases can be inhibited by either the direct interaction of the inhibitor with the enzyme active site or its protein targeting signals, or by selectively interfering with methyltransferase synthesis. The DNA Y-junction can be used to target DNA methyltransferase traps (noncompetitive inhibitors of the enzyme) or DNAzymes targeting the messenger RNA for the methyltransferase itself to the nucleus.

7.3.4.2 First Generation DNA Methyltransferase Traps

Since the target of methylation is deoxycytidine, the first inhibitors of MT activity to be developed were analogs of this nucleoside [47,48]. The compounds in this group are structurally based upon 5-azacytidine. These drugs are phosphorylated in cells and incorporated into RNA and DNA, with the

TABLE 7.1 Electronic Structure Classification of DNA Methyltransferase Targets

Productive Target'

Nonproductive Target

Trapping Target

Attacked Target Intermediate

Target

Intermediate Attacked Target

Intermediate

Cyt+

Cyt Enol 4-ThioU

2-Pyrimidinone+

5-FCyt+

5-AzaCyt+

2-Pyrimidinone Enol 5-FCyt Enol 5-AzaC Enol

5-BrU

0 0

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