Christopher C. Benz

Polyamine inhibition of estrogen receptor (ER) DNA-binding and ligand-binding functions

Polyamines are known to inhibit sequence specific DNA-binding activity of several zinc-finger transcription factors, including estrogen receptor (ER) binding to its cognate estrogen response element (ERE). The mechanism accounting for this disruption of protein-DNA interaction is unknown, although polyamine induction of DNA conformational changes has been suggested. To determine if polyamines can directly impair ER action, we compared the effects of putrescine (Putr), spermidine (Spd), and spermine (Spm) on ER DNA-binding (ER-ERE complex formation), ER ligand-binding (estradiol), ER structure (circular dichroism and sucrose gradient sedimentation), and the capacity of ER to transactivate an ERE-tk-CAT reporter in transient transfection assays. Polyamine concentrations causing 50% inhibition of ER-ERE formation (IC50 values) were found to be 1 mM for Putr, 4 mM for Spd, and 3 mM for Spm. This loss of ER DNA-binding was associated with a direct and irreversible effect on the ER DNA-binding domain (ER-DBD). Additionally, polyamines were observed to inhibit ER ligand-binding with IC50 values of 10 mM for Putr, 2 mM for Spd, and < 0.1 mM for Spm; and this correlated with a measureable change in higher-order ER structure (5S to 3.5S sedimentation) and inhibition of intracellular ER transactivation. These findings suggest that in ER-positive human breast tumors with increased polyamine (especially Spm) content, ER structure and function may be directly altered by tight-ion polyamine complexing that results in loss of ER-mediated gene regulation.

Genomic aberrations in normal tissue adjacent to HER2-amplified breast cancers: field cancerization or contaminating tumor cells?

Field cancerization effects as well as isolated tumor cell foci extending well beyond the invasive tumor margin have been described previously to account for local recurrence rates following breast conserving surgery despite adequate surgical margins and breast radiotherapy. To look for evidence of possible tumor cell contamination or field cancerization by genetic effects, a pilot study (Study 1: 12 sample pairs) followed by a verification study (Study 2: 20 sample pairs) were performed on DNA extracted from HER2-positive breast tumors and matching normal adjacent mammary tissue samples excised 1–3xa0cm beyond the invasive tumor margin. High-resolution molecular inversion probe (MIP) arrays were used to compare genomic copy number variations, including increased HER2 gene copies, between the paired samples; as well, a detailed histologic and immunohistochemical (IHC) re-evaluation of all Study 2 samples was performed blinded to the genomic results to characterize the adjacent normal tissue composition bracketing the DNA-extracted samples. Overall, 14/32 (44xa0%) sample pairs from both studies produced genome-wide evidence of genetic aberrations including HER2 copy number gains within the adjacent normal tissue samples. The observed single-parental origin of monoallelic HER2 amplicon haplotypes shared by informative tumor–normal pairs, as well as commonly gained loci elsewhere on 17q, suggested the presence of contaminating tumor cells in the genomically aberrant normal samples. Histologic and IHC analyses identified occult 25–200xa0μm tumor cell clusters overexpressing HER2 scattered in more than half, but not all, of the genomically aberrant normal samples re-evaluated, but in none of the genomically normal samples. These genomic and microscopic findings support the conclusion that tumor cell contamination rather than genetic field cancerization represents the likeliest cause of local clinical recurrence rates following breast conserving surgery, and mandate caution in assuming the genomic normalcy of histologically benign appearing peritumor breast tissue.

Liposomal Drug Delivery Systems for Cancer Therapy

Liposomes are currently one of the most well-studied drug delivery systems used in the treatment of cancer. They are being employed in the treatment of a wide variety of human malignancies (1–4). Their large size relative to the gaps in the vasculature of healthy tissues inhibits their uptake by these tissues, thus avoiding certain nonspecific toxicities. However, the “leaky” microvasculature supporting solid tumors allows for the uptake of these large (~ 100 nm) drug carriers (5–8) and their subsequent interaction with cancer cells (9), or release of the encapsulated drug specifically near the tumor, where it can diffuse into the tumor in its free form (10,11). Liposomes have many other potential advantages over the corresponding free drugs, including favorable pharmacokinetic properties, where encapsulation of a usually rapidly cleared drug results in a considerable increase in the circulation lifetime for the drug (12–14). In addition, encapsulation or complexation of a normally labile therapeutic agent, such as DNA, antisense oligonucleotides, or the lactone ring of camptothecins, can protect the agent from premature degradation by enzymes in the plasma or from simple hydrolysis. The result of liposome formulation can thus be a substantial increase in antitumor efficacy when compared to the free drug or standard chemotherapy regimens (15–17).