Optimizing buccal cell DNA yields in mothers and infants for human leukocyte antigen genotyping.

Buccal cells provide a convenient source of DNA for epidemiologic studies. Mouthwash rinses yield a higher quality and quantity of DNA than cytobrushes but are not practical for collection from infants. Although cytobrushes yield sufficient DNA for most genotyping analyses, human leukocyte antigen (HLA) analysis can require 1,000-fold more DNA. In Iowa City, Iowa, in 2002, the authors tested two cytobrush collection methods to optimize total DNA yield and purity for HLA genotyping in mothers and infants: 1) brushing the left and right inner cheeks (standard method) and 2) brushing the upper and lower "gutters", that is, the space between the gums and the inner lips/cheeks along the front and sides of the mouth (test method). Storage and mailing experiments were performed to define conditions for optimizing DNA yield and purity. Mothers' gutter samples yielded significantly higher total amounts of DNA (mean yield = 15.0 micro g/two brushes) than cheek samples (mean yield = 7.6 micro g/two brushes) (paired t test: p < 0.001), while DNA yields from cheek and gutter collections from infants were equivalent. Cytobrushes stored and/or mailed in paper envelopes yielded significantly more and higher-purity DNA than brushes in plastic bags or tubes. Cytobrush sampling of the mouth's gutter areas can enhance DNA yield in mothers but not in young infants. DNA yields can be further optimized by controlling mailing and storage conditions.

Antitumor mechanisms of oligodeoxynucleotides with CpG and polyG motifs in murine prostate cancer cells: decrease of NF-kappaB and AP-1 binding activities and induction of apoptosis.

Previous studies have shown that CpG oligodeoxynucleotides (ODNs) have substantial immunostimulatory effects with anticancer applications. The antitumor applications that have been described previously are mediated through the CpG-induced activation of the host immune system, not through direct antitumor effects. Using cytostasis and cell proliferation assays, we demonstrated that specific ODNs inhibit the proliferation of RM-1 cells, a murine prostate cancer cell line. Flow cytometry analysis using propidium iodide (PI) nuclear staining confirmed the direct proapoptotic effect of ODNs on prostate cancer cells. This effect was dose dependent. Further studies using Western blot analysis and electrophoresis mobility shift assay (EMSA) revealed that the treatment of prostate cancer cells with specific ODNs activated the caspase pathway(s) and decreased the binding activities of AP-1 and NF-kappaB in a time-dependent manner. Evaluation of a panel of ODNs containing different DNA motifs demonstrated that the optimal proapoptotic sequences required polyG sequences but that CpG motifs were not essential. Finally, in vivo antitumor studies showed that the proapoptotic polyG motifs significantly inhibited prostate tumor growth. PolyG motifs inhibited tumor growth, and the effects were enhanced by CpG immune activating sequences. ODN containing both polyG and CpG motifs may have enhanced efficacy in tumor therapy through multiple mechanisms of action, including direct antitumor activities and immune activation.