Figure 7

HB-19 and related multivalent Nucant pseudopeptides induce cell death associated with internucleosomal DNA fragmentation. A. Inhibition of cell multiplication by HB-19 and related multivalent Nucant pseudopeptides. T29 cells at 150,000 cells/ml in 2 ml of culture medium were seeded (Day 0) in the absence (Control) or presence of 20 μM of pentavalent (HB-19, N3) and 10 μM of hexavalent (N6 and N7) pseudopeptides. Cell cultures (in triplicate) were monitored for viable cell number daily for 3 days. The histograms show the mean number of cells/ml ± SD of triplicate samples. The multiplication index of cells at day 3 compared to day 0 (the day of seeding) is given at the top of histogram. At 3 days post passage of T29 cells, the cell multiplication index of cells is 17.5-fold compared to 8.9-, 7.2-, 2.2-, and 1.7-fold in the presence of treatment with HB-19, N3, N6, and N7, respectively. B. N7 inhibits T29 cell multiplication in a dose dependent manner. T29 cells seeded at 100,000/ml in 2 ml in the absence (0 μM) or presence of N7 at various concentrations 2.5, 5, 10 and 20 μM were cultured for 3 days and the number of viable cells was determined at day 1, 2, and 3 post-seeding. The ordinate gives the cell number/ml (the mean of 2 samples) in each culture at day 1, 2, and 3 post-seeding. C. Partial cleavage of surface/cytoplasmic nucleolin. T29 cells were cultured for 45 min with 0 (for control; lanes C), 2 and 10 μM of N6 before preparation of cytoplasmic (that contains both surface and cytoplasmic nucleolin) and nuclear extract and analysis by immunoblotting using rabbit polyclonal antibodies that are directed against the first 26 (panel anti-Nt Ab) and the last 16 amino acids (panel anti-Ct Ab) of human nucleolin, respectively. The nuclear extracts were analyzed by immunoblotting using anti-Nt Ab (Methods). The cleavage products, p60 and p70, are revealed with the anti-Ct Ab only. D. Internucleosomal DNA cleavage in Nucant treated Cells. T29 cells, untreated (lane 0) or treated at 2.5, 5, 10, and 20 μM of N6, or 10 μg/ml of cycloheximide (CHX) or 20 μM of bisphosphonate (BisP) for 24 hours, were processed for extraction of the low molecular weight DNA from the nucleoplasm (Methods). Lane M shows the electrophoresis mobility of the DNA marker composed of a 100 base-pair ladder. On the right is the position of nucleosomal fragments, starting from the monomer, dimer, trimer and tetramer unit of 180, 360, 540, and 720 bp, respectively. Statistical significance: *p < 0.1, **p < 0.01, ***p < 0.001.