c-myc antisense PS-ODN induces accumulation of 46- and 50-kD NH₂-terminally truncated c-Myc proteins in anti-CD3 and anti-CD28 activated CD4⁺ T cells. Human PBL CD4⁺ T cells were stimulated with CD3 and CD28 mAbs for 24 h followed by incubation with PS-ODNs for another 24 h at indicated concentrations. c-Myc proteins were detected by Western blotting with 9E10 mAb (top), or with an antibody specific to phosphorylated Thr58/Ser62 of c-Myc (middle), or with an antibody specific to the NH₂-terminal region of c-Myc, respectively (bottom). The migration of mol wt markers is indicated on the left. Similar results were obtained in three additional experiments.

c-myc antisense PS-ODN has no effect on cell cycle progression or proliferation of T cells under conditions of inhibition of HIV-1 infection. Human PBL CD4⁺ T cells were stimulated with CD3 plus CD28 mAbs for 24 h and then incubated with PS-ODNs or the cell cycle inhibitor mimosine as indicated. (A) At day 4, cells were stained with propidium iodide and DNA content was detected by flow cytometry. The percentages of cells in each cell cycle phase were determined with the use of MCycle plus software (Phoenix Flow Systems). (B) Cells were pulsed with 0.5 μCi of [³H]thymidine for 16 h before harvesting at day 3 and incorporated [³H]thymidine was monitored by beta counter. One of three representative experiments is shown.

A c-myc antisense PS-ODN, but not sense or non-sense PS-ODNs, inhibits HIV-1 LTR circle formation in primary CD4⁺ T cells. (A) Induction of c-Myc expression by CD3 and CD28 costimulation in primary CD4⁺ T cells is shown in the top panel. CD4⁺ T cells purified from human PBLs were stimulated with 20 μg/ml of anti-CD28 (αCD28), 10 μg/ml of anti-CD3 (αCD3) alone, or anti-CD3 plus anti-CD28 (αCD3+αCD28), or were left unstimulated (media) for the times indicated. c-Myc protein expression was evaluated by Western blot analysis. The same blot was stripped and reprobed with ERK1 antisera as a protein loading control. (Bottom panel) CSA inhibited c-Myc expression. Primary CD4⁺ T cells were stimulated with CD3 and CD28 mAbs in the presence or absence of CSA (1 μg/ml) for 24 h. Cell lysates were performed by Western blot analysis to detect c-Myc protein expression. Similar results were obtained in three additional experiments. (B) c-myc antisense, but not sense or non-sense, PS-ODN inhibits HIV-1 LTR circle formation in primary CD4⁺ T cells. Human peripheral blood CD4⁺ T cells were stimulated with anti-CD3 mAb plus anti-CD28 mAbs and infected with HIV-1 at the same time. After 24 h, cells were treated with graded doses of c-myc antisense, sense, or non-sense PS-ODNs or were left untreated. DNA was extracted at 3 d after infection, and levels of initiation of reverse transcription (LTR/LTR), full-length viral DNA synthesis (LTR/gag), and formation of LTR circles were monitored by PCR. DNA from heat-inactivated HIV-1 (HI–HIV-1)–treated cells was run in parallel. Different amounts of DNA extracted from HIV-1–infected C8166 T cells were run as a positive control. One of three similar experiments is shown. (C) c-myc antisense PS-ODN inhibits HIV-1 replication and apoptosis of host T cells. Human CD4⁺ T cells were infected with HIV-1, and PS-ODNs (1 μM) were added to the cultures 24 h later and refreshed every 2 d. At day 6, intracellular double-staining with PE-conjugated anti-HIV p24 protein mAb and FITC-conjugated TUNEL were performed to detect HIV-1 replication and induction of apoptosis. One of two representative experiments is shown.