T-oligo exposure induces phospho-p53 (S15) and phospho-Chk2 (T68) in CEM cells. (a) Extracts were prepared from human CEM T leukemic cells treated with 20 µM T-oligo for the indicated times in hours (h). Extracted proteins were separated by SDS-PAGE, then immunoblotted with polyclonal antibody against p-p53 (S15), pChk2 (T68) or cyclophilin B as a loading control. The positive control (+) was exposure to 0.25 µM adriamycin for 6 hr. (b) Cell cycle profiles of CEM cells exposed to 20 µM T-oligo as above, then fixed with ethanol, stained with propidium iodide and analyzed by flow cytometry. Data were collected on the FL2 channel with the detector set on logarithmic scale for visualization of the apoptotic population. Percentages indicated ungated populations of cells with apoptotic, sub-G₁ DNA content. Results are representative of 3 independent experiments. T-oligo DNA (histograms i–iv) is compared to C-oligo DNA (complementary DNA sequence (1–3, 5–7; histograms v–vii), both at 20 µM.

T-oligo treatment does not cause apoptosis in normal primary B or T cells. (a) Primary B-2 cells were purified from mouse spleens, cultured and stimulated with 1 µg/ml goat anti-mouse IgM F(ab′)₂ antibody (μ chain specific), 1 µg/ml rat anti-mouse CD40 and 10 ng/ml murine interleukin-4 (iv–vi), or left unstimulated as controls (i–iii). The proliferating cells after 48 hr mitogenic stimulation (iv–vi), and resting cells (i–iii) immediately after plating, were exposed for 72 hr to 20 µM T-oligo (ii, v); saline vehicle (i, iv) or 20 µM of a telomere-unrelated (scrambled) oligo (iii, vi) as negative controls, then harvested, fixed and analyzed for DNA content by propidium iodide staining and flow cytometry as in Fig. 1b. Percentages of ungated cells are shown. (b) Human primary PBLs were mitogenically stimulated for 48 hr with 10 µg/ml PHA (dot plot i), then exposed to T-oligo (dot plot ii) and assayed for Annexin V induction after an additional 6 hr. Annexin V was detected with a PE conjugated reagent; data were collected on the FL2 channel with the detector in logarithmic mode. T lineage was assayed by anti-CD3-FITC (FL1 channel, logarithmic mode). As a positive control, cells were mitogenically stimulated, then exposed to 0.25 µM adriamycin (dot plot iii). Percentages of gated lymphocytes in each quadrant are shown. (c) As a control, human primary PBLs were assayed for T-cell activation by anti-CD69-PE (FL2 channel, logarithmic mode; dot plot ii) compared to unstimulated control (dot plot i). T lineage was assayed by anti-CD3 as in (b). Percentages of gated lymphocytes in each quadrant are shown. Stimulated cells were then exposed to 20 µM T-oligo (iv) or left unexposed as control (iii) and assayed for DNA content at 72 hr as in panel A. Percentages of ungated sub-G₁ events are indicated. (d) PHA-stimulated human primary lymphocytes were exposed to 20 µM T-oligo and assayed by immunoblot for induction of pp53 (S15) and pChk2 (T68) for the indicated times (hr) as in Fig. 1a. Adriamycin once again was the positive control for DNA damage response (+) and cyclophilin B was the loading control.

Ability of T-CHOP to reduce tumor burden of lymphoid organs in vivo. (a) Spleen weight was measured after completion of in vivo combination chemotherapy studies (n = 6; SD shown). Dosages of anti-cancer chemotherapeutic drugs were as specified in Material and methods. Normal refers to mice not inoculated with tumor cells. Untreated refers to tumor-bearing mice with PBS injection i.p.; CHOP, T oligo or T-CHOP refers to tumor-bearing mice treated i.p. with CHOP alone, T-oligo alone or T-oligo and CHOP together. Significant difference between means is indicated (*) (p < 0.01). (b) Percentage of CD19⁺ (B lineage) cells in bone marrow, as a function of all cells in bone marrow measured by flow cytometry (n = 3; SD shown). (c) Caspase-3 activity in spleen (n = 3; SD shown). (d) Percentage of sub-G₁ (apoptotic) events in peritoneal cavity (n = 5; SD shown). (e) Proliferation of malignant B cells in mesenteric lymph nodes after i.p. treatment with T-oligo alone (dot plot iii) compared to untreated (dot plot ii) and normal (dot plot i). Mice were labelled by i.p. injection of BrdU at the end of the course of treatment and sacrificed 1 hr later. Mesenteric lymph nodes were then harvested, immunophenotyped with anti-B220-Pacific Blue (as a pan-B cell marker), fixed, permeabilized, and then doubly stained with anti-BrdU-FITC antibody and 7-aminoactinomycin D (7-AAD) for DNA content. A total of 40,000 events within a double gate for lymphocytes and B220⁺ cells were analyzed; one representative experiment of 3 is shown. Percentages of cells within each stage of the cell cycle are shown within each panel: lower left rectangle defines G₀/G₁-phase population, upper central rectangle defines S-phase population and lower right rectangle defines G₂/M-phase population.

T-oligo causes apoptosis in B cell lymphoma lines. (a) Human RL B cell lymphoma cells were treated with T-oligo and analyzed as in Fig. 1b. Results are representative of 3 independent experiments. (i–iv, ix and x, FL2 detector was in linear mode; v–viii, FL2 detector was in logarithmic mode). For positive control (ix, x), RL cells were treated with 0.25 µM adriamycin. DNA content at 0 hr (i, v), 24 hr (ii, vi, ix), 48 hr (iii, vii, x) and 72 hr (iv, viii) after exposure is shown. Percentages of ungated sub-G₁ events are indicated (v–viii). (b) RL cell extracts were immunoblotted for total p53 at the indicated times after exposure to T-oligo. (c) T-oligo dose-response assay of caspase-3 activity in RL cells (●) and p53-null MOLT-4 cells (○) treated with T-oligo for 4 hr (n = 3). Fluorescence values are shown as relative fluorescence units.

Vincristine cooperates with T-oligo at low doses to induce apoptosis of B cell lymphoma. (a) Assay of caspase-3 activity 12 hr after addition of low dose vincristine (25 nM) and low dose T-oligo (2 µM), together or separately, to human B cell lymphoma cells in vitro. Control was saline alone (SD shown, n = 3; filled bars, RL cells; open bars, Toledo cells). (p < 0.001; Holm-Sidak pairwise comparison of combination to saline control) (b) Induction of Annexin V after 6 hr exposure to T-oligo and vincristine, as in A, of murine primary DLCL B cells.21,22 (i, vehicle control; ii, vincristine alone; iii, T-oligo alone; iv; both agents together). B lineage was identified with anti-CD19-FITC and data were collected on the FL1 channel with the detector in logarithmic mode. Annexin V was detected as in Fig. 3. Percentages of gated malignant lymphocytes are indicated in quadrants. Double positive cells are therefore Annexin V-staining B lymphocytes.

Tumor burden in lung after chemotherapy. Lungs were removed from chemotherapy-treated mice after euthanasia, fixed in formalin and stained with H&E. (i) lung of normal, irradiated mouse (arrow indicates bronchiole); (ii) lung of untreated lymphoma mouse (arrow emphasizes heavy accumulation of malignant cells around a bronchiole); (iii) lung of mouse treated with CHOP only; (iv) lung of mouse treated with T-CHOP combination (arrow emphasizes reduced tumor infiltration at bronchiole) (bar, 100 µm).