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Flavopiridol Induces Apoptosis of Normal Lymphoid Cells, Causes Immunosuppression, and Has Potent Antitumor Activity In Vivo Against Human Leukemia and Lymphoma Xenografts

Francisco Arguello, Mark Alexander, Judith A. Sterry, Gabriela Tudor, Erik M. Smith, Naina T. Kalavar, John F. Greene Jr, William Koss, C. David Morgan, Sherman F. Stinson, Timothy J. Siford, W. Gregory Alvord, Richard L. Klabansky and Edward A. Sausville

Article Figures & Data

Figures

  • Fig. 1.

    Effect of flavopiridol on normal cells and tissues. (a) Histological section (10×) of the spleen of an untreated immunocompetent C57BL/6 mouse, showing a rich population of lymphocytes forming the germinal centers and marginal zone of the folliculi of the white pulp, surrounded by the blood-filled sinusoids of the red pulp. (b) Histological section (10×) of the spleen of an immunocompetent mouse 96 hours after initiation of treatment with daily IV bolus injection of flavopiridol, showing a marked depletion of lymphocytes, and only remnants of the white pulp. (c) Histological section of the thymus (10×) of a nontreated immunocompetent mouse showing the densely populated cortex by lymphocytes, surrounding the medullary areas of the lobules. (d) Histological section of a thymus (10×) of a flavopiridol-treated mouse showing an atrophic thymus, in which most lymphoid areas have disappeared. (e) ApopTag immunohistochemistry of the spleen (50×) of a nontreated mouse showing the rare presence of apoptotic brown-stained cells. (f) ApopTag immunohistochemistry of the spleen (50×) of a mouse 48 hours after initiation of treatment with flavopiridol, showing multiple brown-stained apoptotic lymphocytes in the follicular centers of the white pulp. (g) ApopTag immunostaining of the thymus (10×) of a nontreated immunocompetent mouse showing the lack of apoptosis in the lymphocyte-formed cortex. (h) ApopTag immunostaining of the thymus (10×) of a flavopiridol-treated mouse, showing a brown-stained, atrophic cortex caused by the death of thymocytes through apoptosis.

  • Fig. 2.

    Apoptotic death of human promyelocytic leukemia HL-60 cells in vivo after flavopiridol therapy. (a) Histological section of a s.c. HL-60 tumor (100×) removed from a nontreated control nude mouse, showing a large number of predominantly viable and dividing malignant blasts. (b) ApopTag immunostaining of the same HL-60 tumor as (a), showing the sporadic presence of brown-stained cells indicative of rare spontaneous apoptosis. (c) Histological section of an HL-60 tumor (100×) 96 hours after initiation of treatment with daily IV bolus injection of flavopiridol, showing multiple fragments of condensed chromatin (“apoptotic bodies”) indicative of cell death through apoptosis. (d) ApopTag immunostaining of an HL-60 tumor 96 hours after initiation of treatment with bolus IV flavopiridol. Multiple cells densely stained brown indicate DNA fragmentation. (e) Agarose gel analysis of DNA isolated from SUDHL-4 nontreated lymphoma (lane 2), and DNA isolated from a tumor 72 hours after initiation of treatment with flavopiridol (lane 3), which shows the typical “ladder” pattern of internucleosomal cleavage of DNA into multiples of 180 to 250 bp. Lane 1 includes DNA molecular mass markers.

  • Fig. 3.

    Effect of flavopiridol therapy in systemic leukemia and lymphoma xenografts. (a) Kaplan and Meier survival curve of SCID mice bearing systemic human acute lymphoblastic leukemia (Nalm/6) treated with flavopiridol 7.5 mg/kg IV every other day × 5 at days 3 to 7, and repeated again at days 17 to 21 posttransplantation of cells (•). Control mice (▪) received the vehicle 1% DMSO in NaCl. (b) Kaplan and Meier survival curve of SCID mice bearing systemic human AS283 human lymphoma after one 5-day cycle of flavopiridol bolus IV therapy from days 3 to 7 posttransplantation of cells (•). Control mice received the vehicle (▪).

  • Fig. 4.

    Plasma concentrations of flavopiridol observed in combined groups receiving 1, 3, and 5 daily IV injections of 5 mg/kg in mice. The experimental data points (•), representing the geometric mean of the assayed plasma concentrations for each time point, and the best-fit curve generated by nonlinear regression analysis, are shown.

Tables

  • Table 1.

    Thymidine Incorporation of Flavopiridol-Treated Human Lymphocytes After Mitogen Stimulation in Vitro

    Pretreatment Stimulation Indices From 3H-Methyl Thymidine Incorporation According to the Mitogen Used
    CON-A 1.56 μg/mL PHA-P 1.56 μg/mLPWM 1.56 μg/mL Anti-CD3 10 ng/mL
    Flavopiridol (nmol/L)
     0 252.2 155.8 86.4 35.3
     10068.1 89.9 23.2 −0.7
     250 1.0 4.7 2.8−0.9
     500 −0.3 −0.4 0.1 −0.8
    Actinomycin D (μg/mL)
     10 −0.5 −0.1 −0.1−0.9
  • Table 2.

    Effect of Flavopiridol on s.c. Human Leukemia and Lymphoma Xenografts

    Human Cell Line Mouse Strain Flavopiridol Treatment (days of treatment) 100% Tumor Regressions (50% regression) Tumor- Free >3 months Growth Delay (prob. value) Day Euthanasia (no. of mice)
    HL-60 Promyelocytic Leukemia nu/nu Sham controls 1% DMSO0/6 (0/6) 0/6 20 (6/6)
    20.6 mg/kg/d 72-h continuous infusion (days 10-13) 0/6 (0/6) 0/6 20.5% (P = .0248) 20 (6/6)
    10.8 mg/kg/d 72-h continuous infusion (days 10-13) 0/6 (0/6) 0/6 17.2% (P = .0014) 20 (6/6)
    Sham controls 1% DMSO 0/5 (0/5) 0/5 21 (4/5)
    7.5 mg/kg/d bolus IV × 5 (days 13-17) 4/5 (1/5) 4/5 N/A >90 (5/6)
    7.5 mg/kg/d IP × 5 (days 13-17) 6/6 (N/A) 6/6 N/A>90 (6/6)
    SUDHL-4 Follicular Lymphoma SCIDSham controls 1% DMSO 0/5 (0/5) 0/5 38 (5/5)
    7.5 mg/kg/d bolus IV × 5 (2 cycles) (days 25-30), and 38-42) 4/8 (2/8) 2/8 >73% (P = <.0001) 52 (8/8)
    AS283 AIDS-Associated Lymphoma SCID Sham control 1% DMSON/A Early tumor 0/5 32 (5/5)
    7.5 mg/kg/d bolus IV × 5 (2 cycles) (days 3-8, and 18-22)N/A Early tumor 0/7 >84% (P = <.0001) 32 (5/7)
    Sham control 1% DMSO 0/5 0/5 20 (5/5)
    7.5 mg/kg/d bolus IP × 5 (days 13-17) 1/6 (2/6) 0/6 45.8% (P = .013)  20 (6/6)
    • Abbreviation: N/A, not applicable.