Fig. 5.
Fig. 5. Partial rescue of leukemic cells from chemotherapy-induced death. / (A) HEL and THP-1 cells (1 × 106 per well) were exposed to different chemotherapy agents (Ara-C, 200 ng/mL; etoposide, 1 μM; daunorubicin, 200 ng/mL) for 48 hours, and the number of apoptotic cells was determined by annexin V staining, as described in “Materials and methods.” In some conditions, cells were pretreated with 100 ng/mL VEGF-C, mutVEGF-C, or VEGF (100 ng/mL) for 24 hours, before addition of the chemotherapeutic agents. The cytokines, as with the chemotherapeutic agent, were readded to the cultures daily. As shown, VEGF-C and mutVEGF-C exerted a significant protective effect against the 3 agents used in this study. VEGF also protected HEL cells from Ara-C–induced apoptosis. Results shown were obtained from 3 independent experiments, and each condition was assayed in triplicate. *P < .05 (significant difference compared with chemotherapeutic agent alone)., indicates untreated; □, chemotherapy;, chemotherapy + VEGF-C; ■, Arg-C+VEGF. (B) Similar to the cell lines, 6 primary leukemia samples were cultured in serum-free conditions for 48 hours in the presence of different chemotherapeutic agents (Ara-C, etoposide, daunorubicin). In some conditions the cells were pretreated with VEGF-C (100 ng/mL) or mutVEGF-C (200 ng/mL) for 24 hours, prior to the addition of the chemotherapeutic agent. In these conditions, the cells received cytokines plus chemotherapeutic agent daily. Apoptotic cells were determined by annexin V staining, as described in “Materials and methods.” As shown, VEGF-C and mutVEGF-C protected FLT-4+ primary cells from chemotherapy-induced apoptosis (samples1, 3, 4, 5, and 6). Importantly, neither VEGF-C nor mutVEGF-C protected a FLT-4− sample (number 2) from chemotherapy-induced apoptosis. Results shown were obtained from 3 independent experiments, and each condition was assayed in triplicate. *P < .05 (significant difference compared with chemotherapeutic agent alone). nd: due to sample constraints, some conditions could not be assayed in triplicate and were therefore removed from the final graph., indicates untreated; □, chemotherapy;, chemotherapy + VEGF-C; ■, Arg-C+mutVEGF-C.

Partial rescue of leukemic cells from chemotherapy-induced death.

(A) HEL and THP-1 cells (1 × 106 per well) were exposed to different chemotherapy agents (Ara-C, 200 ng/mL; etoposide, 1 μM; daunorubicin, 200 ng/mL) for 48 hours, and the number of apoptotic cells was determined by annexin V staining, as described in “Materials and methods.” In some conditions, cells were pretreated with 100 ng/mL VEGF-C, mutVEGF-C, or VEGF (100 ng/mL) for 24 hours, before addition of the chemotherapeutic agents. The cytokines, as with the chemotherapeutic agent, were readded to the cultures daily. As shown, VEGF-C and mutVEGF-C exerted a significant protective effect against the 3 agents used in this study. VEGF also protected HEL cells from Ara-C–induced apoptosis. Results shown were obtained from 3 independent experiments, and each condition was assayed in triplicate. *P < .05 (significant difference compared with chemotherapeutic agent alone)., indicates untreated; □, chemotherapy;, chemotherapy + VEGF-C; ■, Arg-C+VEGF. (B) Similar to the cell lines, 6 primary leukemia samples were cultured in serum-free conditions for 48 hours in the presence of different chemotherapeutic agents (Ara-C, etoposide, daunorubicin). In some conditions the cells were pretreated with VEGF-C (100 ng/mL) or mutVEGF-C (200 ng/mL) for 24 hours, prior to the addition of the chemotherapeutic agent. In these conditions, the cells received cytokines plus chemotherapeutic agent daily. Apoptotic cells were determined by annexin V staining, as described in “Materials and methods.” As shown, VEGF-C and mutVEGF-C protected FLT-4+ primary cells from chemotherapy-induced apoptosis (samples1, 3, 4, 5, and 6). Importantly, neither VEGF-C nor mutVEGF-C protected a FLT-4− sample (number 2) from chemotherapy-induced apoptosis. Results shown were obtained from 3 independent experiments, and each condition was assayed in triplicate. *P < .05 (significant difference compared with chemotherapeutic agent alone). nd: due to sample constraints, some conditions could not be assayed in triplicate and were therefore removed from the final graph., indicates untreated; □, chemotherapy;, chemotherapy + VEGF-C; ■, Arg-C+mutVEGF-C.

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