![Fig. 1. A3D8 anti-CD44 mAb inhibits drug-induced apoptosis. / The inhibitory effect of CD44-specific A3D8 mAb on drug-induced apoptosis was determined in HL60, as shown in A, C, and D, and NB4 (B) myeloid cells. Cells were preincubated for 16 hours with 2.5 μg/mL anti-CD44 mAbs A3D8 or J173, control mouse IgG1, or culture medium only, then treated with DNR (0.5 μmol/L), mitoxantrone (0.3 μmol/L), or etoposide (10 μmol/L) for 24 hours (A-C) or 6 hours (D). Apoptosis was evaluated by measuring the percentage of annexinV-FITC-positive, propidium iodide-negative cells, by flow cytometry (A and B); the percentage of apoptotic cells, by microscopical observation of May-Grünwald-Giemsa stained cytosmears (C); and the percentage of fragmented DNA, in which the spontaneous DNA fragmentation of untreated cells (routinely < 5% DNA fragmentation) has been subtracted, by the [3H]-thymidine release assay (D), as described.16 Panels A and B show results from 1 representative experiment of 3. The mean ± SEM from triplicate samples are shown for panels C and D. (E) Analysis of cell survival in DNR-treated cultures preincubated with CD44-specific mAbs. HL60, HL60/Bcl-2 and NB4 cells were preincubated for 16 hours with culture medium, A3D8 or J173 mAbs, as indicated, then treated with 0.5 μmol/L DNR or culture medium alone (DNR-free control) for 24 hours. The total number of viable cells per milliliter was evaluated by trypan blue dye exclusion. For each preincubation, the percentage of surviving cells was calculated as follows: [number of viable cells/mL in DNR-treated culture] / [number of viable cells/mL in DNR-free culture] × 100. Results represent 1 of 2 independent experiments. (F) A3D8 and J173 anti-CD44 mAbs do not cross-block each other's binding to HL60 cells. HL60 cells were preincubated with a saturating concentration (20 μg/mL) of A3D8 or J173 mAbs, or IgG1 control isotype, and subsequently stained with 2 μg/mL FITC-conjugated A3D8 or J173. Cell fluorescence (black histograms) was analyzed by flow cytometry. Nonspecific background fluorescence was determined on cells incubated with control IgG1, followed by GAM-FITC (gray histograms).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/96/3/10.1182_blood.v96.3.1187/5/bloo01501001x.jpeg?Expires=1735854073&Signature=sOtOj7af8F8ufvcbbKRQUan9tr4DZJZiRnYbSpr1oAMEy-5HWOIfH-3V8J50pqVCZxsXlS1P3N7ZDG1yQc2Gt8FbekVD0ngN2NmfnmhPAkZF5DosY2BZzWU~Nk~p3HEmkbvq1o76iV5ToeuuKCR5H-jEu1p9yihaDng4wteNZtT5hajMAxzq4Bvg8tTA9MMGy1ojUH1trJNwXKMGAw1ptzrZZLCsDsI67v0XDtSZuRGWlYbRgIZRhj-ukSzF0IUsJspLuL4p7bkFwpCIXCUqs-qAhVuXbGtOTJWdgd9AvWIxmAfOVoiYykphfjfCxil29aV-MMFCFIQ3qmbUF7wUzA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
A3D8 anti-CD44 mAb inhibits drug-induced apoptosis.
The inhibitory effect of CD44-specific A3D8 mAb on drug-induced apoptosis was determined in HL60, as shown in A, C, and D, and NB4 (B) myeloid cells. Cells were preincubated for 16 hours with 2.5 μg/mL anti-CD44 mAbs A3D8 or J173, control mouse IgG1, or culture medium only, then treated with DNR (0.5 μmol/L), mitoxantrone (0.3 μmol/L), or etoposide (10 μmol/L) for 24 hours (A-C) or 6 hours (D). Apoptosis was evaluated by measuring the percentage of annexinV-FITC-positive, propidium iodide-negative cells, by flow cytometry (A and B); the percentage of apoptotic cells, by microscopical observation of May-Grünwald-Giemsa stained cytosmears (C); and the percentage of fragmented DNA, in which the spontaneous DNA fragmentation of untreated cells (routinely < 5% DNA fragmentation) has been subtracted, by the [3H]-thymidine release assay (D), as described.16 Panels A and B show results from 1 representative experiment of 3. The mean ± SEM from triplicate samples are shown for panels C and D. (E) Analysis of cell survival in DNR-treated cultures preincubated with CD44-specific mAbs. HL60, HL60/Bcl-2 and NB4 cells were preincubated for 16 hours with culture medium, A3D8 or J173 mAbs, as indicated, then treated with 0.5 μmol/L DNR or culture medium alone (DNR-free control) for 24 hours. The total number of viable cells per milliliter was evaluated by trypan blue dye exclusion. For each preincubation, the percentage of surviving cells was calculated as follows: [number of viable cells/mL in DNR-treated culture] / [number of viable cells/mL in DNR-free culture] × 100. Results represent 1 of 2 independent experiments. (F) A3D8 and J173 anti-CD44 mAbs do not cross-block each other's binding to HL60 cells. HL60 cells were preincubated with a saturating concentration (20 μg/mL) of A3D8 or J173 mAbs, or IgG1 control isotype, and subsequently stained with 2 μg/mL FITC-conjugated A3D8 or J173. Cell fluorescence (black histograms) was analyzed by flow cytometry. Nonspecific background fluorescence was determined on cells incubated with control IgG1, followed by GAM-FITC (gray histograms).
