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Induction of Apoptosis in Rat Peripheral Blood Lymphocytes by the Anticancer Drug CI-994 (Acetyldinaline)(*).

Graziano MJ, Spoon TA, Cockrell EA, Rowse PE, Gonzales AJ - J. Biomed. Biotechnol. (2001)

Bottom Line: Morphological and biochemical features of apoptosis were evaluated using several techniques, and lactate dehydrogenase (LDH) release was measured as an indicator of cell necrosis.After 24 hours, concentration-dependent increases in apoptosis characterized by DNA condensation, DNA fragmentation, and/or externalization of phosphatidyl serine were seen at CI-994 concentrations as low as 1 &mgr;M and were statistically significant beginning at 10 &mgr;M.Ultrastructural analysis confirmed the presence of DNA condensation, DNA fragmentation, cell shrinkage, and membrane blebbing in cells exposed to 30 &mgr;M CI-994.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
CI-994 (acetyldinaline) is an investigational anticancer drug currently in clinical trials. In preclinical safety studies in rats and dogs, CI-994 resulted in significant toxicity to bone marrow and lymphoid tissue. To determine if apoptosis was involved in CI-994 toxicity, peripheral blood lymphocytes were isolated from untreated male Wistar rats and exposed to CI-994 (1, 3, 10, or 30 &mgr;M) in vitro for up to 24 hours. Morphological and biochemical features of apoptosis were evaluated using several techniques, and lactate dehydrogenase (LDH) release was measured as an indicator of cell necrosis. No evidence of apoptosis or necrosis was detected in lymphocytes exposed to CI-994 for 4 hours. After 24 hours, concentration-dependent increases in apoptosis characterized by DNA condensation, DNA fragmentation, and/or externalization of phosphatidyl serine were seen at CI-994 concentrations as low as 1 &mgr;M and were statistically significant beginning at 10 &mgr;M. Ultrastructural analysis confirmed the presence of DNA condensation, DNA fragmentation, cell shrinkage, and membrane blebbing in cells exposed to 30 &mgr;M CI-994. After 24 hours, the percent of maximum LDH release from lymphocytes treated with 10 and 30 &mgr;M CI-994 was 7% and 15%, respectively, compared with 0% in the controls. In comparison, morphological changes of apoptosis detected by fluorescent microscopy were observed in 79% of the lymphocytes at these two concentrations. Additionally, apoptosis was seen in more than 24% of lymphocytes exposed to 1 and 3 &mgr;M CI-994, whereas maximum LDH release was less than or equal to 1% at these concentrations. These results show that apoptosis is the primary mode of cell death in rat lymphocytes exposed to CI-994 in vitro.

No MeSH data available.


Related in: MedlinePlus

DNA fragmentation in rat peripheral blood lymphocytes exposed to CI-994 for 24 hours. Mitogen (concanavalin A, 0.63 μg/ml) was added to all lymphocyte cultures at time zero. DNA (0.6 μg) was separated on a 1% agarose gel stained with ethidium bromide. Arrows indicate molecular weight markers. Positive control = 1 μM staurosporine (lane 6).
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Figure 6: DNA fragmentation in rat peripheral blood lymphocytes exposed to CI-994 for 24 hours. Mitogen (concanavalin A, 0.63 μg/ml) was added to all lymphocyte cultures at time zero. DNA (0.6 μg) was separated on a 1% agarose gel stained with ethidium bromide. Arrows indicate molecular weight markers. Positive control = 1 μM staurosporine (lane 6).

Mentions: Apoptotic cells fixed and permeabilized in ethanol lose fragmented DNA resulting in DNA histograms with a sub-G1 DNA content [23, 24, 25]. No significant increase in sub-G1 cells was detected in rat lymphocytes treated with CI-994 for 4 hours when analyzed by flow cytometry and Hoechst 33352stain (Figure 5). However, exposure to CI-994 for24 hours led to a dose-related increase in the number of sub-G1 cells relative to the untreated controls (Figure 5). The increase in sub-G1 cells was statistically significant at 30 μM CI-994. Without mitogen, the percentage of apoptotic cells detected by flow cytometry and Hoechst 33352 stain were 18%, 15%, and 55%, at CI-994 concentrations of 0, 1, and 30 μM, respectively. A distinct DNA fragmentation pattern consisting of multiples of 180 bp was also observed in rat lymphocytes exposed to CI-994 for 24 hours when analyzed by gel electrophoresis (Figure 6). DNA fragmentation was evident at CI-994 concentrations as low as 3 μM with a dose-related increase in band intensity.


Induction of Apoptosis in Rat Peripheral Blood Lymphocytes by the Anticancer Drug CI-994 (Acetyldinaline)(*).

Graziano MJ, Spoon TA, Cockrell EA, Rowse PE, Gonzales AJ - J. Biomed. Biotechnol. (2001)

DNA fragmentation in rat peripheral blood lymphocytes exposed to CI-994 for 24 hours. Mitogen (concanavalin A, 0.63 μg/ml) was added to all lymphocyte cultures at time zero. DNA (0.6 μg) was separated on a 1% agarose gel stained with ethidium bromide. Arrows indicate molecular weight markers. Positive control = 1 μM staurosporine (lane 6).
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Related In: Results  -  Collection

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Figure 6: DNA fragmentation in rat peripheral blood lymphocytes exposed to CI-994 for 24 hours. Mitogen (concanavalin A, 0.63 μg/ml) was added to all lymphocyte cultures at time zero. DNA (0.6 μg) was separated on a 1% agarose gel stained with ethidium bromide. Arrows indicate molecular weight markers. Positive control = 1 μM staurosporine (lane 6).
Mentions: Apoptotic cells fixed and permeabilized in ethanol lose fragmented DNA resulting in DNA histograms with a sub-G1 DNA content [23, 24, 25]. No significant increase in sub-G1 cells was detected in rat lymphocytes treated with CI-994 for 4 hours when analyzed by flow cytometry and Hoechst 33352stain (Figure 5). However, exposure to CI-994 for24 hours led to a dose-related increase in the number of sub-G1 cells relative to the untreated controls (Figure 5). The increase in sub-G1 cells was statistically significant at 30 μM CI-994. Without mitogen, the percentage of apoptotic cells detected by flow cytometry and Hoechst 33352 stain were 18%, 15%, and 55%, at CI-994 concentrations of 0, 1, and 30 μM, respectively. A distinct DNA fragmentation pattern consisting of multiples of 180 bp was also observed in rat lymphocytes exposed to CI-994 for 24 hours when analyzed by gel electrophoresis (Figure 6). DNA fragmentation was evident at CI-994 concentrations as low as 3 μM with a dose-related increase in band intensity.

Bottom Line: Morphological and biochemical features of apoptosis were evaluated using several techniques, and lactate dehydrogenase (LDH) release was measured as an indicator of cell necrosis.After 24 hours, concentration-dependent increases in apoptosis characterized by DNA condensation, DNA fragmentation, and/or externalization of phosphatidyl serine were seen at CI-994 concentrations as low as 1 &mgr;M and were statistically significant beginning at 10 &mgr;M.Ultrastructural analysis confirmed the presence of DNA condensation, DNA fragmentation, cell shrinkage, and membrane blebbing in cells exposed to 30 &mgr;M CI-994.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
CI-994 (acetyldinaline) is an investigational anticancer drug currently in clinical trials. In preclinical safety studies in rats and dogs, CI-994 resulted in significant toxicity to bone marrow and lymphoid tissue. To determine if apoptosis was involved in CI-994 toxicity, peripheral blood lymphocytes were isolated from untreated male Wistar rats and exposed to CI-994 (1, 3, 10, or 30 &mgr;M) in vitro for up to 24 hours. Morphological and biochemical features of apoptosis were evaluated using several techniques, and lactate dehydrogenase (LDH) release was measured as an indicator of cell necrosis. No evidence of apoptosis or necrosis was detected in lymphocytes exposed to CI-994 for 4 hours. After 24 hours, concentration-dependent increases in apoptosis characterized by DNA condensation, DNA fragmentation, and/or externalization of phosphatidyl serine were seen at CI-994 concentrations as low as 1 &mgr;M and were statistically significant beginning at 10 &mgr;M. Ultrastructural analysis confirmed the presence of DNA condensation, DNA fragmentation, cell shrinkage, and membrane blebbing in cells exposed to 30 &mgr;M CI-994. After 24 hours, the percent of maximum LDH release from lymphocytes treated with 10 and 30 &mgr;M CI-994 was 7% and 15%, respectively, compared with 0% in the controls. In comparison, morphological changes of apoptosis detected by fluorescent microscopy were observed in 79% of the lymphocytes at these two concentrations. Additionally, apoptosis was seen in more than 24% of lymphocytes exposed to 1 and 3 &mgr;M CI-994, whereas maximum LDH release was less than or equal to 1% at these concentrations. These results show that apoptosis is the primary mode of cell death in rat lymphocytes exposed to CI-994 in vitro.

No MeSH data available.


Related in: MedlinePlus