Limits...
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

Electron micrograph of (A) untreated rat peripheral blood lymphocyte and (B) rat peripheral blood lymphocyte exposed to 30 μM CI-994 for 24 hours. Mitogen (concanavalin A, 0.63 μg/ml) was added to all lymphocyte cultures at time zero. Treated lymphocyte shows morphological changes of apoptosis including chromatin condensation and fragmentation, membrane blebbing, and cell shrinkage.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC113775&req=5

Figure 4: Electron micrograph of (A) untreated rat peripheral blood lymphocyte and (B) rat peripheral blood lymphocyte exposed to 30 μM CI-994 for 24 hours. Mitogen (concanavalin A, 0.63 μg/ml) was added to all lymphocyte cultures at time zero. Treated lymphocyte shows morphological changes of apoptosis including chromatin condensation and fragmentation, membrane blebbing, and cell shrinkage.

Mentions: There was no significant increase in the number of apoptoticcells detected by fluorescent microscopy after 4 hours of exposure to CI-994 (Figure 2). After 24 hours of exposure, the number of apoptotic lymphocytes at 10 and 30 μM CI-994 was significantly increased relative to the untreated controls: 79% at 10 and 30 μM CI-994versus 16% in the controls (Figure 2).Although not statistically significant, the percentage of apoptotic cells was also increased at 1 and 3 μM CI-994: 28% and 34%, respectively. For lymphocytes incubated without mitogen, the percentage of apoptotic cells were 12%, 15%, and 51%, at CI-994 concentrations of 0, 1, and 30 μM, respectively. Condensed chromatin, apoptotic bodies, and membrane blebbing were characteristic of apoptotic cells (Figure 3). Ultrastructural examination of rat lymphocytes exposed to 30 μM CI-994 for 24 hours also showed typical morphological features of apoptosis including chromatin condensation and fragmentation, membrane blebbing, andcell shrinkage (Figure 4).


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)

Electron micrograph of (A) untreated rat peripheral blood lymphocyte and (B) rat peripheral blood lymphocyte exposed to 30 μM CI-994 for 24 hours. Mitogen (concanavalin A, 0.63 μg/ml) was added to all lymphocyte cultures at time zero. Treated lymphocyte shows morphological changes of apoptosis including chromatin condensation and fragmentation, membrane blebbing, and cell shrinkage.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC113775&req=5

Figure 4: Electron micrograph of (A) untreated rat peripheral blood lymphocyte and (B) rat peripheral blood lymphocyte exposed to 30 μM CI-994 for 24 hours. Mitogen (concanavalin A, 0.63 μg/ml) was added to all lymphocyte cultures at time zero. Treated lymphocyte shows morphological changes of apoptosis including chromatin condensation and fragmentation, membrane blebbing, and cell shrinkage.
Mentions: There was no significant increase in the number of apoptoticcells detected by fluorescent microscopy after 4 hours of exposure to CI-994 (Figure 2). After 24 hours of exposure, the number of apoptotic lymphocytes at 10 and 30 μM CI-994 was significantly increased relative to the untreated controls: 79% at 10 and 30 μM CI-994versus 16% in the controls (Figure 2).Although not statistically significant, the percentage of apoptotic cells was also increased at 1 and 3 μM CI-994: 28% and 34%, respectively. For lymphocytes incubated without mitogen, the percentage of apoptotic cells were 12%, 15%, and 51%, at CI-994 concentrations of 0, 1, and 30 μM, respectively. Condensed chromatin, apoptotic bodies, and membrane blebbing were characteristic of apoptotic cells (Figure 3). Ultrastructural examination of rat lymphocytes exposed to 30 μM CI-994 for 24 hours also showed typical morphological features of apoptosis including chromatin condensation and fragmentation, membrane blebbing, andcell shrinkage (Figure 4).

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