1. by Jin Gao, and Des R. Richardson
The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents, IV: the mechanisms involved in inhibiting cell-cycle progression
by Jin Gao, and Des R. Richardson
Blood
Volume 98(3):
August 1, 2001
©2001 by American Society of Hematology

2. Structures of 311 and DFO. Structures of 311 and DFO.
Jin Gao, and Des R. Richardson Blood 2001;98:
©2001 by American Society of Hematology

3. The iron chelator 311 is more effective than DFO at decreasing the expression of the cyclins A, B1, D1, D2, and D3 and increasing the expression of cyclin E.SK-N-MC cells were incubated with DFO (5-150 μM) or 311 (1-25 μM) for 30 hours at 37°C, and the cell...
The iron chelator 311 is more effective than DFO at decreasing the expression of the cyclins A, B1, D1, D2, and D3 and increasing the expression of cyclin E.SK-N-MC cells were incubated with DFO (5-150 μM) or 311 (1-25 μM) for 30 hours at 37°C, and the cells were harvested. Western blot analysis was then performed as described in “Materials and methods.” Results shown are representative of 3 separate experiments performed.
Jin Gao, and Des R. Richardson Blood 2001;98:
©2001 by American Society of Hematology

4. Saturation of DFO or 311 with iron prevents their effects at reducing the protein levels of cyclin A, B1, D1, D2, and D3 and increasing cyclin E protein levels.The SK-N-MC neuroepithelioma cell line was incubated with DFO (150 μM), the DFO-Fe complex (150 μ...
Saturation of DFO or 311 with iron prevents their effects at reducing the protein levels of cyclin A, B1, D1, D2, and D3 and increasing cyclin E protein levels.The SK-N-MC neuroepithelioma cell line was incubated with DFO (150 μM), the DFO-Fe complex (150 μM), 311 (25 μM), or the 311-Fe complex (25 μM) for 30 hours at 37°C. Western blot analysis was then performed as described in “Materials and methods.” Results shown are representative of 3 separate experiments performed.
Jin Gao, and Des R. Richardson Blood 2001;98:
©2001 by American Society of Hematology

5. Ribonucleotide reductase inhibitor HU acts differently to DFO and 311 as it increases the protein levels of cyclins A, B1, D1, D2, and D3 but has similar action to chelators in terms of increasing cyclin E.The SK-N-MC neuroepithelioma cell line was incubate...
Ribonucleotide reductase inhibitor HU acts differently to DFO and 311 as it increases the protein levels of cyclins A, B1, D1, D2, and D3 but has similar action to chelators in terms of increasing cyclin E.The SK-N-MC neuroepithelioma cell line was incubated for 30 hours at 37°C with DFO (150 μM), 311 (25 μM), or HU (1-300 μM). Western blot analysis was then performed as described in “Materials and methods.” Results shown are representative of 3 separate experiments performed.
Jin Gao, and Des R. Richardson Blood 2001;98:
©2001 by American Society of Hematology

6. Chelator 311 markedly reduced the expression of cyclin-dependent kinase 2 (cdk-2) but not cdk4.The SK-N-MC neuroepithelioma cell line was incubated for 30 hours at 37°C with DFO (5-150 μM) or 311 (1-25 μM).
Chelator 311 markedly reduced the expression of cyclin-dependent kinase 2 (cdk-2) but not cdk4.The SK-N-MC neuroepithelioma cell line was incubated for 30 hours at 37°C with DFO (5-150 μM) or 311 (1-25 μM). Western blot analysis was then performed as described in “Materials and methods.” Results shown are representative of 3 separate experiments performed.
Jin Gao, and Des R. Richardson Blood 2001;98:
©2001 by American Society of Hematology

7. The chelator 311 has no effect on p53 or mdm-2 mRNA levels but increases the expression of WAF1 mRNA in a variety of cell types with wild-type or mutant p53.The SK-N-MC neuroepithelioma, BE-2 neuroblastoma, IMR-32 neuroblastoma, SK-N-SH neuroblastoma, and M...
The chelator 311 has no effect on p53 or mdm-2 mRNA levels but increases the expression of WAF1 mRNA in a variety of cell types with wild-type or mutant p53.The SK-N-MC neuroepithelioma, BE-2 neuroblastoma, IMR-32 neuroblastoma, SK-N-SH neuroblastoma, and MCF-7 breast cancer cell lines were exposed to control medium or 311 (25 μM) for 30 hours at 37°C. Cells were harvested, and the total mRNA was isolated using standard techniques.20 (A) Ethidium bromide staining of the agarose gel; (B) p53; (C) mdm-2; (D) WAF-1; (E) β-actin. The result is representative of 2 separate experiments performed.
Jin Gao, and Des R. Richardson Blood 2001;98:
©2001 by American Society of Hematology

8. Effect of DFO and 311 concentration on cyclin-dependent kinase inhibitors and other cell-cycle inhibitory molecules.The SK-N-MC neuroepithelioma cell line or SH-SY-5Y neuroblastoma cell line* was incubated for 30 hours at 37°C with DFO (5-150 μM) or 311 (1-...
Effect of DFO and 311 concentration on cyclin-dependent kinase inhibitors and other cell-cycle inhibitory molecules.The SK-N-MC neuroepithelioma cell line or SH-SY-5Y neuroblastoma cell line* was incubated for 30 hours at 37°C with DFO (5-150 μM) or 311 (1-25 μM). Western blot analysis was then performed, as described in “Materials and methods.” Results shown are representative of 3 to 5 separate experiments performed.
Jin Gao, and Des R. Richardson Blood 2001;98:
©2001 by American Society of Hematology

9. Effect of chelator concentration, chelator incubation time, and complexation with Fe on the phosphorylation status of the retinoblastoma susceptibility gene product (pRb).(A) Chelator concentration.
Effect of chelator concentration, chelator incubation time, and complexation with Fe on the phosphorylation status of the retinoblastoma susceptibility gene product (pRb).(A) Chelator concentration. The SK-N-MC neuroepithelioma cell line was incubated for 30 h at 37°C with DFO (5-150 μM) or 311 (1-25 μM). (B) Chelator incubation time. Cells were incubated for 2-30 h with DFO (150 μM) or 311 (25 μM). (C) Complexation with Fe. Cells were incubated with DFO (150 μM) or 311 (25 μM) or their preformed Fe complexes for 30 hours at 37°C. Western blot analysis was then performed after each of these incubation conditions, as described in “Materials and methods.” Results shown are representative of 3 separate experiments performed.
Jin Gao, and Des R. Richardson Blood 2001;98:
©2001 by American Society of Hematology

10. Schematic illustration showing multiple effects of Fe chelators on the expression of molecules involved in cell-cycle control.Iron chelators deplete intracellular Fe pools that can lead to a number of effects.
Schematic illustration showing multiple effects of Fe chelators on the expression of molecules involved in cell-cycle control.Iron chelators deplete intracellular Fe pools that can lead to a number of effects. In SK-N-MC neuroepithelioma cells, these compounds act through a p53-independent pathway to markedly increase the levels ofWAF1 and GADD45 mRNAs. However, the mRNAs of these cell-cycle inhibitory molecules are not efficiently translated, leading to possible cell-cycle dysregulation. Iron chelation also markedly reduces the expression of cyclins D1, D2, and D3, each of which forms a complex with cyclin-dependent kinase 4 (cdk4). The cyclin D-cdk4 complex is involved in progression through the G1phase and the phosphorylation of the retinoblastoma susceptibility gene product (pRb). Phosphorylation of this latter molecule is required for it to release the E2F family of transcription factors critical for the transcription of genes essential for cell-cycle progression. Chelators also depress the expression of cdk2, which combines with cyclin E to further phosphorylate pRb that is essential for G1/S progression (see “Discussion” for further details).
Jin Gao, and Des R. Richardson Blood 2001;98:
©2001 by American Society of Hematology