Blackwell Publishing Asia
CR229, a novel derivative of ββββ -carbolin-1-one,
induces cell cycle arrest and apoptosis in HeLa cells via p53 activation
Min Kyoung Kim,1 Ha Lim Oh,1 Bu-Young Choi,2 Haeyoung Lim,1 Youl-Hee Cho1 and Chul-Hoon Lee1,3
1Department of Medical Genetics, College of Medicine, Hanyang University, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791; 2Department of Molecular Biology and Pharmacology, C&C Research Laboratories, 146-141 Annyung-dong, Hwasung-City, Kyunggi-Do 445-970, Korea
(Received December 5, 2006/Revised March 19, 2007/2nd Revised April 20, 2007/Accepted April 28, 2007/Online publication July 9, 2007)
In the course of screening for novel anticancer compounds, CR229 (6-Bromo-2,3,4,9-tetrahydro-carbolin-1-one), a novel derivative of ββββ-carbolin-1-one, was generated as a new scaffold candidate. For the first time, the authors demonstrate that CR229 inhibited the growth of HeLa cells by the induction of cell cycle arrest and apoptosis.
Analysis of flow cytometry and western blots of HeLa cells treated with 2.5 µµµµM CR229 revealed an appreciable cell cycle arrest in the G1, G2/M phase and apoptotic induction via the p53-dependent pathway. Furthermore, the release of cytochrome c from mitochondria was detected using confocal microscopy in HeLa cells treated with CR229. Accordingly, these data demonstrate that the anticancer activity of CR229 is associated with: (i) the down-regulation of cyclins and cyclin-dependent kinase; (ii) the induction of p53, p21, and p16; and (iii) the activation of caspase-3. (Cancer Sci 2007; 98: 1402–1407)
T
he discovery of natural β-carboline, a class of indole alkaloid, from marine invertebrates as antitumor and antiviral agents has stimulated great interest in the synthetic and pharmaceutical studies of β-carboline derivatives. A particularly interesting target includes the derivatives of β-carbolin-1-one, which have been found to possess potent bioactivities on the central nervous system,(1) and significant antiserotonin,(2) as well as antioxidative activity.(3) Recently, β-carbolin-1-one derivatives have been reported to inhibit colon and lung tumor,(4) and the proliferation of HeLa cells.(5,6) However, the detailed anticancer mechanisms of β-carbolin-1-one derivatives can not be elucidated.The cell cycle is coordinated by three families of molecules;
cyclins, CDK, and CKI. The function of CDK-cyclin complexes is regulated by the phosphorylation of CDK at different positions and proteolysis of cyclin.(7) In mammalian cells, CDK2, 3, 4, and 6 play a role in the G1 and/or S phase, while CDK1 appears to be restricted to mitosis. To antagonize the actions of the CDK-cyclin complexes in the cell cycle, mammalian cells also express CKI, such as p21 and p27, which bind to the complexes, thereby preventing their activation and inhibiting previously activated complexes. The expression of these CKI is induced by inhibitory regulators of the cell cycle, and occurs during the normal state of growth arrest.(8)
Apoptosis can occur at any stage of the cell cycle. In particular, many cells regulate the cell progression from the late G1 phase into the S phase using p53 and the activation of CDK.(9) The growth suppressor p53 is a tightly regulated transcription factor that can induce either cell cycle arrest or apoptosis according to its expression level.(10) In addition, p53 is particularly important for cell protection when the DNA is damaged by radiation, chemicals, or viral infection. p53 normally responds to different forms of cellular stress by targeting the activation of checkpoint genes that inhibit progression of the cell cycle (such as p21WAF1/CIP1) and/or trigger apoptotic cell death.(11,12) Stimuli that activate p53 include radiation, hypoxia, and chemotherapeutic drugs. Modu- lation of the p53 molecule is in part achieved by post-translational
modifications, such as phosphorylation and acetylation, which promote the formation of specific interactions with other proteins and target gene regulatory elements.(13) Meanwhile, the loss of p53 activity via mutation, deletion, or inactivation by endogenous or viral oncogenes leads to the propagation of DNA damage, resulting in genetic instability.
Accordingly, the current study was designed to elucidate the anticancer mechanisms of a novel compound, CR229 (6-Bromo- 2,3,4,9-tetrahydro-carbolin-1-one; Fig. 1), in HeLa cells. When investigating the chemopreventive potential of CR229, it was found that CR229 induced apoptosis and cell cycle arrest in can- cer cells accompanied by: (i) the down-regulation of cyclins and CDK; (ii) the induction of p53, p21, and p16; and (iii) the activation of caspase-3.
Materials and Methods
Cell culture and reagents.The human normal and cancer cell lines were all purchased from the American Type Culture Collection (Rockville, MD, USA). The antibodies were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), except for p53-phospho-Ser15, that was obtained from Cell Signaling Technology (Beverly, MA, USA). All other materials were obtained from Sigma (St Louis, MO, USA).
Cell growth inhibition assay. The viability test was assessed using the conventional colorimetric dye reduction method based on the reduction of MTT (Promega, Madison, WI, USA.), while the viable cell number was measured at 570 nm using an ELISA reader (Molecular Device, Sunnyvale, CA, USA).
Flow cytometric analysis for cell cycle distribution. After treatment with CR229, the cells (1 × 106 cells/mL) were washed and fixed in 70% ethanol. Immediately before the analysis, the cells were stained with a solution containing 0.2 mg/mL PI for 1 h at 4°C and 0.1 mg/mL RNase A for 30 min at 37°C, then analyzed with a fluorescent-activated cell sorter FACScan (Becton Dickinson, San Diego, CA, USA) using Cell Quest software (version 5.1.1).
TUNEL assay for apoptotic induction. The apoptosis detection system (TUNEL) developed by Promega was used according to the supplier’s recommended protocol. After reaction, the cells were analyzed using FACScan with Cell Quest software.
Nuclear staining with DAPI.The cells were fixed with 4%
neutral buffered formalin. Thereafter, the cell suspension was smeared on slides and dried at RT, and then the fixed cells were
3To whom correspondence should be addressed. E-mail: [email protected] Abbreviations: CDK, cyclin-dependent kinase; CKI, cyclin-dependent kinase inhibi- tor; DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; FITC, fluoroscein-5-isothiocyanate; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5- dimphenyltetrazolium bromide; PARP, poly(ADP-ribose) polymerase; PBS, phosphate- buffered saline; PI, propidium iodide; pRb, retinoblastoma protein; PVDF, polyvi- nylidene fluoride; RT, room temperature; RT-PCR, reverse transcription–polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis;
TUNEL, Tdt-mediated dUTP nick-end labeling.
stained with DNA-specific fluorochrome DAPI for 30 min at 37°C. Finally, the slides were observed under a fluorescence microscope.
Immunocytochemistry using confocal microscopy. Cells were un- treated or treated with CR229 for 24 h. After fixation with 2%
formaldehyde, cells were permeabilized for 10 min in 100 mM glycine in PBS, and DNA was stained using 1 µg/mL PI in PBS and washed twice for 5 min with PBS. Incubation with the mouse anticytochrome c antibody was performed for 4 h at room temperature after dilution in PBS and visualized with donkey antirabbit IgG FITC antibody. Images were obtained using confocal laser scanning microscopy (LSM 410; Zeiss, Jena, Germany).
Western immunoblotting. The protein extracts (30–50 µg) were analyzed based on 8–14% SDS-PAGE and transferred to a PVDF membrane (Millipore; Billerica, MA, USA). The membranes were blocked with 5% (w/v) non-fat dry milk, then incubated with the indicated antibodies in Tris-buffered saline (10 mM Tris-HCl, 150 mM NaCl, pH 7.6) containing 0.1% Tween-20 with gentle shaking at 4°C for 2–12 h. As a secondary antibody, peroxidase-conjugated goat antimouse, rabbit antibody was used. The signals were detected using an ECL western blotting kit (GE Healthcare, Buckinghamshire, UK).
Real-time RT-PCR. The total RNA was extracted from the cells using an Rneasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. RT-PCR for the mRNA quantification was then performed with an iCycler iQ multi-color real-time PCR detection system (Bio-Rad, USA). Specific primers were used for the human p53 (5′-GTTCCGAGAGCTGAATGAGG- 3′ and 5′-TGAGTCAGGCCCTTCTGTCT-3′), p21 (5′-CGCGA- CTGTGAYGCGCTA-3′ and 5′-AAGTCGAAGTTCCATCG CTCA- 3′), p16 (5′-ACGCACCGAATAGTTACGG.-3′ and 5′-CCAGGTCC ACGGGCAGA-3′), and β-actin (5′-CCAACCGCGAGAAG- ATGACC-3′ and 5′-TGCCAATGGTGATGACCTGC-3′). The relative quantization was calculated using the comparative threshold cycle method.(14) The housekeeping gene β-actin was used to confirm the homogeneity of the DNA products.
RNA interference assay. The cells were plated at 5 × 104 cells/
mL in a 12-well plate. 24 h after plating, the cells were transfected with either the control siRNA (against fluorescein conjugate) or the p53 siRNA (p53 siRNA 825, 5′-GCATCTTATCCGAGTG- GAA-3′ and p53 siRNA 1026, 5′-GACTCCAGTGGTATCTAC- 3′). The transfection of the siRNA was carried out according to the manufacturer’s instructions (Cell Signaling Technology).
Subsequently, the cells were collected and analyzed using flow cytometry and western blotting.
Statistical analysis. The data are reported as the mean ± standard deviation of three independent experiments and were evaluated using Student’s t-test. Values of P < 0.05 were considered to be statistically significant.
Results
Anti-proliferative effect of CR229 on human cancer cells. To deter- mine the inhibition of CR229-induced cell growth, various human cancer cells treated with CR229 were assessed using an MTT assay. As shown in Table 1, among the cancer cells treated
with 0.5–100 µM of CR229 for 24 h, the cell growth of HeLa (GI50: 1 µM) and PA-1 (GI50: 3.4 µM) was strongly inhibited, while the growth of the normal stomach and lymphocyte cells treated with CR229 was not significantly inhibited.
CR229-induced apoptosis in HeLa cells. To verify whether the growth inhibitory effect of CR229 was due to apoptosis, a TUNEL assay was used to examine the DNA fragmentation in the nuclei of the cells treated with 2.5 µM CR229 for 24 and 48 h. As shown in Fig. 2, the DAPI and TUNEL assay revealed apoptotic induction in the HeLa cells.
As shown in Fig. 2c, CR229 induced the activation of caspase-3. Furthermore, the cleavage of PARP was used as an indicator of caspase activation in response to 10 µM of CR229 treatment, which became obvious after 24 h (Fig. 2c). These results were also consistent with the results of a flow cytometric analysis, which revealed apoptotic induction after 24 h of treatment with CR229 (Fig. 3).
Release of cytochrome c induced by CR229. In response to a variety of apoptosis-inducing agents, cytochrome c is released from mitochondria into the cytosol. As shown in Fig. 4, cytochrome c (green) release was observed significantly in HeLa cells treated with 2.5 µM CR229 for 24 h. However, cytochrome c release in non-treated control cells was not observed. Therefore, we conclude that cytochrome c release is induced by CR229.
Effect of CR229 on cell cycle progression in HeLa cells. To elucidate the mechanism of the CR229-induced inhibition of HeLa cell growth, the cell cycle progression was analyzed using flow cytometry, which revealed an appreciable arrest in the G2/M and G1 phase after treatment with 2.5 µM CR229. As shown in Fig. 3, the HeLa cell population gradually increased from 21%
at 0 h to 24% after 12 h in the G2/M phase and from 40% at 0 h to 48% after 24 h in the G1 phase after exposure to CR229.
Furthermore, an eventual progression to apoptosis was observed after 48 h (25%).
Furthermore, as shown in Fig. 5, CR229 suppress the hyper- phosphorylation of pRb with a commensurate increase in the hypophosphorylated form. The expression levels of E2F-1 were also reduced. The levels of cyclin D1 and cyclin E decreased at 12 h, yet there was almost no change in the CDK2 and CDK4 proteins. To determine the CR229-induced G2/M phase arrest, the expression levels of CDK1, cyclin B1, and cyclin A were assessed.
As expected, the expression levels were significantly reduced 24 and 48 h after treatment with 2.5 µM CR229.
Effect of CR229 on the cellular level of p53 and its downstream genes. Post-translational modification of p53 by phosphorylation has been proposed to be an important mechanism by which p53 stabilization and function are regulated. Phosphorylation on Ser- 15 is believed to affect interaction with the negative regulator, MDM2, and hence contribute to the stabilization of p53.
Phosphorylation on Ser-392 is believed to enhance the specific DNA binding of p53.
Fig. 1. Chemical structure of CR229.
Table 1. GI50 value of CR229 on human cancer and normal cells
Cell line GI50/µM Cell line GI50/µM
Normal lymphocyte (+/+ p53) >100 HT-29 (+/+ p53) 72 Normal stomach (+/+ p53) >100 HL-60 (–/– p53) 64
HeLa (+/+ p53) 1 SK-OV-3 (–/– p53) 43
PA-1 (+/+ p53) 3.4 SK-MEL-2 (+/– p53) 64
MCF-7 (+/+ p53) 40 A549 (+/+ p53) 78
LNCaP (+/+ p53) 50 HCT-116 (+/+ p53) 48
Cells were seeded in 96-well plates and incubated with 1–100 µM CR229 for 24 h. The cell viability was determined based on an MTT assay.
Fig. 2. Induction of apoptosis by CR229 in HeLa cells. The cells were treated with 2.5 µM CR229 for 24 or 48 h, then stained with d-UTP fluoroscein-5-isothiocyanate (FITC) and propidium iodide (PI) and the nuclei were analyzed for their DNA content using flow cytometry. (a) terminal dUTP nick end labeling (TUNEL) assay. (b) Micro- scopic examination of CR229-treated cells stained with DAPI. (c) Western blots of caspase-3 activations and poly(ADP-ribose) polymerase (PARP) cleavage.
FPO
Fig. 3. Quantification of cell cycle arrest by CR229 in HeLa cells. The cells were treated with 2.5 µM CR229 for the indicated time, then stained with propidium iodide (PI) and the nuclei analyzed for their DNA content by flow cytometry. *P < 0.05, **P < 0.01. Ap, apoptosis.
As shown in Fig. 6a, phosphorylation of p53 at Ser-392 and Ser-15 increased after 3 h and 12 h, respectively, in HeLa cells treated with CR229. Furthermore, the cellular level of p21WAF1/CIP1 and p16INK4A increased after treatment with 2.5 µM CR229.
Accordingly, a real-time RT-PCR analysis demonstrated that the mRNA level of p21WAF1/CIP1 and p16INK4A increased 8- and 2-fold compared with the control after 24 h treatment
(Fig. 6b). However, the mRNA level of p53 in the HeLa cells treated with CR229 was not increased (Fig. 6b), whereas the cellular protein level of p53 was increased (Fig. 6a).
Therefore, these results indicate that CR229 induced the stabilization and activation of p53 by phosphorylation, followed by the transcriptional activation of the p21WAF1/CIP1 and p16 INK4A genes.
Inhibition of CR229-induced apoptosis by knock-down of p53 using siRNA transfection. To directly address the role of p53 in CR229-induced cell death, the siRNA technique was used to selectively knock-down the p53 gene. When the HeLa cells were transfected with siRNA of p53, then treated with 2.5 µM CR229 for 24 h, the apoptotic induction was decreased by 67%
compared to that without siRNA transfection (Fig. 7a,b).
Furthermore, the levels of p53 and p21 in HeLa cells transfected with p53 siRNA were significantly decreased by 90–100%, as demonstrated using western blotting (Fig. 7c). Accordingly, these data demonstrated the involvement of p53 in CR229- induced apoptosis in HeLa cells.
Discussion
Recently, the derivatives of β-carbolin-1-one have been reported to possess various bioactivities, such as antiserotonin, and antioxidative activity, as well as antiproliferative effects on cancer cells. However, the detailed anticancer mechanisms of β-carbolin-1-one derivatives have not be elucidated.
The current study was designed to elucidate the anticancer mechanisms of a novel derivative of β-carbolin-1-one, CR229 (6-Bromo-2,3,4,9-tetrahydro-carbolin-1-one), in HeLa cells. The authors demonstrate that CR229 exhibits two key biochemical effects, accumulation of cells in the G1 and G2/M phase of the cell cycle progression and the induction of apoptosis, both of which might be important in anticancer activity.
Interestingly, as shown in Table 1, among the cancer cells treated with CR229, only the proliferation of HeLa (GI50: 1 µM) and PA-1 cells (GI50: 3.4 µM) was strongly inhibited, while the growth of the rest of the cancer cells tested so far, and normal stomach and lymphocyte cells was not significantly inhibited.
Unfortunately, this extremely selective antiproliferative activity of CR229 on HeLa and PA-1 cells is still unclear. A further detailed study on this selectivity is needed.
The induction of cell cycle arrest in HeLa cells treated with CR229 was analyzed using flow cytometry and western blotting, which revealed an appreciable arrest in the G2/M and G1 phase
Fig. 4. Effect of CR229 on cytochrome c release from the mitochondria. (a) HeLa cells with confocal medium and (b) cells treated with 2.5 µM CR229 for 24 h were fixed and labeled for cytochrome c (green) and DNA (red). Images were obtained using confocal laser scanning microscopy.
Fig. 5. Effect of CR229 on cell cycle regulatory proteins in HeLa cells. Proteins from HeLa cells treated with 2.5 µM CR229 for 24 h were analyzed using western blotting. β-actin served as the internal control. CDK, cyclin-dependent kinase.
through down-regulation of cyclins and CDK, and up-regulation of downstream genes from p53, such as p21 and p16, which play a pivotal role in the control of cell cycle progression by opposing the activities of CDK.
Furthermore, the authors have elucidated that CR229 resulted in the cleavage of PARP, and subsequent DNA degradation through the activation of caspase-3, which was induced by cytochrome c released from the mitochondria in HeLa cells.
Therefore, the present data indicated that the apoptotic induction by CR229 in HeLa cells was involved apparently in a p53- dependent mitochondrial pathway.
Commonly used chemotherapeutic agents, which in many cases act by inducing DNA damage, activate cellular pathways that inhibit proliferation and lead to growth arrest or apoptosis.
Recently, it was also reported that several CDK inhibitors induced the activation (phosphorylation) of p53 in various human cancer cells.(15–17)
In the present study, CR229 was found to induce cell cycle arrest and apoptosis through the activation of p53 by phosphorylation at Ser-15 and Ser-392. Ser-15 is a functionally important residue within the p53 amino-terminal region, and the phosphorylation of Ser-15 represents an early cellular response to a variety of genotoxic stresses.
Furthermore, the p53 phosphorylated at Ser-15 displays reduced binding to the inhibitor protein, MDM2, which was targeted for proteasome-mediated degradation that inhibits its transactivating function.(18–20)
Accordingly, the phosphorylation on Ser-15 could be explained as one of the reasons for the increased cellular protein level of p53 without up-regulation of the p53 gene expression in the HeLa cells treated with CR229 (Fig. 6). The phosphorylation on Ser-392 is believed to enhance the specific DNA binding of p53. Therefore, the phosphorylation of p53 at Ser-392 is altered in human tumors and has been reported to influence growth suppressor function, DNA binding and the transcriptional activation of p53.(21)
The authors demonstrated that the inhibition of p53 expression by transient transfection with siRNA resulted in significant decrease of apoptotic induction by 67% in HeLa cells treated with CR229 when compared with control cells, and decrease of the cellular level of p21 protein. Therefore, this data provides direct evidence of the involvement of p53 in the CR229-induced cell cycle arrest and apoptosis in HeLa cells.
In summary, CR229 may have a cancer chemopreventive/
antiproliferative effect on HeLa cells, while having no cytotoxic effect on normal cells. CR229-induced apoptosis and cell cycle arrest were mediated by the p53 pathway and cytochrome c release, respectively. Consequently, CR229 would appear to have potential as a novel anticancer drug candidate.
Acknowledgments
This work was supported mainly by Seoul R & BD Program (10580) and partly by grant No. R01-2003-000-10594-0 from the Basic Research Program of the Korea Science and Engineering Foundation.
Fig. 6. CR229-induced activation of p53, p21, and p16 proteins in HeLa cells. The cells were treated with 2.5 µM CR229 for the indicated times, then the total protein and RNA were extracted. To detect the level of mRNA, real-time quantitative reverse transcription–
polymerase chain reaction was performed. (a) Protein levels of p53 and its downstream genes (p21 and p16) based on western blotting in HeLa cells. (b) p53, p21, and p16 mRNA levels and relative threshold cycle (Ct) value for each gene compared with the reference gene (β-actin). *P < 0.05.
Fig. 7. Inhibition of CR229-induced apoptosis by transfection of p53 siRNA in HeLa cells. The cells were transfected with a control and p53 siRNA duplex for 24 h plus recovery, followed by treatment with 2.5 µM CR229 for 24 h. (a,b) Apoptosis and cell cycle distributions assessed using propidium iodide (PI) staining with flow cytometry, as described in materials and methods. (c) p53 and p21 protein levels checked based on western blot analysis. *P < 0.05, **P < 0.01.
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