help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, H.-L.
Right arrow Articles by Yeung, S.-C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, H.-L.
Right arrow Articles by Yeung, S.-C. J.
The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 2 763-772
Copyright © 2003 by The Endocrine Society

p21 Waf-1 (Cip-1) Enhances Apoptosis Induced by Manumycin and Paclitaxel in Anaplastic Thyroid Cancer Cells

Hui-Ling Yang, Jing-Xuan Pan, Lily Sun and Sai-Ching Jim Yeung

Departments of General Internal Medicine, Ambulatory Treatment, and Emergency Care (H.-L.Y., S.-C.J.Y.), Experimental Therapeutics (J.-X.P.), and Endocrine Neoplasia and Hormonal Disorders (L.S., S.-C.J.Y.), The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Address all correspondence and requests for reprints to: S. Jim Yeung, M.D., Ph.D., Assistant Professor, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 437, Houston, Texas 77030. E-mail: syeung{at}notes.mdacc.tmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our previous studies demonstrated that manumycin (a farnesyltransferase inhibitor) enhanced the antineoplastic activity and induction of apoptosis when combined with paclitaxel against anaplastic thyroid cancer cells. We found that manumycin induces endogenous expression of p21 Waf-1 in anaplastic thyroid cancer cells. Manumycin increased the activity of the p21promoter, the level of p21mRNA, and the amount of p21 protein. We hypothesized that p21 had a proapoptotic effect in cells treated with manumycin, or paclitaxel, or both agents. By measuring viability and caspase-3 activity, we found that stably transfected KAT-4 cells with p21 cDNA under the control of a metallothionein promoter were more sensitive to manumycin alone, paclitaxel alone, and manumycin plus paclitaxel when p21was induced. The increased sensitivity of the cells with induced p21 was associated with an increase in caspase-3 activity (i.e. apoptosis). We also found that cells with both p21 alleles deleted were less sensitive to manumycin plus paclitaxel than its wild-type parent cells. Expression of p21 per se did not induce apoptosis but enhanced the cytotoxic effects of manumycin and paclitaxel. These findings suggested that p21 might be required to maintain cell sensitivity to the cytotoxic effects of manumycin and paclitaxel.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANAPLASTIC THYROID CANCER (ATC) is one of the most aggressive solid tumors (1). Despite the current multimodality approach to treatment of ATC, patients with the disease have a very poor prognosis, with a mean survival of 3–7 months (2, 3, 4, 5). Thus, investigating new therapeutic modalities against ATC is necessary.

Apoptosis is a form of programmed cell death regulated by genetic programs and by signaling and regulatory pathways initiated by various extracellular stimuli/agents. Tumor growth is a dynamic balance between cellular proliferation and apoptosis is a general concept applicable to thyroid carcinomas. ATCs have both rapid proliferation (short doubling time; Refs. 6 and 7) and resistance to apoptosis (perhaps conferred by mutations in the p53 tumor suppressor gene; Refs. 8, 9, 10). However, the biomolecular mechanisms behind the regulation of apoptosis in thyroid cancer have not been well defined (11).

Paclitaxel, an inhibitor of tubulin depolymerization (12, 13) that has been used to treat ovarian cancer, breast cancer, lung cancer, and head and neck cancer (14), has been shown to have antineoplastic activity against ATC in vitro (15) and in a clinical trial (16). It has also recently been shown that combining a farnesyltransferase inhibitor (FTI) with a taxane can result in enhanced antitumor activity (17, 18, 19). The FTI manumycin A, a natural production of Streptomyces parvulus, inhibits farnesyltransferase by competing with the farnesyl pyrophosphate substrate (20, 21) and has antitumor activity in vitro (22) and in xenograft models against a variety of cancers (23, 24, 25, 26).

We previously demonstrated that manumycin enhanced the antineoplastic activity of paclitaxel against ATC cells in vitro (25) and in a nude mouse xenograft model (25, 26). The enhanced effect on ATC cells was due to enhanced induction of apoptosis, and the point of interaction between manumycin and paclitaxel was at the release of cytochrome C or further upstream in the apoptosis regulatory pathway (27). Our studies also suggested that inhibition of tumor angiogenesis played a significant role in the antineoplastic effect of the combination of manumycin plus paclitaxel (26). This finding is in agreement with several other studies showing that FTIs inhibit tumor angiogenesis by down-regulating vascular endothelial growth factor expression (28, 29, 30, 31, 32) and inhibiting endothelial cell proliferation (28).

The antineoplastic action of FTIs may involve multiple mechanisms—antiproliferative, proapoptotic, and antiangiogenic (31). The intracellular pathways involved in the antiproliferative and proapoptotic effects of FTIs are complex. It is widely accepted that one mechanism by which FTIs exert an antineoplastic effect is interference with the Ras signaling pathway (33, 34). A second mechanism is increase in geranylgeranylated RhoB, which inhibits progression through the cell cycle (35, 36). A third mechanism is inhibition of the phosphatidylinositol 3-kinase/AKT2-mediated cell survival and adhesion pathway (37).

The cell cycle regulating protein p21 Waf-1 may be involved in the antineoplastic effect of FTIs. In tumor cells with wild-type p53 tumor suppressor, an FTI induces p21 Waf-1 expression and G1 arrest in the cell cycle (38), and the p21 Waf-1induction may be due to increase in geranylgeranylated RhoB (35). However, the role of p21 Waf-1 in apoptosis is controversial. There is evidence that it is antiapoptotic. G1 arrest by p21 Waf-1 has been suggested to protect cells from apoptosis induced by DNA damage (39). In U937 cells (human myelomonocytic leukemia), p21 Waf-1 prevents apoptosis induced by sodium butyrate (40); nuclear localization of p21 Waf-1 is associated with G1 arrest, and subsequent differentiation into monocytes is associated with cytoplasmic localization of p21 Waf-1 where p21 Waf-1 confers resistance to apoptosis by binding to apoptosis signal-regulating kinase 1 (41). In RKO cells (human colorectal carcinoma), p21 Waf-1 also appears to protect against prostaglandin A2-mediated apoptosis (42). On the other hand, there is evidence that p21 Waf-1 is proapoptotic. In Hep3B cells (human hepatoma), retinoic acid-induced apoptosis is associated with increased p21 Waf-1 expression (43). Increased apoptosis is observed with a three-drug combination treatment of leukemic T-cells in association with increased expression of p53 and p21 Waf-1 (44). In HeLa, SiHa, Z172, and C33A cells (all cervical cancer cell lines), adenovirus-mediated expression of p21 Waf-1 induces apoptosis (45). In ATC cells, we discovered that manumycin induced expression of p21 Waf-1. We hypothesized that p21 Waf-1 had a proapoptotic effect in cells treated with manumycin or paclitaxel or both agents. To address the controversial role of p21 Waf-1 in apoptosis, we investigated this hypothesis by evaluating the response to manumycin and paclitaxel in an ATC cell line (KAT-4) stably transfected with an inducible p21 Waf-1construct. In addition to investigating the effect of forced/ectopic expression of p21 Waf-1, we also investigated the effect of loss of p21 Waf-1on the antineoplastic effect of manumycin and paclitaxel using a colon cancer cell line with both p21 Waf-1 alleles deleted (HCT-116 p21-/-; Ref. 46).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Manumycin A, paclitaxel, tissue-culture-grade dimethylsulfoxide (DMSO), Tween 20, zinc sulfate (ZnSO4 · 7 H2O, cell culture grade), and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO). All tissue culture media, trypsin/EDTA, and additives were purchased from Life Technologies, Inc. (Gaithersburg, MD). Monoclonal antiactin antibody and anti-p21 antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Hygromycin B (cell culture tested) was purchased from Roche Diagnostic (Indianapolis, IN) and Sigma Chemical Co. The EnzChek caspase-3 assay kit no. 2, propidium iodide, and sulforhodamine B were purchased from Molecular Probes, Inc. (Eugene, OR).

Cell culture

The colon cancer cell line with loss of both p21alleles (HCT-116 p21-/-) and its parent cell line (HCT-116 p21+/+) were kindly provided by Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD; Ref. 46). The KAT-4 ATC cell line was provided by Dr. K. Ain (University of Kentucky, Lexington, KY; Ref. 15). The ARO cell line was provided by Dr. J. Fagin (University of Cincinnati, Cincinnati, OH; Ref. 8). All of the cells were incubated at 37 C in a water-saturated atmosphere with 5% CO2. The human ATC cell lines (KAT-4, ARO, and cell lines derived by stable transfection) were cultured in RPMI-1640 medium. The HCT-116 p21+/+ and HCT-116 p21-/- cell lines were cultured in McCoy’s medium. The media were supplemented with heat-treated bovine serum (10%), penicillin (50 U/ml), streptomycin (50 µg/ml), nonessential amino acids (1x), pyruvate (1 mM), glutamine (2 mM), and Amphotericin B (2.5 µg/ml). The medium in which the stably transfected cell lines were cultured also contained hygromycin B (200 µg/ml).

Drug treatments

Manumycin and paclitaxel were dissolved in DMSO. The stock solutions of these drugs were diluted in complete culture medium with 10% bovine serum (as described above) to appropriate concentrations. The final concentration of DMSO in the culture medium was 0.1% (vol/vol) or less.

Late log-phase cultures were trypsinized, and 4000–8000 cells were plated in each well of a 96-well tissue culture plate. The next morning, medium in each well was replaced with fresh medium or medium containing various concentrations of the test drugs. The cells were then incubated for specific periods of time.

Insertion of p21 cDNA into expression vector

The full-length wild-type p21 cDNA plasmid (pCEP4-p21/waf-1) was kindly provided by Dr. B. Vogelstein (47). The p21 cDNA was released from the plasmid by restriction digestion with NotI. The NotI fragment was then ligated into the NotI site in the polylinker cloning region of pMEP4 (Invitrogen Life Technologies, Inc., Carlsbad, CA), which is an expression vector containing the human metallothionein promoter, the simian virus 40 polyadenylation site, and a hygromycin B-resistant element. The NotI fragment has only one XhoI restriction site near the 3' end. XhoI restriction digestion of plasmids from ligation of pMEP4 and p21 cDNA will produce a 2-kb fragment and a 10-kb fragment if and only if the p21 cDNA was ligated in the antisense direction. After confirming the correct size of the insertion fragment by digestion with NotI, plasmids with p21 cDNA ligated in the sense direction were selected by restriction digestion with XhoI.

Stable transfection of expression plasmids

KAT-4 cells in exponential growth were cultured to 50–80% confluence and transfected using Lipofectamine Plus (Invitrogen Life Technologies, Inc.) according to the manufacturer’s recommended procedure with the plasmids described above. The KAT-4 cell line was chosen for the transfection experiments because the transfection efficiency using Lipofectamine Plus is higher in KAT-4 cells than in the ARO cell line (Yeung, S.-C. J., unpublished data). Cells were then selected in the presence of 800 µg/ml hygromycin B for 3 wk, after which resistant colonies were maintained in the presence of 200 µg/ml hygromycin B and expanded for further analysis.

RT-PCR analysis of p21Waf-1 expression

ARO cells were treated with control culture medium, 54 µM manumycin alone, 21 µM paclitaxel alone, or both manumycin and paclitaxel for 6 h. Total RNA was purified from the cell samples using the RNEasy kit (QIAGEN Inc., Valencia, CA), and mRNA was purified using the Oligotex kit (QIAGEN Inc.). RT was performed with oligo (dT) and random hexamers as primers and Moloney murine leukemia virus reverse transcriptase (SuperScript II, Invitrogen Life Technologies, Inc.). PCR was performed with melting at 95 C for 1 min, annealing at 55 C for 1 min, and extension at 72 C for 2 min for 30 cycles. The primers used for amplifying p21were: forward, 5'-GTTCCTTGTGGAGCCGGAGC-3'; reverse, 5'-GGTACAAGACAGTAGCAGATC-3'. Control PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed with the following primers: forward, 5'-TGGTATCGTGGAAGGACTCATGAC-3'; reverse, 5'-ATGCCAGTGAGCTTCCCGTTCAGC-3'. The PCR products are evaluated by agarose gel electrophoresis. Images of ethidium bromide-stained agarose gels were captured using the Foto/Analyst Visionary Gel Documentation System (Fotodyne, Inc., Hart land, WI).

Gene expression analysis with DNA array

We used cDNA microarray hybridization to screen for possible pathways or genes involved in the effect of manumycin and paclitaxel on ARO cells. ARO cells were treated with control culture medium, 54 µM manumycin alone, 21 µM paclitaxel alone, or both manumycin and paclitaxel for 6 h. Three independent experiments were done for each treatment group. After treatment, the cell pellets were snap-frozen in liquid nitrogen. For each treatment group, the cell pellets from different experiments were pooled together, and RNA was extracted and reverse-transcribed to produce the probes for array hybridization. The Clontech Human Cancer 1.2k Array (BD Biosciences Clontech Laboratories, Inc., Palo Alto, CA) was used. The intensity of the hybridization signal was quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and the results were calculated using AtlasImage 2.0c (BD Biosciences Clontech Laboratories, Inc.).

Luciferase assay of p21 promoter activity

ARO cells were transfected with the WWP-Luc plasmid (kindly provided by Dr. B. Vogelstein; Ref. 47) using Lipofectamine Plus. After transfection overnight, the cells were trypsinized and split equally into 24-well plates and then treated with control culture medium, 54 µM manumycin alone, 21 µM paclitaxel alone, or both manumycin and paclitaxel for 6 h. Each treatment was performed in triplicate. The cells were lysed and assayed for luciferase activity using the Steady-Glo Luciferase Assay System (Promega Corp., Madison, WI) according to the procedures recommended by the manufacturer. The luminescence was measured using a Spectrafluor Plus microplate reader (Tecan, Maennedorf, Switzerland).

Preparation of total cell lysates

After experimental treatments, cells floating in the culture medium were pelleted by centrifugation. Cells that attached to the dish were rinsed with PBS. Both the cell pellet and the cells attached to the dish were then lysed in a total of 200–400 µl of RIPA buffer [1x PBS, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml phenylmethylsulfonyl fluoride, and Complete Protease Inhibitor Mix (Roche), one tablet per 50 ml]. The DNA in the lysate was sheared by rapidly passing the lysate five times through a 23-gauge needle or by sonication with eight 1-sec bursts at medium power.

SDS-PAGE and immunoblotting

SDS-PAGE was performed using standard methods. The protein concentrations of samples were measured using a modified Lowry method (DC Protein Assay, Bio-Rad Laboratories, Inc., Hercules, CA). Equal amounts of total protein from each sample were loaded onto the SDS-polyacrylamide gel. Kaleidoscope Prestained Standards (Bio-Rad Laboratories, Inc.) were used for molecular weight calibration. Immunoblotting was performed using polyvinylidine fluoride membranes (Hybond-P, Amersham Pharmacia Biotech, Piscataway, NJ). Images of the immunoblots are generated by enhanced chemiluminescence using the ECL kit (Amersham Pharmacia Biotech) or the SuperSignal West Dura extended duration substrate (Pierce Chemical Co., Rockford, IL) and recorded on Kodak X-omatic AR films (Eastman Kodak Co., Rochester, NY).

Colorimetric measurement of viable cells

The number of viable cells was measured using a tetrazolium dye method. In metabolically active cells, cleavage of the yellow tetrazolium salt MTT formed a colored formazan product by the succinate-tetrazolium reductase system of the mitochondrial respiratory chain. Standard protocols were followed. In brief, 20 µl MTT (3 mg/ml) were added to 100 µl culture medium in each well. After 4 h of incubation at 37 C, the amount of formazan dye was measured as absorbance at wavelength 590 nm with reference at wavelength 635 nm using a microtiter plate spectrophotometer (Spectrafluor Plus). All of the experiments were performed with the absorbance values within the linear range of this colorimetric assay.

Percentage viability was defined as 100% times the ratio of absorbance above background in the sample to the average absorbance above background in the control (DMSO-treated) samples. Background absorbance was measured in wells with cells that had been killed by exposure to 70% ethanol for 10 min.

The inhibitory concentration 50 (IC50) was calculated by linear regression of dose-response curves and interpolation based on the linear regression formula.

Soft agar clonogenic assay

KAT-4 p21S cells were trypsinized and counted using a hemocytometer. Equal numbers of cells were subcultured in 60-mm Petri dishes. After an overnight incubation, the dishes were divided into groups and treated with DMSO 0.1%, manumycin 6 µM, paclitaxel 2.3 µM, or both in the presence or absence of Zn2+ 100 µM for 24 h. The drug concentrations were reduced (with the same manumycin-to-paclitaxel ratio) such that a reasonable number of colonies survived after drug treatments. After treatments, the floating cells were collected by centrifugation, and the attached cells were collected by trypsinization. After the attached and floating cells from each dish were combined, the cells were washed twice with sterile PBS at ambient temperature. The cells from each Petri dish were resuspended in an equal volume of culture media. Tissue culture dishes (diameter, 60 mm) were prepared with a 2-ml base layer of 0.5% Difco Bacto agar. An equal volume of cell suspension from each treatment Petri dish was mixed with 0.8% Difco Bacto agar in RPMI 1640 culture medium and overlaid above the 0.5% agar. The plates were then incubated at 37 C for 18 d. Then, plates were stained with crystal violet (0.005%), and the colonies of more than 50 cells were counted. Colonies were counted by taking a digital picture of the Petri dish and then processing the digital image with Scion Image (a version of NIH Image adapted for Windows PC).

Fluorogenic assay of caspase-3 activity

The EnzChek caspase-3 assay kit no. 2 was used to measure caspase-3 activity. Two million cells were plated in each of 12 dishes. After attachment to the dish overnight, the experimental groups were treated with manumycin alone (54 µM, 6 h, 3 dishes), paclitaxel alone (21 µM, 6 h, 3 dishes), manumycin plus paclitaxel (3 dishes), or 0.1% DMSO in tissue culture medium (3 dishes; control). After cell lysis subjected to a freeze-thaw cycle, the protein concentrations of samples were measured using a modified Lowry method (DC Protein Assay, Bio-Rad Laboratories). Then, equal amounts of total protein from each sample were added into wells. Caspase-3 activity was measured in each cytosolic extract according to the manufacturer’s protocol using Z-DEVD-R110 as a substrate. Fluorescence was measured at excitation and emission wavelengths of 485 nm and 535 nm, respectively.

Statistical analysis

One-way ANOVA with the Tukey test was used to assess the statistical significance of differences between groups. Differences associated with P values less than 0.05 were considered significant. SPSS for Windows computer software (SPSS, Inc., Chicago, IL) was used to facilitate calculations. Results were reported as means ± 95% confidence intervals.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
p21 Waf-1 expression was induced by manumycin and by manumycin plus paclitaxel in ARO and KAT-4 cells

In ARO cells, cDNA microarray hybridization and quantitative measurement using a PhosphorImager (Molecular Dynamics) showed that the level of p21mRNA was increased in cells treated with manumycin alone (2.7 times the level in control cells) and in cells treated with manumycin plus paclitaxel (2.5 times the level in control cells). Treatment with paclitaxel alone was associated with a slight (10%) decrease in p21mRNA compared with the level in control cells (data not shown).

Whether the induction of p21 Waf-1expression is mediated by induction of promoter activity and increased transcription of the p21 Waf-1gene was tested by a transfection experiment. ARO cells were transfected with the WWP-Luc plasmid, which has the luciferase reporter gene under the control of the p21 Waf-1promoter. After transfection overnight, the cells were treated with control culture medium, 54 µM manumycin, 21 µM paclitaxel, or both manumycin and paclitaxel for 6 h. Assay of luciferase activity in the cell lysates showed that p21 Waf-1promoter activity was increased by treatment with manumycin or manumycin plus paclitaxel (Fig. 1Go).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 1. Activation of p21 Waf-1promoter by manumycin. ARO cells transfected with WWP-Luc plasmid were treated with DMSO 0.1% (control), manumycin 54 µM, paclitaxel 21 µM, or manumycin plus paclitaxel for 6 h and then assayed for luciferase activity. The error bars represent 95% confidence intervals. Statistically significant differences (P < 0.05) between manumycin and control and between manumycin plus paclitaxel and control are labeled with an asterisk.

 
Using immunoblotting, we documented changes in the level of p21 Waf-1 protein in ARO cells and another ATC cell line KAT-4 when they were treated with manumycin alone (54 µM), paclitaxel alone (21 µM), or manumycin plus paclitaxel for 4, 8, 12, or 18 h. Anti-p21 Waf-1 immunoblotting of the cell lysates revealed a time-dependent increase in p21 Waf-1 protein level in cells treated with manumycin or manumycin plus paclitaxel (Fig. 2AGo). Surprisingly, the increase in p21 Waf-1 in cells treated with the drug combination was less than the increase in cells treated with manumycin alone. The antiactin immunoblot was included to control for overall differences in protein content.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 2. Increase in p21 Waf-1 protein level with manumycin treatment in ATC cells. A, Anti-p21 Waf-1 immunoblots of KAT-4 and ARO cells treated for different durations. The names of the cell lines are shown on the right. The detected antigens are shown on the left. The corresponding treatment is labeled above the blots. B, Anti-p21 Waf-1 immunoblots of subcellular fractions. ARO and KAT-4 cells were treated with manumycin 54 µM (M) or DMSO 0.1% (C). The cells were sequentially extracted with detergents for fractionation (labeled at the top from left to right).

 
We also investigated p21 Waf-1 protein levels using immunoblotting and differential detergent extraction, an alternative method of cell fractionation (48) in which sequential extraction with buffered detergents is used to separate proteins into distinct partitions. By immunoblotting and differential detergent extraction, we demonstrated that manumycin (54 µM, 6 h) increased the amount of p21 Waf-1 protein in the organellar, nuclear, and detergent-resistant fractions but not in the cytosolic fraction (Fig. 2BGo). The increase of p21 Waf-1 in cellular compartments other than the nucleus raised the possibility of actions of p21 Waf-1 that were not related to regulation of cell cycling.

Because p21 Waf-1 protein was also increased by manumycin and manumycin plus paclitaxel in KAT-4 cells, we went back to confirm that the level of p21 Waf-1mRNA was also increased in KAT-4 cells. We assessed the level of p21 Waf-1mRNA in KAT-4 cells after treatment with manumycin alone, paclitaxel alone, or manumycin plus paclitaxel semiquantitatively by RT-PCR. Ethidium bromide staining of PCR products in agarose gels demonstrated that there was more p21 Waf-1mRNA in cell samples treated with manumycin and manumycin plus paclitaxel than in control cells (Fig. 3Go, top panel). The GAPDH control gel showed bands of equal intensities (Fig. 3Go, bottom panel).



View larger version (41K):
[in this window]
[in a new window]
  		Figure 3. RT-PCR of p21 Waf-1in KAT-4 cells. The ethidium bromide-stained agarose gels of PCR products are shown. Top panel, This gel shows the p21 Waf-1PCR products. The lanes are labeled at the top. The cells were treated with DMSO 0.1% (control), manumycin 54 µM, paclitaxel 21 µM, or manumycin plus paclitaxel for 6 h. The arrows indicate the location of the PCR products. Bottom panel, This gel shows the GAPDHPCR products.

 
Stably transfected KAT-4 cells overexpressed p21 Waf-1 when induced with zinc

As a tool for specifically perturbing the intracellular level of p21 Waf-1, we inserted the human cDNA of p21 Waf-1in the expression plasmid pMEP4 under the control of a metallothionein promoter. To explore the effect of p21 Waf-1 on the cytotoxic effects of manumycin alone, paclitaxel alone, and the two agents in combination, we stably transfected the pMEP4 p21S plasmid and the empty plasmid pMEP4 (control) into KAT-4 cells. As demonstrated by immunoblotting, expression of p21 Waf-1 in KAT-4 p21S was inducible by the presence of Zn2+ in the culture medium. The induction was concentration-dependent (Fig. 4AGo), time-dependent (Fig. 4BGo), sustained, and specific (i.e. no induction of p21 Waf-1by Zn2+ was observed in KAT-4 cells with pMEP4 empty plasmid). Strong ectopic expression of p21 Waf-1was induced at a zinc concentration of 100 µM, in agreement with reported experience with the metallothionein promoter (49).



View larger version (50K):
[in this window]
[in a new window]
  		Figure 4. Dependence on Zn2+ concentration and time of p21 Waf-1 expression in KAT-4 p21S cells. A, The immunoblot of KAT-4 p21S cells incubated for 6 h with Zn2+. The Zn2+ concentration is labeled above each lane. B, The immunoblots of KAT-4 p21S and KAT-4 pMEP4 cells incubated with Zn2+ 100 µM. The cell line and duration of induction are labeled above each lane. The locations of actin and p21 Waf-1 bands are indicated by arrows in both panels.

 
Ectopic expression of p21 Waf-1 enhanced the cytotoxicity of manumycin and paclitaxel

The hypothesis that p21 Waf-1 has a proapoptotic effect in cells treated with manumycin or paclitaxel was tested by evaluating the response to manumycin and paclitaxel when p21 Waf-1 was induced by Zn2+ in KAT-4 p21S cells. KAT-4 p21S and KAT-4 pMEP4 (control) cells (pretreated for 4 h with Zn2+) were treated with DMSO (0.1%, vol/vol), manumycin (54 µM), paclitaxel (21 µM), or manumycin plus paclitaxel for 24 h in the presence or absence of zinc sulfate (100 µM). Using the MTT assay, we measured the percentage viability of each sample relative to the viability of the respective DMSO control. Although the presence of Zn2+ 100 µM did not significantly affect the viability of KAT-4 pMEP4 cells, the presence of Zn2+ was associated with a significant (P < 0.05, ANOVA, Tukey test) decrease in viability in KAT-4 p21S cells treated with manumycin, paclitaxel, or both agents (Fig. 5AGo). The lack of significant change in viability in KAT-4 pMEP4 cells provided evidence that the change in viability was not a direct effect of Zn2+. The lack of significant change in viability of the DMSO-treated KAT-4 p21S cells suggested that induced expression of p21 Waf-1 per se did not decrease viability; rather, induced ectopic expression of p21 Waf-1 enhanced the cytotoxic effect of antineoplastic agents. The enhancement of the cytotoxic effect of antineoplastic agents was also supported by the decreases of IC50 values for manumycin-treated and paclitaxel-treated KAT-4 p21S cells in the presence of Zn2+ (Table 1Go).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 5. Enhanced cytotoxicity of manumycin and paclitaxel when p21 Waf-1expression was induced by Zn2+. A, Relative viability. KAT-4 p21S and KAT-4 pMEP4 cells were treated with DMSO 0.1% (control), manumycin 54 µM, paclitaxel 21 µM, or manumycin plus paclitaxel. The viability of the cells expressed as the percentage of the control viability is presented in the bar chart. The white bars represent cells treated in the presence of Zn2+ 100 µM, and the black bars represent cells treated in the absence of Zn2+. The error bars represent the 95% confidence intervals. Pairs with statistically significant differences (P < 0.05) are indicated with asterisks. B, Clonogenicity. Equal numbers of KAT-4 p21S cells were treated with DMSO 0.1% (control), manumycin 6 µM, paclitaxel 2.3 µM, or manumycin plus paclitaxel for 24 h in the presence or absence of Zn2+ 100 µM. Then, all of the cells in each treatment group were plated in soft agar. The average number of clones formed on soft agar is plotted for each group (n = 2 for each). The black bars are the groups without Zn2+, and the white bars are the groups with Zn2+. C, Caspase-3 activation. KAT-4 p21S cells were treated with DMSO 0.1% (control), manumycin 54 µM, paclitaxel 21 µM, or manumycin plus paclitaxel. The caspase-3 activity expressed as relative fluorescent units (rfu) is presented in the bar chart. The white bars represent cells treated in the presence of Zn2+ 100 µM, and the black bars represent cells treated in the absence of Zn2+. The error bars represent the 95% confidence intervals. Pairs with statistically significant differences (P < 0.05) are indicated with asterisks.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Half-maximal inhibitory concentration (IC50) for KAT-4 p21S cells

 
Expression of p21 Waf-1 enhanced the inhibitory effect of manumycin and paclitaxel on clonogenicity

Using the soft agar clonogenic assay, we measured the number of cells capable of forming clones in soft agar after drug treatments. While the presence of Zn2+ 100 µM did not decrease the number of clones in untreated KAT-4 p21S cells, Zn2+ 100 µM decreased the number of clones in KAT-4 p21 cells treated with manumycin or paclitaxel (Fig. 5BGo). In the cells treated with both drugs, the number of clones was so small that the presence of Zn2+ 100 µM did not make a big difference. These results suggested that induced expression of p21 Waf-1 per se did not decrease clonogenicity but enhanced the inhibitory effect of antineoplastic agents on clonogenicity.

Overexpression of p21 Waf-1 increased the activation of caspase-3 by manumycin and paclitaxel

To investigate whether the decrease in cell viability was due to induction of apoptosis, we measured the enzyme activity of caspase-3 in KAT-4 p21S cells. KAT-4 p21S cells were treated for 18 h with DMSO (0.1%, vol/vol), manumycin (54 µM), paclitaxel (21 µM), or manumycin plus paclitaxel in the presence or absence of Zn2+ 100 µM. Three independent experiments were done in each treatment group. The 18-h treatment duration was chosen on the basis of prior experience (25) and to ensure an easily detectable activation of caspase-3. The presence of Zn2+ significantly (P < 0.05; ANOVA, Tukey test) increased caspase-3 activity when the cells were treated with manumycin, paclitaxel, or both but did not significantly change caspase-3 activity when the cells were treated with DMSO (Fig. 5CGo).

p21 Knock-out cells were less sensitive to 48-h treatments of manumycin and paclitaxel

The hypothesis that p21 Waf-1 has a proapoptotic effect in cells treated with manumycin or paclitaxel was also tested by evaluating the response to manumycin and paclitaxel in the p21 Waf-1 knock-out (p21-/-) HCT-116 cells (46). HCT-116 cells are more sensitive to manumycin and paclitaxel than are ATC cells. Therefore, we tested the effects of manumycin and paclitaxel at both high doses (manumycin 54 µM and paclitaxel 21 µM) and low doses (manumycin 3 µM and paclitaxel 1.2 µM). Equal numbers of HCT-116 p21-/- cells and the wild-type HCT-116 p21+/+ cells were seeded in tissue culture wells at the beginning of the experiments. After incubation overnight, each cell line was divided into groups of wells to be treated with 0.1% DMSO (control), manumycin, paclitaxel, or both at low or high doses for 24 or 48 h. The numbers of viable cells at the end of the experiments were measured using the MTT method. Each reported data point is the average of results from six wells, and the error bars on the graphs represent the 95% confidence intervals.

When the relative viability of the cells was expressed as percentage relative to the respective controls, the two cell lines exhibited markedly different responses to treatment with manumycin and paclitaxel over time (Fig. 6Go). The responses to low doses (Fig. 6AGo) and the responses to high doses (Fig. 6BGo) were qualitatively the same. After 24 h of drug treatment, HCT-116 p21-/- cells have lower viability than the wild-type cells (P < 0.05; ANOVA, post hoc pairwise comparison, Tukey test). HCT-116 p21-/- cells that survived after the first 24 h of drug treatment must have continued to grow despite continued drug exposure, resulting in higher relative viability at 48 h for the HCT-116 p21-/- cells than the wild-type cells (P < 0.05; ANOVA, post hoc pairwise comparison, Tukey test). Wild-type cells that survived after the first 24 h of drug treatment continued to show sensitivity to the drugs, resulting in further lowering of viability between 24 and 48 h. In the end, after 48 h of drug treatments (both low dose and high dose), the wild-type cells have lower relative viability (i.e. more sensitive to manumycin and paclitaxel) than the p21-/- cells (ANOVA, post hoc pairwise comparison, Tukey test; P < 0.05).



View larger version (31K):
[in this window]
[in a new window]
  		Figure 6. Differences in cytotoxic responses to manumycin and paclitaxel between HCT-116 p21-/- and wild-type cells. A and B, Results with low doses of manumycin and paclitaxel and high doses (concentrations as labeled), respectively. In each chart, the relative viability (expressed as percentage of control) is plotted on the y-axis. The black bars represent the HCT-116 p21-/- cells, and the white bars represent the wild-type cells. The treatment groups were: DMSO 0.1% (C), manumycin (M), paclitaxel (P), and manumycin plus paclitaxel (M+P). The error bars represent the 95% confidence intervals. The asterisks indicate statistically significant (P < 0.05) difference between the HCT-116 p21-/- cells and the wild-type cells treated with the same treatment (ANOVA, post hoc pairwise comparison, Tukey test). C, The logarithms of the ratios of mean viability after a 48-h treatment relative to that of a 24-h treatment are plotted against the treatment groups. The dosage levels and the cell lines are labeled in the key above the graph.

 
Another way to present the data visually was to plot the logarithm of the ratio of mean relative viability at 48 h to that at 24 h against the treatment groups (Fig. 6CGo). The difference in the response to drug treatments between HCT-116 p21-/- and wild-type cells was obvious. The mean relative viability increased between 24 and 48 h (i.e. positive logarithm) in HCT-116 p21-/- cells treated with drugs at low doses and at high doses. In contrast, the mean relative viability continued to decrease between 24 and 48 h (i.e. negative logarithm) in wild-type cells treated with drugs at low doses and at high doses. These data suggested that in the absence of p21 Waf-1, HCT-116 cells that initially survived the cytotoxic effects of manumycin and paclitaxel could proliferate to increase the number of viable cells, and that in the presence of p21 Waf-1, sensitivity to the cytotoxic effects of manumycin and paclitaxel persisted.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
For differentiated thyroid carcinomas that retain the ability to take up radioactive iodine, surgery and radioactive iodine remain the primary treatments. In addition to ATC and undifferentiated thyroid cancers, a significant number of disease-specific deaths in thyroid cancer patients are from differentiated thyroid carcinomas (papillary and follicular thyroid carcinomas) that turned aggressive and lost the ability to concentrate iodide. Undifferentiated thyroid cancers and ATC are thought to develop from preexisting differentiated thyroid carcinoma following a second hit mutation (probably a p53 tumor suppressor gene mutation) that leads to aggressive behavior. Advances in chemotherapy against ATC may also be applicable to other aggressive thyroid tumors. Because ATC is perhaps the most aggressive solid tumor, ATC may be used as a model experimental system to investigate pharmacological therapeutics, and effective chemotherapy against ATC may be applicable to other types of aggressive solid tumors as well.

The p21cDNA was cloned independently by several groups, and thus p21 has many names: Waf-1 (wild-type p53 activated factor), Cip-1 (cyclin-dependent kinase interacting protein), Sdi-1 (senescent cell-derived inhibitor), and MDA-6 (melanoma differentiation associated) factor. p21 is a cyclin kinase inhibitor that binds to and inhibits the activity of the cyclin-dependent protein kinases. Tumor suppressor p53 activation of p21 gene expression is the best-known mechanism regulating transcription of p21 Waf-1. Other factors that regulate expression of p21 Waf-1 include E2F and a variety of cytokines and growth factors (e.g. interferon-ß and TGF-ß). Mitsuuchi’s study (50) indicated that the phosphatidylinositol 3-kinases/AKT signal transduction pathway plays a critical role in the expression of p21 Waf-1 induced by cisplatin and paclitaxel.

The p53 gene is mutated in a wide variety of human cancers. In contrast, mutations in the p21 Waf-1 gene are rare (51). In tumor cells with wild-type p53, an FTI induces p21 Waf-1 expression (38). In a rat mammary carcinoma animal model, perillyl alcohol (also an FTI) induces p21 Waf-1without significant change in expression of p53 (52). The ARO cell line has a p53 mutation (8), and the KAT-4 cell line is likely to have a p53 mutation as well because of intense immunostaining of these cells with anti-p53 antibody (Yeung, S.-C. J., unpublished data). We found that manumycin, another FTI, induced p21 Waf-1in these cell lines. At least in the ARO cell line, it is certain that manumycin-induced p21 Waf-1 was independent of p53 function. The mechanism by which manumycin induced expression of p21 Waf-1 is unknown at this time, but preliminary data suggest that other members of the p53 gene family (e.g. p73; Ref. 53) were up-regulated by manumycin (Yeung, S.-C. J., unpublished data).

Our conclusion that manumycin induced expression of p21 Waf-1 was supported by several lines of evidence. First, manumycin treatment led to the activation of p21 Waf-1 promoter activity. Second, manumycin treatment increased the mRNA level of p21 Waf-1. Third, manumycin treatment induced a rise in the p21 Waf-1 protein level. Finally, manumycin treatment increased p21 Waf-1 protein in several subcellular fractions and not just the nuclear fraction.

The protein level of p21 Waf-1 in cells treated with manumycin plus paclitaxel was less than that in cells treated with manumycin alone, despite similar levels of gene expression. There are two possible explanations for this phenomenon. Our previous results showed that manumycin plus paclitaxel induced apoptosis significantly earlier than did manumycin alone (27). Early in apoptosis, general inhibition of protein translation has been observed (54). Thus, the first possible explanation is general inhibition of protein translation caused by apoptosis. Because apoptosis occurs with activation of caspase-3 and because p21 Waf-1 is known to be a substrate for caspase-3 (55, 56), the second explanation is proteolysis of p21 Waf-1 by caspase-3.

The effects of p21 Waf-1 on the cell cycle have been well studied. p21 Waf-1 exists in quaternary complexes with cyclin, cyclin-dependent kinases, and proliferating cell nuclear antigen, and p21 Waf-1 can induce G1 arrest and block entry into S phase by inactivating cyclin-dependent protein kinases or by inhibiting the activity of proliferating cell nuclear antigen. In some experimental systems, p21 Waf-1 mediates G2 arrest in the cell cycle (57). However, the effect of p21 Waf-1 on apoptosis is controversial. Although overexpression of p21 Waf-1 results in growth arrest and has been shown to effectively suppress tumor growth in vitro and in vivo, growth arrest by p21 Waf-1 has been suggested to protect cells from apoptosis (39, 40, 42, 58, 59, 60). There is also experimental evidence that cytoplasmic p21 Waf-1 may be antiapoptotic (41). In other experimental systems, p21 Waf-1 may be proapoptotic (43, 44, 45). It has been proposed that p21 Waf-1 can be antiapoptotic or proapoptotic in different experimental systems, depending on the genetic makeup of the cells or the presence of trans-acting response modifying factors (58).

Our hypothesis that p21 Waf-1 expression was proapoptotic in cells treated with manumycin and paclitaxel was supported by two sets of experiments. The first set of experiments involved KAT-4 cells stably transfected with a plasmid that expressed p21 Waf-1 upon induction by Zn2+. By using the MTT method, we demonstrated that KAT-4 cells with induced p21 Waf-1 expression were more sensitive to treatment with manumycin and paclitaxel than were cells incubated without Zn2+. The increased cytotoxicity of manumycin and paclitaxel when p21 Waf-1was induced was associated with an increase in caspase-3 activity (i.e. apoptosis). In agreement with similar experiments in urothelial carcinoma cell lines (61), the induction of p21 Waf-1alone (in the absence of treatment with a chemotherapy drug) did not result in significant apoptosis.

The second set of experiments involved the HCT-116 p21 knock-out (-/-) cells and the wild-type cells. In the absence of p21 Waf-1, HCT-116 p21-/- cells that survived after 24 h of drug treatment could proliferate. In the presence of endogenous p21 Waf-1, the viability of the p21 wild-type (+/+) cells continued to decrease with continued drug treatment. Overall, after drug treatments for 48 h, the HCT-116 p21-/- cells were less sensitive to manumycin and paclitaxel than the wild-type HCT-116 cells, so we concluded that endogenous p21 Waf-1 was an important factor influencing the sensitivity of human colon cancer cells to treatment with manumycin and paclitaxel.

Obviously, the genetic background of the ATC cell lines and the HCT-116 cell lines are different. Because the effect of p21 Waf-1 on apoptosis can be context dependent (58), we would emphasize the limitation of our data from these different experimental systems and caution against overgeneralization. In conclusion, we found that manumycin induced expression of p21 Waf-1 in a p53-independent manner in ATC cells. Ectopic expression of p21 Waf-1 was proapoptotic when ATC cells were treated with manumycin and paclitaxel. Endogenous p21 Waf-1 was an important factor influencing the sensitivity of HCT-116 cells to treatment with manumycin and paclitaxel.

How is p21 Waf-1 linked to the apoptosis regulation mechanisms? This is an important question to address next. The Gadd45/p38 MAPK pathway may be a candidate. At this point, our data are very preliminary, and we shall refrain from speculation.


   Acknowledgments
 


   Footnotes
 
This research was supported by a grant to S.-C.J.Y. from the American Cancer Society (RPG-99-154-01-CDD). H.-L.Y. is a visiting scientist from Zhongshan Medical College, Sun Yat-Sen University, Guangzhou, China.

H.-L.Y. and J.-X.P. contributed equally to this work.

Abbreviations: ATC, Anaplastic thyroid cancer; DMSO, dimethylsulfoxide; FTI, farnesyltransferase inhibitor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium.

Received June 25, 2002.

Accepted November 15, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sherman SI, Brierley JD, Sperling M, Ain KB, Bigos ST, Cooper DS, Haugen BR, Ho M, Klein I, Ladenson PW, Robbins J, Ross DS, Specker B, Taylor T, Maxon 3rd HR 1998 Prospective multicenter study of treatment of thyroid carcinoma: Initial analysis of staging and outcome. Cancer 83:1012–1021[CrossRef][Medline]
  2. Ain K 1998 Anaplastic thyroid carcinoma: behavior, biology, and therapeutic approaches. Thyroid 8:715–726[Medline]
  3. Sherman SI 2000 Anaplastic carcinoma: clinical aspects. In: Wartofsky L, ed. Thyroid cancer: a comprehensive guide to clinical management. Totowa, NJ: Humana Press Inc.; 319–325
  4. Giuffrida D, Gharib H 2000 Anaplastic thyroid carcinoma: current diagnosis and treatment. Ann Oncol 11:1083–1089[Abstract/Free Full Text]
  5. Sweeney PJ, Haraf DJ, Recant W, Kaplan EL, Vokes EE 1996 Anaplastic carcinoma of the thyroid. Ann Oncol 7:739–744[Free Full Text]
  6. Oka Y, Kobayashi T, Fujita S, Matsuura N, Okamoto S, Asakawa H, Murata A, Mori T 1993 Establishment of a human anaplastic thyroid cancer cell line secreting granulocyte colony-stimulating factor in response to cytokines. In Vitro Cell Dev Biol Anim 29A:537–542
  7. Palyi I, Peter I, Daubner D, Vincze B, Lorincz I 1993 Establishment, characterization and drug sensitivity of a new anaplastic thyroid carcinoma cell line (BHT-101). Virchows Arch B Cell Pathol Incl Mol Pathol 63:263–269[Medline]
  8. Fagin JA, Matsuo K, Karmakar A, Chen DL, Tang SH, Koeffler HP 1993 High prevalence of mutations of the p53 gene in poorly differentiated human thyroid carcinomas. J Clin Invest 91:179–184
  9. La Perle KM, Jhiang SM, Capen CC 2000 Loss of p53 promotes anaplasia and local invasion in ret/PTC1-induced thyroid carcinomas. Am J Pathol 157:671–677[Abstract/Free Full Text]
  10. Gotz C, Montenarh M 1996 p53: DNA damage, DNA repair, and apoptosis. Rev Physiol Biochem Pharmacol 127:65–95[Medline]
  11. Lumachi F, Basso S 2002 Apoptosis: life through planned cellular death regulating mechanisms, control systems, and relations with thyroid diseases. Thyroid 12:27–34[CrossRef][Medline]
  12. Yvon AM, Wadsworth P, Jordan MA 1999 Taxol suppresses dynamics of individual microtubules in living human tumor cells. Mol Biol Cell 10:947–959[Abstract/Free Full Text]
  13. Amos LA, Lowe J 1999 How Taxol stabilises microtubule structure. Chem Biol 6:R65—R69
  14. Rowinsky EK 1997 The development and clinical utility of the taxane class of antimicrotubule chemotherapy agents. Annu Rev Med 48:353–374[CrossRef][Medline]
  15. Ain KB, Tofiq S, Taylor KD 1996 Antineoplastic activity of taxol against human anaplastic thyroid carcinoma cell lines in vitro and in vivo. J Clin Endocrinol Metab 81:3650–3653[Abstract]
  16. Ain KB, Egorin MJ, DeSimone PA 2000 Treatment of anaplastic thyroid carcinoma with paclitaxel: phase 2 trial using ninety-six-hour infusion. Collaborative Anaplastic Thyroid Cancer Health Intervention Trials (CATCHIT) Group. Thyroid 10:587–594[Medline]
  17. Moasser MM, Sepp-Lorenzino L, Kohl NE, Oliff A, Balog A, Su DS, Danishefsky SJ, Rosen N 1998 Farnesyl transferase inhibitors cause enhanced mitotic sensitivity to taxol and epothilones. Proc Natl Acad Sci USA 95:1369–1374[Abstract/Free Full Text]
  18. Nielsen LL, Gurnani M, Shi B, Terracina G, Johnson RC, Carroll J, Mathis JM, Hajian G 2000 Derivation and initial characterization of a mouse mammary tumor cell line carrying the polyomavirus middle T antigen: utility in the development of novel cancer therapeutics. Cancer Res 60:7066–7074[Abstract/Free Full Text]
  19. Shi B, Yaremko B, Hajian G, Terracina G, Bishop WR, Liu M, Nielsen LL 2000 The farnesyl protein transferase inhibitor SCH66336 synergizes with taxanes in vitro and enhances their antitumor activity in vivo. Cancer Chemother Pharmacol 46:387–393[CrossRef][Medline]
  20. Hara M, Akasaka K, Akinaga S, Okabe M, Nakano H, Gomez R, Wood D, Uh M, Tamanoi F 1993 Identification of Ras farnesyltransferase inhibitors by microbial screening. Proc Natl Acad Sci USA 90:2281–2285[Abstract/Free Full Text]
  21. Yang W, Del Villar K, Urano J, Mitsuzawa H, Tamanoi F 1997 Advances in the development of farnesyltransferase inhibitors: substrate recognition by protein farnesyltransferase. J Cell Biochem Suppl 27:12–19[Medline]
  22. Wang W, Macaulay RJ 1999 Apoptosis of medulloblastoma cells in vitro follows inhibition of farnesylation using manumycin A. Int J Cancer 82:430–434[CrossRef][Medline]
  23. Hara M, Han M 1995 Ras farnesyltransferase inhibitors suppress the phenotype resulting from an activated ras mutation in Caenorhabditis elegans. Proc Natl Acad Sci USA 92:3333–3337[Abstract/Free Full Text]
  24. Ito T, Kawata S, Tamura S, Igura T, Nagase T, Miyagawa JI, Yamazaki E, Ishiguro H, Matasuzawa Y 1996 Suppression of human pancreatic cancer growth in BALB/c nude mice by manumycin, a farnesyl:protein transferase inhibitor. Jpn J Cancer Res 87:113–116[CrossRef][Medline]
  25. Yeung SC, Xu G, Pan J, Christgen M, Bamiagis A 2000 Manumycin enhances the cytotoxic effect of paclitaxel on anaplastic thyroid carcinoma cells. Cancer Res 60:650–656[Abstract/Free Full Text]
  26. Xu G, Pan J, Martin C, Yeung SC 2001 Angiogenesis inhibition in the in vivo antineoplastic effect of manumycin and paclitaxel against anaplastic thyroid carcinoma. J Clin Endocrinol Metab 86:1769–1777[Abstract/Free Full Text]
  27. Pan J, Xu G, Bamiagis AE, Yeung SC 2001 Cytochrome c release is upstream to activation of caspase-9, caspase-8, and caspase-3 in the enhanced apoptosis of anaplastic thyroid cancer cells induced by manumycin and paclitaxel. J Clin Endocrinol Metab 86:4731–4740[Abstract/Free Full Text]
  28. Gu WZ, Tahir SK, Wang YC, Zhang HC, Cherian SP, O’Connor S, Leal JA, Rosenberg SH, Ng SC 1999 Effect of novel CAAX peptidomimetic farnesyltransferase inhibitor on angiogenesis in vitro and in vivo. Euro J Cancer 35:1394–1401
  29. White FC, Benehacene A, Scheele JS, Kamps M 1997 VEGF mRNA is stabilized by ras and tyrosine kinase oncogenes, as well as by UV radiation: evidence for divergent stabilization pathways. Growth Factors 14:199–212[Medline]
  30. Feldkamp MM, Lau N, Rak J, Kerbel RS, Guha A 1999 Normoxic and hypoxic regulation of vascular endothelial growth factor (VEGF) by astrocytoma cells is mediated by Ras. Int J Cancer 81:118–124[CrossRef][Medline]
  31. Feldkamp MM, Lau N, Guha A 1999 Growth inhibition of astrocytoma cells by farnesyl transferase inhibitors is mediated by a combination of antiproliferative, pro-apoptotic and anti-angiogenic effects. Oncogene 18:7514–7526[CrossRef][Medline]
  32. Charvat S, Duchesne M, Parvaz P, Chignol MC, Schmitt D, Serres M 1999 The up-regulation of vascular endothelial growth factor in mutated Ha-ras HaCaT cell lines is reduced by a farnesyl transferase inhibitor. Anticancer Res 19:557–561[Medline]
  33. Lerner EC, Hamilton AD, Sebti SM 1997 Inhibition of Ras prenylation: a signaling target for novel anti-cancer drug design. Anticancer Drug Des 12:229–238[Medline]
  34. Lobell RB, Kohl NE 1998 Pre-clinical development of farnesyltransferase inhibitors. Cancer Metastasis Rev 17:203–210[CrossRef][Medline]
  35. Du W, Prendergast GC 1999 Geranylgeranylated RhoB mediates suppression of human tumor cell growth by farnesyltransferase inhibitors. Cancer Res 59:5492–5496[Abstract/Free Full Text]
  36. Liu A, Du W, Liu JP, Jessell TM, Prendergast GC 2000 RhoB alteration is necessary for apoptotic and antineoplastic responses to farnesyltransferase inhibitors. Mol Cell Biol 20:6105–6113[Abstract/Free Full Text]
  37. Jiang K, Coppola D, Crespo NC, Nicosia SV, Hamilton AD, Sebti SM, Cheng JQ 2000 The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis. Mol Cell Biol 20:139–148[Abstract/Free Full Text]
  38. Sepp-Lorenzino L, Rosen N 1998 A farnesyl-protein transferase inhibitor induces p21 expression and G1 block in p53 wild-type tumor cells. J Biol Chem 273:20243–20251[Abstract/Free Full Text]
  39. Bissonnette N, Hunting DJ 1998 p21-induced cycle arrest in G1 protects cells from apoptosis induced by UV-irradiation or RNA polymerase II blockage. Oncogene 16:3461–3469[CrossRef][Medline]
  40. Rosato RR, Wang Z, Gopalkrishnan RV, Fisher PB, Grant S 2001 Evidence of a functional role for the cyclin-dependent kinase-inhibitor p21WAF1/CIP1/MDA6 in promoting differentiation and preventing mitochondrial dysfunction and apoptosis induced by sodium butyrate in human myelomonocytic leukemia cells (U937). Int J Oncol 19:181–191[Medline]
  41. Asada M, Yamada T, Ichijo H, Delia D, Miyazono K, Fukumuro K, Mizutani S 1999 Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic differentiation. EMBO J 18:1223–1234[CrossRef][Medline]
  42. Gorospe M, Wang X, Guyton KZ, Holbrook NJ 1996 Protective role of p21(Waf1/Cip1) against prostaglandin A2-mediated apoptosis of human colorectal carcinoma cells. Mol Cell Biol 16:6654–6660[Abstract]
  43. Hsu SL, Chen MC, Chou YH, Hwang GY, Yin SC 1999 Induction of p21(CIP1/Waf1) and activation of p34(cdc2) involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells. Exp Cell Res 248:87–96[CrossRef][Medline]
  44. Avramis VI, Nandy P, Kwock R, Solorzano MM, Mukherjee SK, Danenberg P, Cohen LJ 1998 Increased p21/WAF-1 and p53 protein levels following sequential three drug combination regimen of fludarabine, cytarabine and docetaxel induces apoptosis in human leukemia cells. Anticancer Res 18:2327–2338[Medline]
  45. Tsao YP, Huang SJ, Chang JL, Hsieh JT, Pong RC, Chen SL 1999 Adenovirus-mediated p21((WAF1/SDII/CIP1)) gene transfer induces apoptosis of human cervical cancer cell lines. J Virol 73:4983–4990[Abstract/Free Full Text]
  46. Waldman T, Kinzler KW, Vogelstein B 1995 p21 is necessary for the p53-mediated G1 arrest in human cancer cells. Cancer Res 55:5187–5190[Abstract/Free Full Text]
  47. el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B 1993 WAF1, a potential mediator of p53 tumor suppression. Cell 75:817–825[CrossRef][Medline]
  48. Ramsby ML, Makowski GS 1999 Differential detergent fractionation of eukaryotic cells. In: Link AJ, ed. 2-D proteome analysis protocols. Totowa, NJ: Humana Press; 53–66
  49. Lang D, Miknyoczki SJ, Huang L, Ruggeri BA 1998 Stable reintroduction of wild-type P53 (MTmp53ts) causes the induction of apoptosis and neuroendocrine-like differentiation in human ductal pancreatic carcinoma cells. Oncogene 16:1593–1602[CrossRef][Medline]
  50. Mitsuuchi Y, Johnson SW, Selvakumaran M, Williams SJ, Hamilton TC, Testa JR 2000 The phosphatidylinositol 3-kinase/AKT signal transduction pathway plays a critical role in the expression of p21WAF1/CIP1/SDI1 induced by cisplatin and paclitaxel. Cancer Res 60:5390–5394[Abstract/Free Full Text]
  51. Shiohara M, el-Deiry WS, Wada M, Nakamaki T, Takeuchi S, Yang R, Chen DL, Vogelstein B, Koeffler HP 1994 Absence of WAF1 mutations in a variety of human malignancies. Blood 84:3781–3784[Abstract/Free Full Text]
  52. Ariazi EA, Satomi Y, Ellis MJ, Haag JD, Shi W, Sattler CA, Gould MN 1999 Activation of the transforming growth factor ß signaling pathway and induction of cytostasis and apoptosis in mammary carcinomas treated with the anticancer agent perillyl alcohol. Cancer Res 59:1917–1928[Abstract/Free Full Text]
  53. Yu J, Zhang L, Hwang PM, Rago C, Kinzler KW, Vogelstein B 1999 Identification and classification of p53-regulated genes. Proc Natl Acad Sci USA 96:14517–14522[Abstract/Free Full Text]
  54. Jeffrey IW, Bushell M, Tilleray VJ, Morley S, Clemens MJ 2002 Inhibition of protein synthesis in apoptosis: differential requirements by the tumor necrosis factor {alpha} family and a DNA-damaging agent for caspases and the double-stranded RNA-dependent protein kinase. Cancer Res 62:2272–2280[Abstract/Free Full Text]
  55. Gervais JL, Seth P, Zhang H 1998 Cleavage of CDK inhibitor p21(Cip1/Waf1) by caspases is an early event during DNA damage-induced apoptosis. J Biol Chem 273:19207–19212[Abstract/Free Full Text]
  56. Park JA, Kim KW, Kim SI, Lee SK 1998 Caspase 3 specifically cleaves p21WAF1/CIP1 in the earlier stage of apoptosis in SK-HEP-1 human hepatoma cells. Eur J Biochem 257:242–248[Medline]
  57. Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP, Sedivy JM, Kinzler KW, Vogelstein B 1998 Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282:1497–1501[Abstract/Free Full Text]
  58. Polyak K, Waldman T, He TC, Kinzler KW, Vogelstein B 1996 Genetic determinants of p53-induced apoptosis and growth arrest. Genes Dev 10:1945–1952[Abstract/Free Full Text]
  59. Bellido T, O’Brien CA, Roberson PK, Manolagas SC 1998 Transcriptional activation of the p21(WAF1, CIP1, SDI1) gene by interleukin-6 type cytokines. A prerequisite for their pro-differentiating and anti-apoptotic effects on human osteoblastic cells. J Biol Chem 273:21137–21144[Abstract/Free Full Text]
  60. Gorospe M, Cirielli C, Wang X, Seth P, Capogrossi MC, Holbrook NJ 1997 p21(Waf1/Cip1) protects against p53-mediated apoptosis of human melanoma cells. Oncogene 14:929–935[CrossRef][Medline]
  61. Makri D, Schulz WA, Grimm M, Clasen S, Bojar H, Schmitz-Drager BJ 1998 WAF1/p21 regulates proliferation, but does not mediate p53-dependent apoptosis in urothelial carcinoma cell lines. Int J Oncol 12:621–628[Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
R. Aneja, A. M. Ghaleb, J. Zhou, V. W. Yang, and H. C. Joshi
p53 and p21 Determine the Sensitivity of Noscapine-Induced Apoptosis in Colon Cancer Cells
Cancer Res., April 15, 2007; 67(8): 3862 - 3870.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Pan, M. She, Z.-X. Xu, L. Sun, and S.-C. J. Yeung
Farnesyltransferase Inhibitors Induce DNA Damage via Reactive Oxygen Species in Human Cancer Cells
Cancer Res., May 1, 2005; 65(9): 3671 - 3681.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. I. Geller, K. Szekely-Szucs, I. Petak, B. Doyle, and J. A. Houghton
P21Cip1 Is a Critical Mediator of the Cytotoxic Action of Thymidylate Synthase Inhibitors in Colorectal Carcinoma Cells
Cancer Res., September 1, 2004; 64(17): 6296 - 6303.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Z. Zhang, H. Wang, M. Li, S. Agrawal, X. Chen, and R. Zhang
MDM2 Is a Negative Regulator of p21WAF1/CIP1, Independent of p53
J. Biol. Chem., April 16, 2004; 279(16): 16000 - 16006.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow