Cancer Research Meeting Calendar Frontiers in Basic Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Deguchi, A.
Right arrow Articles by Weinstein, I. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Deguchi, A.
Right arrow Articles by Weinstein, I. B.
[Cancer Research 64, 3966-3973, June 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Activation of Protein Kinase G Is Sufficient to Induce Apoptosis and Inhibit Cell Migration in Colon Cancer Cells

Atsuko Deguchi1, W. Joseph Thompson3 and I. Bernard Weinstein1,2

1 Herbert Irving Comprehensive Cancer Center, 2 Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York, and 3 OSI Pharmaceuticals, Inc., Farmingdale, New York


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The activation of protein kinase G (PKG) by cGMP has become of considerable interest as a novel molecular mechanism for the induction of apoptosis in cancer cells, because sulindac sulfone (exisulind, Aptosyn) and certain derivatives that inhibit cGMP-phosphodiesterases and thereby increase cellular levels of cGMP appear to induce apoptosis via this mechanism. However, other effects of these compounds have not been excluded, and the precise mechanism by which PKG activation induces apoptosis has not been elucidated in detail. To directly examine the effects of PKG on cell growth and apoptosis, we generated a series of mutants of PKG I{alpha}: PKG I{alpha}S65D, a constitutively activated point mutant; PKG I{alpha}{Delta}, a constitutively activated N-terminal truncated mutant; and PKG I{alpha}K390R, a dominant-negative point mutant. A similar series of mutants of PKG Iß were also constructed (Deguchi et al., Mol. Cancer Ther., 1: 803–809, 2002). The present study demonstrates that when transiently expressed in SW480 colon cancer, the constitutively activated mutants of PKG Iß, and to a lesser extent PKG I{alpha}, inhibit colony formation and induce apoptosis. We were not able to obtain derivatives of SW480 cells that stably expressed these constitutively activated mutants, presumably because of toxicity. However, derivatives that stably overexpressed wild-type PKG Iß displayed growth inhibition, whereas derivatives that stably expressed the dominant-negative mutant (KR) of PKG Iß grew more rapidly and were more resistant to Aptosyn-induced growth inhibition than vector control cells. Stable overexpression of PKG Iß was associated with decreased cellular levels of ß-catenin and cyclin D1 and increased levels of p21CIP1. Reporter assays indicated that activation of PKG Iß inhibits the transcriptional activity of the cyclin D1 promoter. We also found that transient expression of the constitutively activated mutants of PKG Iß inhibited cell migration. Taken together, these results indicate that activation of PKG Iß is sufficient to inhibit growth and cell migration and induce apoptosis in human colon cancer cells and that these effects are associated with inhibition of the transcription of cyclin D1 and an increase in the expression of p21CIP1.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
cGMP is an important second messenger that mediates the action of several hormones, neurotransmitters, and drugs (1) by regulating various physiological functions including neurotransmission, platelet aggregation, and smooth muscle tone (2) . There is increasing evidence that it can play an important role in cellular proliferation, differentiation, and apoptosis (3, 4, 5) . The intracellular level of cGMP is regulated through a dynamic balance between its rate of synthesis by guanylyl cyclases and degradation by specific phosphodiesterases (PDEs), especially PDEs 2 and 5 (6) . cGMP has several intracellular targets. Thus, it can bind to specific PDEs, thereby stimulating or inhibiting their activities (7) ; it can bind to and activate cGMP-gated cation channels; it can bind to and activate protein kinase G (PKG); and, under certain conditions, it can bind to and activate protein kinase A (8) .

Two major forms of PKG have been identified in mammalian cells, PKG I and PKG II. In addition, there are two splice variants of PKG I, which are designated I{alpha} and Iß (9) . PKG I is expressed in platelets, vascular smooth muscle cells, fibroblasts, certain endothelial cells, the lung, the cerebellum, and the heart (10 , 11) . In endothelial cells, activation of PKG I by cGMP is associated with a reduction in thrombin-induced calcium transients and paracellular permeability (12 , 13) . Direct evidence for functional roles of PKG I in relaxation of smooth muscle, inhibition of Ca2+ transients, and inhibition of platelet adhesion and aggregation was obtained from studies of PKG I knockout mice (14 , 15) . Vasodilator-stimulated phosphoprotein (VASP) is known to be one of the substrates of PKG I, and its phosphorylation plays a role in inhibiting platelet aggregation and focal adhesion (16) . PKG II is expressed mainly in the brush border of the intestinal mucosa and in specific regions of the brain and plays a role in transepithelial Cl– and Na+ transport in the intestine (17 , 18) .

Expression of PDE 5 has been detected in the normal bladder, colon, pancreas, lung, placenta, prostate, small intestine, and stomach (19) . It is of interest that metastatic breast cancers, colon adenocarcinoma, bladder squamous carcinoma, and lung cancers often express increased cellular levels of PDE 2 or 5 when compared with adjacent normal tissues (20, 21, 22, 23, 24, 25) . In addition, the endogenous polypeptide guanylyl cyclase activators guanylin and uroguanylin are expressed at reduced levels in colon cancer cells (26 , 27) . Taken together, these findings suggest that cGMP-mediated pathways are suppressed in colon cancer cells, presumably to inhibit downstream signaling pathways related to the activation of PKG. There is also supporting evidence that activation of PKG by cGMP in cardiomyocytes (3) , pancreatic B cells (4) , and cultured smooth muscle cells (28 , 29) can cause growth inhibition and apoptosis. In addition, PKG activation negatively regulates interleukin-2 signaling in T cell lines (30) . Furthermore, recent studies indicate that sulindac sulfone (Aptosyn), a metabolite of the nonsteroidal anti-inflammatory drug sulindac, and two potent derivatives of Aptosyn, OSI-248 and OSI-461, specifically inhibit the cGMP-specific PDEs 2 and 5. Evidence has also been obtained that the resulting increase in cellular levels of cGMP in human colon cancer cells leads to activation of PKG and thereby the induction of apoptosis (31, 32, 33) . These novel effects of Aptosyn and related drugs may explain why these compounds exert anticancer effects in a variety of biological systems even though, in contrast to conventional nonsteroidal anti-inflammatory drugs, they do not inhibit cyclooxygenase activity (34) . The precise pathway by which PKG activation leads to apoptosis is not known, although it appears to involve both a decrease in cellular levels of ß-catenin and the activation of c-Jun NH2-terminal kinase 1 (31 , 32 , 35 , 36) .

Cell cycle progression is regulated by the orderly activation and inactivation of a series of cyclins and cyclin-dependent kinases and is coordinated by both internal and external signals that act at key checkpoints during cell cycle progression (37) . Cyclin D1 is a major positive regulator of the G1-S transition. It acts by binding to and activating cyclin-dependent kinase 4 or cyclin-dependent kinase 6, which then phosphorylates and thereby inactivates the tumor suppressor protein pRB (38) . These activities are negatively regulated by p16INK4, p21CIP1 (p21), and p27KIP1 (39) . p21CIP1 was first cloned and characterized as a mediator of p53-induced growth arrest (40) , but its expression can also be up-regulated by p53-independent mechanisms (41) . Although the cyclin D1 gene is not amplified in human colon cancer, the expression of cyclin D1 is induced in about 30% of human adenocarcinoma and also in adenomatous polyps of the colon (42 , 43) . Furthermore, inhibition of cyclin D1 expression with an antisense cDNA causes growth inhibition in colon carcinoma cell lines (44) . Colon cancers also frequently display elevated levels of ß-catenin as a result of inactivation of the adenomatous polyposis coli molecule or mutations in ß-catenin (45) . This results in increased binding of ß-catenin to the transcription element T-cell factor/lymphoid enhancer factor-1 and increased transcription of cyclin D1 and other genes that enhance growth (46 , 47) . Therefore, the high expression levels of cyclin D1 in colon cancer are at least in part due to up-regulation of ß-catenin/T-cell factor transcriptional activity. Recent studies by Li et al. (48) provide evidence that activated PKG directly phosphorylates ß-catenin in the COOH-terminal region of the molecule, in contrast to GSK3ß, which phosphorylates ß-catenin at a characteristic site in the NH2 terminus, and that this phosphorylation by PKG results in proteolytic degradation of ß-catenin.

Previous studies implicating PKG in growth inhibition and apoptosis in colon cancer cells are based on using pharmacological agents (31, 32, 33) . In the present study, we obtained more direct evidence by exploring the effects of expressing a series of wild-type (WT), constitutively active, or dominant-negative mutants of PKG, previously developed in our laboratory (33) , in the SW480 human colon cancer cell line.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and Cell Culture.
The cell-permeable cGMP compound 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP) and the anti-PKG Iß antibody were purchased from Calbiochem (San Diego, CA), an anti-ß-catenin antibody from Transduction Laboratories (San Diego, CA), a polyclonal antibody to cyclin D from Upstate USA, Inc. (Charlottesville, VA), an anti-ß-actin monoclonal antibody from Sigma (St. Louis, MO), an anti-p21CIP1 antibody from Santa Cruz Biotechnology (Santa Cruz, CA), and an anti-hemagglutinin (HA) antibody from Covance (Richmond, CA). Aptosyn and OSI-461 were provided by OSI Pharmaceuticals, Inc. The construction and characteristics of plasmids that encode HA-COOH-terminal tagged WT, constitutively active ({Delta} and SD), and dominant-negative (KR) forms of PKG I{alpha} and PKG Iß are described in our previous publication (33) . Similar vector-only plasmids were used as controls. SW480 and 293T cells were cultured in DMEM with 10% fetal bovine serum.

Colony Formation Assay.
SW480 cells were transfected with various constructs of PKG I{alpha} and PKG Iß as described previously (33) . After 18 h, the cells were replated into 100-mm dishes with 1 x 105 cells/dish, and the cells were cultured in the presence of 1 mg/ml G418 for 3 weeks. The colonies were fixed with formalin, stained with Giemsa, and counted. All assays were done in triplicate, and the results are expressed as "% of control," i.e., the number of colonies obtained compared with the vector control plasmid.

Production and Infection with Retrovirus Constructs.
The above-described HA-tagged PKG constructs were subcloned into the expression vector pMIG upstream of the IRES-enhanced Green Fluorescent Protein (GFP) sequence. pMIG was provided by Dr. Jeremy Luban (Columbia University). 293T cells were cotransfected with pMIG, PMDG, and pCL-Eco as described previously (49 , 50) . After 16 h, cells were incubated with fresh DMEM/10% fetal bovine serum medium. The retrovirus containing medium was harvested after another 24 or 48 h. SW480 cells were infected with the indicated retroviruses encoding PKG for 6 h and then incubated in fresh DMEM/10% fetal bovine serum medium for 72 h.

Determination of Apoptotic Index.
SW480 cells were infected with the indicated PKG retrovirus constructs as described above. After 72 h, the cells were harvested and analyzed on a FACSCalibur instrument using CELLQuest software (Becton Dickinson, Mountain View, CA) with the FL-1 channel to detect GFP. This indicated that the infection efficiency in all samples was about 80%. Therefore, we used the entire cell population to determine the sub-G1 population. The extent of apoptosis was determined using the FL-2 channel after propidium iodide (Sigma) staining and expressed as a percentage, by calculating the number of cells that were in the sub-G1 population divided by the total number of cells examined. For the Annexin V staining assay, the infected SW480 cells were incubated for 72 h. The harvested cells were then stained with phycoerythrin-conjugated Annexin V (PharMingen, San Diego, CA) for 15 min in the dark, according to the manufacturer’s protocol. The stained cells were then analyzed on a FACSCalibur instrument using CELLQuest software (Becton Dickinson). The apoptotic index was expressed as a percentage, by calculating the number of cells that were positive for both phycoerythrin-conjugated Annexin V and GFP divided by the total number of GFP-positive cells examined.

Determination of Cell Doubling Time.
Cells were plated at a density of 2 x 104 in 15.6-mm diameter, 24-well dishes. The numbers of cells per well were counted daily for the next 7 days, using a Coulter counter. The doubling times were measured during the period of exponential growth.

Western Blot Analysis.
SW480 cells were harvested and then sonicated in radioimmunoprecipitation assay (RIPA) buffer, and extracts were examined by Western blot analysis as described previously (33) . The lysates were electrophoresed on a 10% polyacrylamide gel and then electophoretically transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% dry milk and incubated with the indicated antibody. After washing, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences, Piscataway, NJ). Protein bands were visualized with the enhanced chemiluminescence Western blotting system (Amersham Biosciences). Fold expression was determined with NIH Image1.62 software.

Luciferase Reporter Assays.
SW480 cells were plated at 1 x 105 cells per 6-well 35-mm-diameter plates, and 24 h later, they were transfected with 500 ng of the cyclin D1-luc (51) reporter plasmid, the indicated PKG expression plasmids, and 10 ng of cytomegalovirus-ß-galactosidase reporter plasmid (as internal control), using Lipofectin (Invitrogen, Carlsbad, CA). After 18 h of transfection, some of the cells were incubated in fresh growth medium in the presence or absence of 8-pCPT-cGMP for 24 h before harvesting. Luciferase activities were normalized to ß-galactosidase activities, to correct for differences in transfection efficiency. The relative luciferase activity measured in the vector control cells was assigned the value of 100%. All assays were done in triplicate.

Cell Invasion Assays.
SW480 cells were infected with the indicated retroviruses of PKG, as described above. After 18 h, the infected cells were replated into the upper well of transwell chambers (Matrigel; BD Biosciences, Bedford, MA). After a 24 h incubation with or without 100 µM 8-pCPT-cGMP, the cells on the upper surface of the chamber were removed by scraping with a cotton swab. Cells that had migrated through the filter were stained with 1% crystal violet and counted by microscopy. Similar assays were done with cells that stably overexpressed PKG I{alpha} or Iß as described in Fig. 4Citation .



View larger version (42K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Stable overexpression of PKG Iß in SW480 cells decreases the expression of ß-catenin and cyclin D1 but increases the expression of p21CIP1. The levels of expression of PKG Iß, ß-catenin, cyclin D1, p21CIP1, and ß-actin were determined by Western blotting with the respective antibodies, in clonal derivatives of SW480 cells. These included: a vector control clone; two clones that overexpress PKG Iß WT (Iß#3 and Iß#6); and two clones that express the dominant-negative mutant of PKG Iß (IßKR#3 and IßKR#9). Fold expression was analyzed with NIH Image 1.62 software. Similar results were obtained in three independent experiments.

 
Statistical Methods.
Results of the experimental studies are reported as mean ± SD. Differences were analyzed by Student’s t test. A value of P < 0.05 was regarded as significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Constitutively Activated Mutants of PKG I{alpha} and PKG Iß Inhibit Colony Formation in SW480 Cells.
To directly examine the effects of PKG on growth and apoptosis, we constructed a series of expression vectors that encode WT-PKG I{alpha} or the following mutants of PKG I{alpha}: PKG I{alpha}S65D, a constitutively activated point mutant; PKG I{alpha}{Delta}, a constitutively activated N-terminal truncated mutant; and PKG I{alpha}K390R, a dominant-negative point mutant. A similar series of expression vectors of PKG Iß were also constructed, i.e., PKG Iß, PKG IßS80D, PKG Iß{Delta}, and PKG IßK405R (33) . We transfected each of these vectors into SW480 cells, the cells were grown in selection medium containing G418, and afterward, the colonies were stained with Giemsa and counted (Fig. 1)Citation . Transfection with the constitutively activated mutants of PKG I{alpha} {Delta} and SD and the corresponding constitutively activated mutants of PKG Iß caused about 45% inhibition of colony formation (P < 0.001). The WT forms of PKG I{alpha} and PKG Iß produced about 30% inhibition, and the KR forms produced about 10% inhibition, but these values were not statistically significant.



View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Inhibition of colony formation by activated forms of PKG I{alpha} and Iß in SW480 cells. The indicated constructs were transfected into SW480 colon cancer cells, and the cells were grown in the presence of the selection agent G418 for 3 weeks. The colonies were then fixed, stained with Giemsa solution, and counted. The results are representative of triplicate experiments. Assays were done in triplicate, and the data are plotted as mean values with SDs (error bars). *, significant inhibition when compared with the control (P < 0.05). For additional details, see "Materials and Methods."

 
Constitutively Activated Mutants of PKG I Induce Apoptosis in SW480 Cells.
In view of the above results, it was of interest to examine whether activation of PKG I is itself sufficient to induce apoptosis in SW480 cells. To assure that our PKG I constructs were expressed with high efficiency, we incorporated the HA-tagged PKGs into the retrovirus vector pMIG. This vector also encodes eGFP so that we could monitor the infection efficiency (see "Materials and Methods"). Indeed, we found that the infection frequency was about 80% (data not shown). The extent of apoptosis was monitored by determining the sub-G1 fraction of cells by flow cytometry at 72 h after infection with the various retrovirus constructs (Fig. 2A)Citation . We found that when compared with the vector control-infected cells, the PKG I{alpha}, I{alpha}KR, and IßKR vectors did not induce apoptosis. However, the I{alpha}{Delta}, I{alpha}SD, Iß, and IßSD vectors caused about a 1.5-fold increase in apoptosis (P < 0.05, 0.001, 0.001, and 0.001, respectively). The strongest effect was seen with Iß{Delta} vector because it caused about a 2-fold increase in apoptosis (P < 0.001). Similar effects were seen in a repeat experiment (data not shown). The relatively high apoptosis in the vector control cells is probably due to the infection procedure itself, which involved serum starvation. Significantly greater apoptotic effects were seen with the Iß WT, {Delta}, and SD constructs than the corresponding I{alpha} constructs. (P < 0.005, 0.01, and 0.001, respectively). To extend the results obtained with the PKG Iß constructs, a similar experiment was done, but after infection, the extent of apoptosis was assayed by the Annexin V staining procedure (see "Materials and Methods"). In this study, WT-PKG Iß caused a small increase in apoptosis (P < 0.05), but Iß{Delta} and IßSD constructs produced strong increases in apoptosis (P < 0.001 and 0.01, respectively), and again, the IßKR construct did not induce apoptosis (Fig. 2B)Citation . Thus, constitutive activation of PKG I, especially PKG Iß, is sufficient to induce apoptosis in SW480 cells. These results are consistent with the inhibitory effects of activated mutants of PKG I{alpha} and Iß on colony formation, described in Fig. 1Citation .



View larger version (30K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Constitutively activated mutants of PKG can induce apoptosis in SW480 cells. SW480 cells were infected with retroviral constructs encoding the indicated forms of PKG for 6 h. After 72 h, the cells were analyzed by flow cytometry with FL-1 to detect GFP. More than 80% of the cells were positive for GFP (data not shown). A, the sub-G1 fraction of the entire cell population was determined as described in "Materials and Methods." The sub-G1 population of noninfected SW480 cells was shown as a negative control (Non). Error bars indicate SDs; *, significant increases in the apoptotic index when compared with the vector control cells (P < 0.05). B, cells were infected with the indicated PKG constructs as described in A, and after 72 h, the apoptotic index was determined by flow cytometry with phycoerythrin-conjugated Annexin V staining. For additional details, see "Materials and Methods." Error bars indicate SDs; *, significant increases compared with the vector control cells (P < 0.05). These experiments were repeated at least twice and gave similar results.

 
Stable Overexpression of WT-PKG Iß Inhibits Cell Proliferation in SW480 Cells.
Because the above studies suggested that PKG Iß appeared to have greater growth inhibitory activity than PKG I{alpha}, we examined the effects of PKG Iß in greater detail by establishing derivatives of SW480 cells that stably overexpress this enzyme. We could not develop derivatives that stably overexpressed the constitutively activated mutants PKG Iß{Delta} or IßSD, presumably because of their cytotoxicity. We were however, successful in developing pools of cells that stably overexpressed WT-PKG Iß or the KR (dominant-negative) mutant of PKG Iß, as demonstrated by Western blot analysis (Fig. 3A)Citation . The cells that overexpressed the mutant KR were more condensed and more adherent than the vector control cells. Conversely, the cells that overexpressed WT-PKG Iß were less adherent (data not shown). We then compared the growth rates of these two types of derivatives with that of a pool of cells derived from SW480 cells that had been transfected with only the control vector. Whereas, the exponential doubling time for the vector control cells was 21.4 ± 1 h, the corresponding values for the WT-PKG Iß overexpressor cells and the IßKR overexpressor cells were 29.4 ± 0.4 and 16.8 ± 0.7 h, respectively (P < 0.0001 and P < 0.05, respectively). Thus, stable overexpression of PKG Iß inhibits the proliferation of SW480 cells. Because the parental SW480 cells express detectable levels of endogenous PKG Iß (data not shown), the fact that the derivatives that stably express a dominant-negative PKG Iß grow more rapidly suggests that endogenous PKG Iß can itself exert a partial growth inhibitory effect in these cancer cells.



View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Growth curves of PKG derivatives of SW480 cells. SW480 cells were transfected with expression vectors encoding wild-type (IßWT) or dominant-negative (IßKR) forms of PKG Iß or a vector control, and the cells were grown in the presence of the selection agent G418 for 3 weeks. We then did Western blot analysis using an anti-HA antibody (A) and growth curves (B) on pools of the PKG IßWT, PKG IßKR, and vector control cells. The doubling times during the period of exponential growth for vector control, IßWT, and IßKR cells were 21.4 ± 1.0, 29.4 ± 0.4, and 16.8 ± 0.7 h, respectively. P values for differences between the IßWT and IßKR and the vector control cells were <0.0001 and <0.05, respectively. These experiments were repeated at least twice with similar results.

 
Overexpression of PKG Iß Inhibits Expression of Cyclin D1 and ß-Catenin but Increases Expression of p21CIP1.
Because we found that stable overexpression of PKG Iß inhibited cell proliferation, we examined possible effects of PKG Iß on expression of the cell cycle control proteins cyclin D1 and p21CIP1 and on the signaling molecule ß-catenin whose function is often perturbed in colon cancer cells (45) . In these studies, we used two clonal derivatives of SW480 cells that stably expressed WT-PKG Iß (clones Iß#3 and Iß#6), two clonal derivatives that stably overexpressed PKG IßKR (IßKR#3 and IßKR#9), and a vector control clone (v#3). Cell extracts of these clones were obtained from exponentially growing cultures and examined by Western blot analysis. We found that the Iß#3 and Iß#6 cells displayed a marked decrease in the cellular level of cyclin D1 and some decrease in ß-catenin, when compared with the v#3 cells. The IßKR#9 cells had higher levels of cyclin D1 than the v#3 cells, but no apparent change in the level of ß-catenin. There was also a marked increase in the cellular level of p21CIP1 in the Iß#3 and Iß#6 cells when compared with the v#3 cells (Fig. 4)Citation . The latter finding is consistent with our finding that treatment of SW480 cell with Aptosyn, OSI-461, or 8-pCPT-cGMP, agents that activate PKG, causes rapid induction of p21CIP1 (data not shown). Induction of p21CIP1 expression by OSI-461 has also been described in chronic lymphocytic leukemia cells (52) .

Because the SW480 cells that stably overexpress PKG Iß had decreased levels of the cyclin D1 protein (Fig. 4)Citation , we examined whether PKG Iß affects the transcriptional activity of the promoter region of the cyclin D1 gene by carrying out transient transfection reporter assays using a cyclin D1 promoter-luciferase reporter. Cotransfection of the cells with WT-PKG Iß partially inhibited luciferase activity, and this was additionally enhanced by treating the transfected cells with a cell-permeable activator of PKG 8-pCPT-cGMP (Fig. 5A)Citation , cotransfection with PKG Iß{Delta} or PKG IßSD caused marked inhibition, but cotransfection with PKG IßKR had no effect, on luciferase activity (Fig. 5A)Citation . To obtain additional evidence that activation of PKG inhibits the transcriptional activity of the cyclin D1 promoter, we examined the effects of treating parental SW480 cells with two compounds, Aptosyn and OSI-461, that inhibit cGMP PDEs 2 and 5, thereby activating endogenous PKG (31) . It is apparent that both compounds inhibited luciferase activity in these reporter assays (Fig. 5B)Citation .



View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Activation of PKG Iß inhibits the transcriptional activity of the cyclin D1 promoter in SW480 cells. A, SW480 cells were transfected with the –1745 cyclin D1 promoter luciferase (luc) reporter and the pCMV-ß-galactosidase reporter and also cotransfected with PKG WT or PKG mutant expression vectors, as indicated. After 18 h of transfection, the cells were incubated in fresh growth medium, in the presence or absence of 250 µM 8-pCPT-cGMP (8pCPTcGMP) for 24 h prior, and extracts were assayed for relative luciferase activity. B, SW480 cells were transfected with the –1745 cyclin D1-luciferase and pCMV-ß-galactosidase reporters. After 18 h, the cells were incubated in fresh growth medium in the presence or absence of 600 µM Aptosyn or 10 µM OSI-461 for 24 h, and extracts were prepared. The relative luciferase activity measured in the vector control cells was assigned the value of 100%. Error bars indicate SDs of triplicate assays. Similar results were obtained in three independent experiments.

 
Effects of PKG Iß Expression on Sensitivity to Growth Inhibition by Aptosyn.
It was of interest to determine whether derivatives that stably overexpress WT-PKG Iß or the dominant-negative mutant PKG Iß differ in their sensitivity to growth inhibition by Aptosyn, because as described above, the growth inhibitory and apoptotic effects of this compound appear to be mediated, at least in part, through the activation of PKG (31, 32, 33) . Cell viability assays using SW480 cells carried out with increasing concentrations of Aptosyn indicated that with the v#3 control cells, the IC50 for Aptosyn was 300 µM. The Iß#3 clone that stably overexpresses wt-PKG Iß displayed an IC50 of 250 µM (P > 0.05), but this difference was not statistically significant. However, 600 µM Aptosyn, which is twice the IC50 for the parental SW480 and v#3 cells, caused a significantly greater inhibition of the Iß#3 cells than that obtained with the v#3 cells (P < 0.01). On the other hand, the PKG IßKR#9 cells were more resistant to Aptosyn than the v#3 cells because the IC50 with the former cells was greater than 600 µM (P < 0.001; Fig. 6Citation ). Similar results were obtained in repeat studies (data not shown). These results provide additional evidence that PKG plays a role in mediating the growth inhibitory effects of Aptosyn and related compounds, although it is possible that our findings also reflect a more general effect of PKG on cell viability. The possible clinical relevance of these results is mentioned under "Discussion."



View larger version (19K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Effects of expression of PKG Iß on sensitivity to Aptosyn-induced growth inhibition in SW480 cells. Control v#3 cells, PKGIß#3 cells that stably overexpress WT-PKGIß (Iß#3), and PKG IßKR#9 cells that stably overexpress a dominant-negative mutant of PKG Iß (IßKR#9; see Fig. 4Citation ) were treated with Aptosyn (Exisulind) at the indicated concentrations. The numbers of cells were counted after 72 h using a Coulter counter and expressed as the percentage of the respective untreated cells. Error bars indicate means of triplicate assays. The inhibitory effects of 600 µM Aptosyn were significantly greater in the PKG Iß#3 cells (*, P < 0.01) and were significantly less in the PKG IßKR#9 cells (**, P < 0.001) than in the v#3 control cells. Similar results were obtained in three independent experiments.

 
Activation of PKG Inhibits Cell Migration in SW480 Cells.
We previously reported that treatment of SW480 cells with Aptosyn and other compounds that lead to activation of PKG or the overexpression of constitutively activated mutants of PKG results in increased phosphorylation of the protein VASP (33) . Smolenski et al. (16) found that activation of PKG in human endothelial cells caused inhibition of cell migration through the phosphorylation of VASP, presumably because VASP plays a role in regulating focal adhesion complexes. Therefore, it was of interest to examine the effects of our PKG constructs on cell migration in SW480 cells, employing Matrigel-coated transwell chambers. In one set of studies, SW480 cells were infected with the above-described series of retroviruses encoding various forms of PKG I and then assayed 24 h later for cell migration (Fig. 7A)Citation . We found that the WT, {Delta}, and SD constructs of both PKG I{alpha} and PKG Iß, but not the KR constructs, caused significant inhibition of cell migration. These effects were strongest with the PKG Iß constructs, especially the {Delta} and SD constitutively activated mutants. This inhibition is not simply because of loss of cell viability due to induction of apoptosis, because although some of these vectors can induce apoptosis at 72 h after infection (see Fig. 2Citation ), no increase in apoptosis was seen at 24 h (data not shown). Nevertheless, it was important to confirm these effects by also examining the derivatives of SW480 cells that stably overexpress PKG I{alpha} or PKG Iß and yet remain viable. These studies were done with and without the addition of 8-pCPT-cGMP to the medium to enhance the activation of PKG. Fig. 7BCitation indicates that when the vector control derivative was treated with 8-pCPT-cGMP, there was significant inhibition of cell migration. Even in the absence of treatment with this cGMP compound, the PKG I{alpha}#1 clone and the PKG Iß#3 clone displayed significant decreases in cell migration when compared with the untreated vector control cells (P < 0.001). This inhibition was additionally increased when these cells were treated with the cGMP compound (P < 0.001). As in our previous studies (Fig. 1Citation ; Fig. 2Citation ), these effects were greater in the PKG Iß than in the PKG I{alpha} cells. Thus, in addition to causing growth inhibition and activation of apoptosis, activation of PKG can inhibit the migration of colon cancer cells. This effect may be mediated, at least in part, through the phosphorylation of VASP, because VASP phosphorylation can cause disruption of focal adhesions (16) and possibly other changes in the cellular cytoskeleton.



View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. Activation of PKG inhibits cell migration in SW480 cells. A, SW480 cells were infected with the indicated retrovirus constructs of PKG for 6 h. After 24 h, the cells were transferred into Matrigel-coated transwell chambers. After an additional 24 h, cell migration was determined (see "Materials and Methods"), and the results are expressed as the percentage of the vector control. B, migration assays with cells that stably overexpress PKG I{alpha} or Iß. Cells were seeded into Matrigel-coated transwell chambers. After 6 h, 100 µM 8-pCPT-cGMP was added to the medium, as indicated. After 24 h, migration was determined, as described above. Error bars indicate SDs of triplicate assays; *, assays with significant inhibition (P < 0.05). These experiments were repeated and gave similar results.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present studies provide the first direct evidence that activation of PKG is sufficient to induce growth inhibition and apoptosis and also inhibit cell migration in human cancer cells. Our findings are consistent with previous evidence that activation of PKG in vascular smooth muscle cells can have similar effects (28 , 29) and that overexpression of a constitutively activated PKG mutant also inhibits growth and cell migration in the porcine vascular system (29) . It is of interest that in the latter study, vascular surgery decreased the expression of PKG, and this was associated with restenosis. Previous studies indicate that various chemical agents that cause an increase in cellular levels of cGMP lead to induction of apoptosis in human colon cancer cells (31, 32, 33 , 35) , but these findings did not provide direct evidence that activation of PKG was sufficient to produce this effect.

The present study indicates that activation of PKG, specifically PKG Iß, is also sufficient to cause a decrease in cellular levels of cyclin D1 and an increase in cellular levels of the cell cycle inhibitory protein p21CIP1 in SW480 colon cancer cells (Fig. 4)Citation . Transient transfection reporter assays provide evidence that these effects are due to inhibition of the transcriptional activity of the cyclin D1 promoter (Fig. 5)Citation . Our results with cyclin D1 differ from those obtained by Hanada et al. (53) , who found that overexpression of PKG Iß in murine mesangial cells that were also treated with 8-bromo-cGMP inhibited the promoter activity of cyclin E but not cyclin D1. This difference may reflect differences between their cell system and ours or the fact that they used a reporter plasmid encoding the –944 to +139 region of the cyclin D1 promoter, whereas we used a full-length cyclin D1 promoter (–1745CD1; Ref. 51 ). Therefore, it is possible that the region of the cyclin D1 promoter between –1745 and –944 is required for PKG to exert its inhibitory effect. ß-Catenin can enhance the transcription of cyclin D1 by binding to the T-cell factor/lymphoid enhancer factor-1 site of the cyclin D1 promoter (45 , 46) . We found that stable overexpression of WT-PKG Iß decreased the cellular level of ß-catenin in SW480 cells (Fig. 4)Citation . In addition, there is evidence that ß-catenin is a substrate of PKG Iß and that this phosphorylation can contribute to the degradation of ß-catenin through a GSK3ß-independent pathway (47) . This mechanism may explain, at least in part, why activation of PKG causes decreased expression of cyclin D1, but possible effects on other transcription factors may also play a role.

It is known that the expression of p21CIP1 is regulated largely at the level of transcription by both p53-dependent and -independent mechanisms (40) . The promoter of the p21CIP1 gene contains two conserved p53-binding sites, and at least one of these is required for p53 responsiveness after DNA damage (54) . In addition, a variety of transcriptional factors that are induced by a number of different signal pathways can activate p21CIP1 transcription by a p53-independent mechanism, including SP1, SP3, STATs, C/EBP{alpha}, C/EBPß, and Smad3 (55, 56, 57) . p21CIP1 expression is also regulated posttranscriptionally by both ubiquitin-dependent and -independent proteasome-mediated degradation (58 , 59) . Because the SW480 cells that we used carry a mutant p53, the induction of p21CIP1 by PKG appears to be through a p53-independent pathway. The precise mechanism by which PKG leads to increased expression of p21CIP1 remains to be determined. Our results are consistent with the finding of Gu et al. (60) that treatment of cells with nitric oxide, which activates guanylyl cyclase, caused increased expression of p21CIP1 via a cGMP-dependent pathway. In addition, we found that treatment of SW480 cells with Aptosyn or OSI-461, agents that increase cellular levels of cGMP (33) , causes a rapid increase in the expression of both p21CIP1 mRNA and protein.4

The decreased expression of ß-catenin and cyclin D1 and increased expression of p21CIP1 may contribute to the growth inhibitory and apoptotic effects of PKG activation in SW480 cells. However, the role of p21CIP1 in growth inhibition and induction of apoptosis can vary in different cell systems (61) . Thus, p21CIP1 null mice display an increase in tumor incidence (62) , and overexpression of p21CIP1 enhances growth inhibition and apoptosis in glioma (63) and ovarian carcinoma (64) cell lines. On the other hand, up-regulation of p21CIP1 inhibits transforming growth factor ß-induced apoptosis in retinal endothelial cells (65) . In addition, other effects of PKG activation appear to play an important role in the induction of apoptosis by Aptosyn and related compounds, including activation of the MEKK1-SEK1-c-Jun NH2-terminal kinase 1 pathway (36) . Furthermore, Shureiqi et al. (66 , 67) have recently obtained evidence that increased expression of 15-lipoxygenase-1 can play an important role in the induction of apoptosis in human colon cancer cell lines by Aptosyn, and we have recently obtained evidence that PKG activation may also play a role in the induction of 15-lipoxygenase-1.5 Thus, the growth inhibition and apoptosis caused by activation of PKG is probably mediated by a complex series of events.

It is of interest that SW480 cells that stably express the dominant negative (KR) mutant of PKG Iß were more resistant to the growth inhibitory effects of Aptosyn than vector control cells, whereas cells that overexpressed WT-PKG Iß were more sensitive to this drug (Fig. 6)Citation . These results suggest that the sensitivity of different cancer cells to the growth inhibitory effects of this class of compounds may depend, at least in part, on their cellular levels of PKG, a finding that may be relevant to the clinical use of these compounds in cancer chemoprevention and treatment. We should, however, emphasize that the present results do not exclude the possibility that the growth inhibitory and apoptotic effects of these compounds are also mediated via pathways that do not involve the action of PKG.

Our finding that activation of PKG in SW480 cells inhibited their migration (Fig. 7)Citation may also be of clinical relevance because it suggests that Aptosyn and related compounds may exert antitumor effects not only by inhibiting cell proliferation but also by inhibiting invasion and metastasis. We previously reported that activation of PKG in SW480 cells results in rapid and persistent phosphorylation of the focal adhesion-associated protein VASP (33) . Because Smolenski et al. (16) have found that in endothelial cells phosphorylation of VASP by PKG inhibits the attachment of focal adhesions and thus inhibits cell migration, we suspect that a similar mechanism explains the inhibitory effect of PKG activation on cell migration in SW480 cells.

In summary, we believe that the present studies provide evidence that PKG can play a direct role in inducing growth inhibition and apoptosis and inhibiting cell migration in cancer cells. These findings provide a rationale for targeting PKG and related pathways as a novel approach to cancer chemoprevention and therapy. Additional studies are required to determine the precise mechanisms by which PKG activation can inhibit growth and induce apoptosis in colon cancer cells.


   ACKNOWLEDGMENTS
 
We thank T. Jahnsen, S. Orstavik, M. Sandberg, and K. Tasken (Oslo, Norway), and R. Reed, S. Francis, and J. Corbin (Vanderbilt University, Nashville, TN) for WT-PKG I{alpha} and Iß cDNAs. We also thank J. Luban and M. Asmal (Columbia University) for retrovirus vectors and R. G. Pestell (Georgetown University, Washington, DC) for providing the cyclin D1 promoter-luc plasmid.


   FOOTNOTES
 
Grant support: OSI Pharmaceuticals, Inc., Entertainment Industry Foundation-National Colorectal Cancer Research Alliance (I. Weinstein), the T. J. Martell Foundation (I. Weinstein), and the National Foundation for Cancer Research (I. Weinstein).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: I. Bernard Weinstein, the Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University, 701 W. 168th St., HHSC-1509, New York, NY 10032-2704. Phone: (212) 305-6921; Fax: (212) 305-6889; E-mail: ibw1{at}columbia.edu

4 Unpublished observations. Back

5 Unpublished observations. Back

Received 12/ 1/03. Revised 2/23/04. Accepted 3/ 3/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Eigenthaler M, Lohmann SM, Walter U, Pilz RB. Signal transduction by cGMP-dependent protein kinases and their emerging roles in the regulation of cell adhesion and gene expression. Rev Physiol Biochem Pharmacol, 135: 173-209, 1999.[Medline]
  2. Vaandrager AB, de Jonge HR. Signalling by cGMP-dependent protein kinases. Mol Cell Biochem, 157: 23-30, 1996.[Medline]
  3. Shimojo T, Hiroe M, Ishiyama S, Ito H, Nishikawa T, Marumo F. Nitric oxide induces apoptotic death of cardiomyocytes via a cyclic-GMP-dependent pathway. Exp Cell Res, 247: 38-47, 1999.[CrossRef][Medline]
  4. Loweth AC, Williams GT, Scarpello JH, Morgan NG. Evidence for the involvement of cGMP and protein kinase G in nitric oxide-induced apoptosis in the pancreatic B-cell line, HIT-T15. FEBS Lett, 400: 285-8, 1997.[CrossRef][Medline]
  5. Chiche JD, Schlutsmeyer SM, Bloch DB, et al Adenovirus-mediated gene transfer of cGMP-dependent protein kinase increases the sensitivity of cultured vascular smooth muscle cells to the antiproliferative and pro-apoptotic effects of nitric oxide/cGMP. J Biol Chem, 273: 34263-71, 1998.[Abstract/Free Full Text]
  6. Stacey P, Rulten S, Dapling A, Phillips SC. Molecular cloning and expression of human cGMP-binding cGMP-specific phosphodiesterase (PDE5). Biochem Biophys Res Commun, 247: 249-54, 1998.[CrossRef][Medline]
  7. Smolenski A, Burkhardt AM, Eigenthaler M, et al Functional analysis of cGMP-dependent protein kinases I and II as mediators of NO/cGMP effects. Naunyn-Schmiedebergs Arch Pharmacol, 358: 134-9, 1998.[CrossRef][Medline]
  8. Lincoln TM, Cornwell TL. Intracellular cyclic GMP receptor proteins. FASEB J, 7: 328-38, 1993.[Abstract]
  9. Ruth P. Cyclic GMP-dependent protein kinases: understanding in vivo functions by gene targeting. Pharmacol Ther, 82: 355-72, 1999.[CrossRef][Medline]
  10. Lohmann SM, Vaandrager AB, Smolenski A, Walter U, De Jonge HR. Distinct and specific functions of cGMP-dependent protein kinases. Trends Biochem Sci, 22: 307-12, 1997.[CrossRef][Medline]
  11. Lohmann SM, Fischmeister R, Walter U. Signal transduction by cGMP in heart. Basic Res Cardiol, 86: 503-14, 1991.[CrossRef][Medline]
  12. Draijer R, Atsma DE, van der Laarse A, van Hinsbergh VW. cGMP and nitric oxide modulate thrombin-induced endothelial permeability: regulation via different pathways in human aortic and umbilical vein endothelial cells. Circ Res, 76: 199-208, 1995.[Abstract/Free Full Text]
  13. Draijer R, Vaandrager AB, Nolte C, de Jonge HR, Walter U, van Hinsbergh VW. Expression of cGMP-dependent protein kinase I and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin. Circ Res, 77: 897-905, 1995.[Abstract/Free Full Text]
  14. Pfeifer A, Klatt P, Massberg S, et al Defective smooth muscle regulation in cGMP kinase I-deficient mice. EMBO J, 17: 3045-51, 1998.[CrossRef][Medline]
  15. Massberg S, Sausbier M, Klatt P, et al Increased adhesion and aggregation of platelets lacking cyclic guanosine 3',5'-monophosphate kinase I. J Exp Med, 189: 1255-64, 1999.[Abstract/Free Full Text]
  16. Smolenski A, Poller W, Walter U, Lohmann SM. Regulation of human endothelial cell focal adhesion sites and migration by cGMP-dependent protein kinase I. J Biol Chem, 275: 25723-32, 2000.[Abstract/Free Full Text]
  17. Uhler MD. Cloning and expression of a novel cyclic GMP-dependent protein kinase from mouse brain. J Biol Chem, 268: 13586-91, 1993.[Abstract/Free Full Text]
  18. Jarchau T, Hausler C, Markert T, et al Cloning, expression, and in situ localization of rat intestinal cGMP-dependent protein kinase II. Proc Natl Acad Sci USA, 91: 9426-30, 1994.[Abstract/Free Full Text]
  19. Stacey P, Rulten S, Dapling A, Phillips SC. Molecular cloning and expression of human cGMP-binding cGMP-specific phosphodiesterase (PDE5). Biochem Biophys Res Commun, 247: 249-54, 1998.
  20. Singer AL, Sherwin RP, Dunn AS, Appleman MM. Cyclic nucleotide phosphodiesterases in neoplastic and nonneoplastic human mammary tissues. Cancer Res, 36: 60-6, 1976.[Abstract/Free Full Text]
  21. Epstein PM, Hachisu R. Cyclic nucleotide phosphodiesterase in normal and leukemic human lymphocytes and lymphoblasts. Adv Cyclic Nucleotide Protein Phosphorylation Res, 16: 303-24, 1984.[Medline]
  22. Pusztai L, Zhen JH, Arun B, et al Phase I and II study of exisulind in combination with capecitabine in patients with metastatic breast cancer. J Clin Oncol, 21: 3454-61, 2003.[Abstract/Free Full Text]
  23. Piazza GA, Xu S, Klein-Szanto A, et al Overexpression of cGMP phosphodiesterase in colonic neoplasias compared to normal mucosa. Gastroenterology, 118: A282 2000.
  24. Piazza GA, Thompson WJ, Pamukcu R, et al Exisulind, a novel proapoptotic drug, inhibits rat urinary bladder tumorigenesis. Cancer Res, 61: 3961-8, 2001.[Abstract/Free Full Text]
  25. Whitehead CM, Earle KA, Fetter J, et al Exisulind-induced apoptosis in a non-small cell lung cancer orthotopic lung tumor model augments docetaxel treatment and contributes to increased survival. Mol Cancer Ther, 2: 479-88, 2003.[Abstract/Free Full Text]
  26. Shailubhai K, Yu HH, Karunanandaa K, et al Uroguanylin treatment suppresses polyp formation in the Apc(Min/+) mouse and induces apoptosis in human colon adenocarcinoma cells via cyclic GMP. Cancer Res, 60: 5151-7, 2000.[Abstract/Free Full Text]
  27. Notterman DA, Alon U, Sierk AJ, Levine AJ. Transcriptional gene expression profiles of colorectal adenoma, adenocarcinoma, and normal tissue examined by oligonucleotide arrays. Cancer Res, 61: 3124-30, 2001.[Abstract/Free Full Text]
  28. Chiche JD, Schlutsmeyer SM, Bloch DB, et al Adenovirus-mediated gene transfer of cGMP-dependent protein kinase increases the sensitivity of cultured vascular smooth muscle cells to the antiproliferative and pro-apoptotic effects of nitric oxide/cGMP. J Biol Chem, 273: 34263-71, 1998.
  29. Sinnaeve P, Chiche JD, Gillijns H, et al Overexpression of a constitutively active protein kinase G mutant reduces neointima formation and in-stent restenosis. Circulation, 105: 2911-6, 2002.[Abstract/Free Full Text]
  30. Fischer TA, Palmetshofer A, Gambaryan S, et al Activation of cGMP-dependent protein kinase I ß inhibits interleukin 2 release and proliferation of T cell receptor-stimulated human peripheral T cells. J Biol Chem, 276: 5967-74, 2001.[Abstract/Free Full Text]
  31. Thompson WJ, Piazza GA, Li H, et al Exisulind induction of apoptosis involves guanosine 3',5'-cyclic monophosphate phosphodiesterase inhibition, protein kinase G activation, and attenuated ß-catenin. Cancer Res, 60: 3338-42, 2000.[Abstract/Free Full Text]
  32. Soh JW, Mao Y, Kim MG, et al Cyclic GMP mediates apoptosis induced by sulindac derivatives via activation of c-Jun NH2-terminal kinase 1. Clin Cancer Res, 6: 4136-41, 2000.[Abstract/Free Full Text]
  33. Deguchi A, Soh JW, Li H, et al Vasodilator-stimulated phosphoprotein (VASP) phosphorylation provides a biomarker for the action of exisulind and related agents that activate protein kinase G. Mol Cancer Ther, 1: 803-9, 2002.[Abstract/Free Full Text]
  34. Piazza GA, Alberts DS, Hixson LJ, et al Sulindac sulfone inhibits azoxymethane-induced colon carcinogenesis in rats without reducing prostaglandin levels. Cancer Res, 57: 2909-15, 1997.[Abstract/Free Full Text]
  35. Liu L, Li H, Underwood T, et al Cyclic GMP-dependent protein kinase activation and induction by exisulind and CP461 in colon tumor cells. J Pharmacol Exp Ther, 299: 583-92, 2001.[Abstract/Free Full Text]
  36. Soh JW, Mao Y, Liu L, Thompson WJ, Pamukcu R, Weinstein IB. Protein kinase G activates the JNK1 pathway via phosphorylation of MEKK1. J Biol Chem, 276: 16406-10, 2001.[Abstract/Free Full Text]
  37. Sgambato A, Flamini G, Cittadini A, Weinstein IB. Abnormalities in cell cycle control in cancer and their clinical implications. Tumori, 84: 421-33, 1998.[Medline]
  38. Sherr CJ. Cancer cell cycles. Science, 274: 1672-7, 1996.[Abstract/Free Full Text]
  39. Sherr CJ. The Pezcoller lecture: cancer cell cycles revisited. Cancer Res, 60: 3689-95, 2000.[Abstract/Free Full Text]
  40. el-Deiry WS, Tokino T, Velculescu VE, et al WAF1, a potential mediator of p53 tumor suppression. Cell, 75: 817-25, 1993.[CrossRef][Medline]
  41. Gartel AL, Tyner AL. Transcriptional regulation of the p21((WAF1/CIP1)) gene. Exp Cell Res, 246: 280-9, 1999.[CrossRef][Medline]
  42. Arber N, Hibshoosh H, Moss SF, et al Increased expression of cyclin D1 is an early event in multistage colorectal carcinogenesis. Gastroenterology, 110: 669-74, 1996.[CrossRef][Medline]
  43. Bartkova J, Lukas J, Muller H, Lutzhoft D, Strauss M, Bartek J. Cyclin D1 protein expression and function in human breast cancer. Int J Cancer, 57: 353-61, 1994.[Medline]
  44. Arber N, Doki Y, Han EK, et al Antisense to cyclin D1 inhibits the growth and tumorigenicity of human colon cancer cells. Cancer Res, 57: 1569-74, 1997.[Abstract/Free Full Text]
  45. Sparks AB, Morin PJ, Vogelstein B, Kinzler KW. Mutational analysis of the APC/ß-catenin/Tcf pathway in colorectal cancer. Cancer Res, 58: 1130-4, 1998.[Abstract/Free Full Text]
  46. Tetsu O, McCormick F. ß-Catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature, 398: 422-6, 1999.[CrossRef][Medline]
  47. Shtutman M, Zhurinsky J, Simcha I, et al The cyclin D1 gene is a target of the ß-catenin/LEF-1 pathway. Proc Natl Acad Sci USA, 96: 5522-7, 1999.[Abstract/Free Full Text]
  48. Li H, Liu L, David ML, et al Pro-apoptotic actions of exisulind and CP461 in SW480 colon tumor cells involve ß-catenin and cyclin D1 down-regulation. Biochem Pharmacol, 64: 1325-36, 2002.[CrossRef][Medline]
  49. Van Parijs L, Refaeli Y, Lord JD, Nelson BH, Abbas AK, Baltimore D. Uncoupling IL-2 signals that regulate T cell proliferation, survival, and Fas-mediated activation-induced cell death. Immunity, 11: 281-8, 1999.[CrossRef][Medline]
  50. Asmal M, Colgan J, Naef F, et al Production of ribosome components in effector CD4+ T cells is accelerated by TCR stimulation and coordinated by ERK-MAPK. Immunity, 19: 535-48, 2003.[CrossRef][Medline]
  51. Albanese C, Johnson J, Watanabe G, et al Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem, 270: 23589-97, 1995.[Abstract/Free Full Text]
  52. Moon EY, Lerner A. Benzylamide sulindac analogues induce changes in cell shape, loss of microtubules and G(2)-M arrest in a chronic lymphocytic leukemia (CLL) cell line and apoptosis in primary CLL cells. Cancer Res, 62: 5711-9, 2002.[Abstract/Free Full Text]
  53. Hanada S, Terada Y, Inoshita S, et al Overexpression of protein kinase G using adenovirus inhibits cyclin E transcription and mesangial cell cycle. Am J Physiol Renal Physiol, 280: F851-9, 2001.[Abstract/Free Full Text]
  54. el-Deiry WS, Tokino T, Waldman T, et al Topological control of p21WAF1/CIP1 expression in normal and neoplastic tissues. Cancer Res, 55: 2910-9, 1995.[Abstract/Free Full Text]
  55. Huang YQ, Li JJ, Karpatkin S. Thrombin inhibits tumor cell growth in association with up-regulation of p21(waf/cip1) and caspases via a p53-independent, STAT-1-dependent pathway. J Biol Chem, 275: 6462-8, 2000.[Abstract/Free Full Text]
  56. Xiao H, Hasegawa T, Isobe K. Both Sp1 and Sp3 are responsible for p21waf1 promoter activity induced by histone deacetylase inhibitor in NIH3T3 cells. J Cell Biochem, 73: 291-302, 1999.[CrossRef][Medline]
  57. Timchenko NA, Wilde M, Nakanishi M, Smith JR, Darlington GJ. CCAAT/enhancer-binding protein {alpha} (C/EBP {alpha}) inhibits cell proliferation through the p21 (WAF-1/CIP-1/SDI-1) protein. Genes Dev, 10: 804-15, 1996.[Abstract/Free Full Text]
  58. Touitou R, Richardson J, Bose S, Nakanishi M, Rivett J, Allday MJ. A degradation signal located in the C-terminus of p21WAF1/CIP1 is a binding site for the C8 {alpha}-subunit of the 20S proteasome. EMBO J, 20: 2367-75, 2001.[CrossRef][Medline]
  59. Sheaff RJ, Singer JD, Swanger J, Smitherman M, Roberts JM, Clurman BE. Proteasomal turnover of p21Cip1 does not require p21Cip1 ubiquitination. Mol Cell, 5: 403-10, 2000.[CrossRef][Medline]
  60. Gu M, Lynch J, Brecher P. Nitric oxide increases p21(Waf1/Cip1) expression by a cGMP-dependent pathway that includes activation of extracellular signal-regulated kinase and p70(S6k). J Biol Chem, 275: 11389-96, 2000.[Abstract/Free Full Text]
  61. Gartel AL, Tyner AL. The role of the cyclin-dependent kinase inhibitor p21 in apoptosis. Mol Cancer Ther, 1: 639-49, 2002.[Abstract/Free Full Text]
  62. Martin-Caballero J, Flores JM, Garcia-Palencia P, Serrano M. Tumor susceptibility of p21(Waf1/Cip1)-deficient mice. Cancer Res, 61: 6234-8, 2001.[Abstract/Free Full Text]
  63. Kondo S, Barna BP, Kondo Y, et al WAF1/CIP1 increases the susceptibility of p53 non-functional malignant glioma cells to cisplatin-induced apoptosis. Oncogene, 13: 1279-85, 1996.[Medline]
  64. Lincet H, Poulain L, Remy JS, et al The p21(cip1/waf1) cyclin-dependent kinase inhibitor enhances the cytotoxic effect of cisplatin in human ovarian carcinoma cells. Cancer Lett, 161: 17-26, 2000.[CrossRef][Medline]
  65. Yan Q, Sage EH. Transforming growth factor-ß1 induces apoptotic cell death in cultured retinal endothelial cells but not pericytes: association with decreased expression of p21waf1/cip1. J Cell Biochem, 70: 70-83, 1998.[CrossRef][Medline]
  66. Shureiqi I, Chen D, Lee JJ, et al 15-LOX-1: a novel molecular target of nonsteroidal anti-inflammatory drug-induced apoptosis in colorectal cancer cells. J Natl Cancer Inst (Bethesda), 92: 1136-42, 2000.[Abstract/Free Full Text]
  67. Shureiqi I, Jiang W, Zuo X, et al The 15-lipoxygenase-1 product 13-S-hydroxyoctadecadienoic acid down-regulates PPAR-{delta} to induce apoptosis in colorectal cancer cells. Proc Natl Acad Sci USA, 100: 9968-73, 2003.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
B. Cen, A. Deguchi, and I. B. Weinstein
Activation of Protein Kinase G Increases the Expression of p21CIP1, p27KIP1, and Histidine Triad Protein 1 through Sp1
Cancer Res., July 1, 2008; 68(13): 5355 - 5362.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
L. Ying, A. B. Hofseth, D. D. Browning, M. Nagarkatti, P. S. Nagarkatti, and L. J. Hofseth
Nitric Oxide Inactivates the Retinoblastoma Pathway in Chronic Inflammation
Cancer Res., October 1, 2007; 67(19): 9286 - 9293.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
L. Yang, G. Liu, S. I. Zakharov, A. M. Bellinger, M. Mongillo, and S. O. Marx
Protein Kinase G Phosphorylates Cav1.2 {alpha}1c and {beta}2 Subunits
Circ. Res., August 31, 2007; 101(5): 465 - 474.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
G. M. Pitari, T. Li, R. I. Baksh, and S. A. Waldman
Exisulind and guanylyl cyclase C induce distinct antineoplastic signaling mechanisms in human colon cancer cells
Mol. Cancer Ther., May 1, 2006; 5(5): 1190 - 1196.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
D. Xiao, A. Deguchi, G. G. Gundersen, B. Oehlen, L. Arnold, and I. B. Weinstein
The sulindac derivatives OSI-461, OSIP486823, and OSIP487703 arrest colon cancer cells in mitosis by causing microtubule depolymerization
Mol. Cancer Ther., January 1, 2006; 5(1): 60 - 67.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. Deguchi, S. W. Xing, I. Shureiqi, P. Yang, R. A. Newman, S. M. Lippman, S. J. Feinmark, B. Oehlen, and I. B. Weinstein
Activation of Protein Kinase G Up-regulates Expression of 15-Lipoxygenase-1 in Human Colon Cancer Cells
Cancer Res., September 15, 2005; 65(18): 8442 - 8447.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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 reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Deguchi, A.
Right arrow Articles by Weinstein, I. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Deguchi, A.
Right arrow Articles by Weinstein, I. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online