Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

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 Yang, F.
Right arrow Articles by Brüne, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, F.
Right arrow Articles by Brüne, B.
(Journal of Leukocyte Biology. 2000;68:916-922.)
© 2000 by Society for Leukocyte Biology

Modulation of nitric oxide-evoked apoptosis by the p53-downstream target p21WAF1/CIP1

Fan Yang*, Andreas von Knethen{dagger} and Bernhard Brüne{dagger}

{dagger} University of Erlangen-Nürnberg, Faculty of Medicine, Department of Medicine IV-Experimental Division, 91054 Erlangen, Germany; and
* The Fourth Military Medical University, Department of Pathology, Xi’an, 710032, China

Correspondence: Bernhard Brüne, University of Erlangen-Nürnberg, Faculty of Medicine, Loschgestrasse 8, 91054 Erlangen, Germany. E-mail: mfm423{at}rzmail.uni-erlangen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
When produced in excess, the inflammatory mediator nitric oxide (NO) attenuates cell-cycle progression at the G1 phase in tight correlation with p21WAF1/CIP1 expression, provokes accumulation of the tumor suppressor p53, and initiates apoptosis/necrosis as judged on cell accumulation in the sub-G1 phase. To verify the role of p21WAF1/CIP1 in modulating cell-cycle arrest vs. apoptosis, we transfected stably antisense p21WAF1/CIP1-encoding plasmids. Following NO exposure, accumulation of p21WAF1/CIP1, but not p53, was largely attenuated in antisense p21WAF1/CIP1 transfectants. Moreover, the G1 cell-cycle arrest was abrogated, and cells were sensitized toward apoptosis compared with parent macrophages. In contrast, antisense elimination of p53 attenuated p53 as well as p21WAF1/CIP1 expression, abolished the G1 cell-cycle arrest, and prevented apoptosis. We conclude that p21WAF1/CIP1 is a downstream target of p53 in macrophages that modulate the sensitivity toward the immune-modulator NO.

Key Words: p21WAF1/CIP1 • p53 • NO • apoptosis • cell cycle • macrophages


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide (NO) is a versatile pathophysiological mediator. It is appreciated that the production of NO from L-arginine metabolism is an essential determinant of the innate immune system, important for nonspecific host defense, and tumor as well as pathogen killing [1 2 3 ]. Cytotoxicity as a result of a substantial NO formation is now established to initiate apoptosis [4 , 5 ]. Apoptotic features comprise upregulation of the tumor suppressor p53, changes in the expression of pro- and antiapoptotic Bcl-2 family members, cytochrome c relocation, activation of caspases, chromatin condensation, and DNA fragmentation. Proof of the involvement of NO was established by blocking adverse effects by NO-synthase inhibition [6 , 7 ]. However, transducing pathways of NO are not only adopted to toxicity but also signal cell-cycle arrest and protection from death [8 9 10 11 ].

One pivotal target in response to DNA damage, decreased oxygen, oncogenic stimuli, or redox stress, among others, is the tumor suppressor p53 [12 ]. Mammalian cells maintain their genomic integrity by activating G1 or G2 cell-cycle checkpoints or by inducing apoptosis [13 , 14 ]. It is established that p53 may initiate cell-cycle arrest or apoptosis with the former being at least in part mediated by p53-dependent transcriptional activation of p21WAF1/CIP1, a potent inhibitor of cell-cycle kinases [15 16 17 ]. Several observations suggest that the failure to express sufficient levels of p21WAF1/CIP1 converts the normal cell-cycle arrest into apoptotic cell death, and ectopical expression of p21WAF1/CIP1 prevents p53-mediated cell death in human melanoma cells, prevents apoptosis following UV irradiation, and prevents tumor necrosis factor-related apoptosis-inducing ligand death receptor-mediated cell death [18 19 20 21 22 ]. Despite the fundamental evidence for p21WAF1/CIP1 in affecting cell-cycle progression, it is appreciated now that oxidative stress can induce p21WAF1/CIP1 expression also through p53-independent pathways [23 , 24 ].

Considering the potential of radicals to alter the level of p21WAF1/CIP1, we became interested in studying p21WAF1/CIP1 expression in response to nitrogen-derived radical formation, i.e., NO generation. Taking into consideration further that the absence of p21WAF1/CIP1 sensitizes cells toward apoptosis-inducing agents, and its upregulation resembles a protective principle, we sought to correlate p21WAF1/CIP1 expression with the apoptotic role of NO.

We present evidence that NO promotes p21WAF1/CIP1 expression, thereby achieving a G1 arrest, and elimination of the cell-cycle inhibitor sensitizes macrophages toward NO-evoked apoptosis. This may help to dissect the various roles of NO in acting as a damaging or survival-promoting agent.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
RPMI 1640 and medium supplements were ordered from Biochrom (Berlin, Germany). Fetal calf serum (FCS) was purchased from Gibco (Berlin, Germany). The anti-p21WAF1/CIP1 antibody C19 came from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany), and the ß-tubulin antibody was obtained from Roche (Mannheim, Germany). Propidium iodide was bought from Molecular Probes (Leiden, The Netherlands). Enhanced chemiluminescence (ECL)-detection reagents were from Amersham (Braunschweig, Germany). All other chemicals were of the highest grade of purity and commercially available.

Cell culture
The mouse monocyte/macrophage cell line RAW 264.7 as well as th p21- and p53-antisense clones were maintained in RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FCS (complete medium). For selection of transfected clones, 1 mg/ml Zeocin (Invitrogen, Groningen, The Netherlands) was added.

Antisense plasmid construction and transfection
p21WAF1/CIP1-antisense constructs were generated using two primers derived from the murine cDNA of p21WAF/CIP1. The following sequences were used: murine p21WAF/CIP1 (201–680) [25 ], TA=60°C: 5'-ATG TCC AAT CCT GGT GAT GTC CG-3' (201–223); 5'-TCA GGG TTT TCT CTT GCA GAA GAC C3' (656–680).

After polymerase chain reaction (PCR) using murine cDNA as a template, the resultant 480 bp fragment was inserted directly into the pCR2.1-TOPO-vector (Invitrogen), taking advantage of the A-overhang of PCR fragments. After digestion with EcoRI, the fragment was subcloned into the pTracer-CMV2-vector (Invitrogen), which contains a green fluorescent protein-Zeocin fusion for selection. Antisense orientation of the fragment was verified using PstI (antisense orientation, 187 bp fragment; sense orientation, 412 bp fragment). A pTracer-p21asn clone was used to transfect RAW 264.7 macrophages and to generate stable p21WAF/CIP1 antisense-expressing cell clones using the (diethylamino)ethyl (DEAE)-dextran method [26 ]. Briefly, 5 x 105 cells were plated in 35 mm dishes 1 day before transfection. Cells were washed twice with phosphate-buffered saline (PBS) and incubated for 3 h at 37°C in 1 ml RPMI 1640 supplemented with 50 mM Tris-HCl (pH 7.3), 400 µg DEAE-dextran, and 10 µg pTracer-p21asn or pTracer-CMV2 as an internal control. After 48 h, medium was changed, and cells were selected for 4 weeks using 1 mg/ml Zeocin. Single clones were picked according to their expression of the GFP marker protein and further grown in complete medium with the addition of 1 mg/ml Zeocin. Effective p21WAF/CIP1-antisense transfection was determined by Western blot analysis. Construction of p53 antisense-expressing RAW 264.7 cells (Rd{Delta}53-asn-11) was described previously [27 ].

S-nitrosoglutathione synthesis
S-nitrosoglutathione was synthesized and characterized, as previously described [28 ].

Western blots
Cell lysis was achieved with ice-cold lysis buffer [50 mM Tris, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM sodium vanadate, pH 8.0] and sonication with a Branson sonifier (10 sec, 100% duty cycle, 1 output control). Following centrifugation (14,000 g, 15 min), protein content was measured. For equal protein loading, 100 µg protein was mixed with the same vol of 2 x sodium dodecyl sulfate (SDS) sample buffer [125 mM Tris-HCl, 2% SDS, 10% glycerin, 1 mM dithiothreitol (DTT), 0.002% bromophenol blue, pH 6.9] and boiled for 15 min. Proteins were resolved on 15% SDS-polyacrylamide gels and blotted onto nitrocellulose sheets. Molecular weight (MW) of corresponding proteins was determined in relation to MW rainbow markers; equal loading was confirmed by Ponceau S staining. Transblots were washed twice with tris-buffered saline (140 mM NaCl, 50 mM Tris-HCl, pH 7.2) containing 0.06% Tween-20 before blocking unspecific binding with TBS/5% skim milk. Filters were incubated overnight at 4°C with the p21WAF1/CIP1 antibody (1:1000). Proteins were detected by a horseradish peroxidase (HRP)-conjugated polyclonal antibody (1:10,000) using the ECL method. Western blotting of ß-tubulin was performed to demonstrate equal protein loading.

Cell-cycle analysis
Macrophages (1x105 cells) were collected, resuspended in PBS containing 0.1% Triton X-100, 10 mg/ml RNase, and 50 µg/ml propidium iodide, and incubated overnight at 4°C. Cell-cycle analysis was performed with a Coulter-Epics XL (Coulter, Krefeld, Germany). Results were analyzed using MultiCycle software (Phoenix Flow Systems, San Diego, CA), which allowed separation of the G1, S, and G2/M phases. For this purpose, a gate was set on living cells. To determine the amount of apoptotic cells, a gate was set on cells in the sub-G1 phase only.

Statistical analysis
Each experiment was performed at least three times, and statistical analysis was performed using the two-tailed Student’s t-test. Normal distribution of data is ensured. Otherwise, representative data of at least three similar examinations are shown.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NO inhibits cell-cycle progression
In a first set of experiments, we explored cell-cycle distribution in RAW 264.7 macrophages under the influence of increasing concentrations of GSNO. The NO donor was supplied for 24 h at doses ranging from 200 to 1000 µM (Fig. 1 ). GSNO evoked dose-dependently a G1 cell-cycle arrest, which was accompanied by a decreased cell number in the S or G2/M phase, respectively.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 1. Cell-cycle distribution under the influence of NO. RAW 264.7 macrophages were treated for 24 h with 200, 500, or 1000 µM GSNO, or remained as controls. Propidium iodide staining in combination with flow cytometry was used to analyze cell-cycle distribution as described in Materials and Methods. (A) Mean values ± SD of at least three determinations are shown. (B) Examples of original data are presented.

 
Accumulation of cells in the G1 phase was noticed at 200 µM GSNO, became substantial at 500 µM GSNO, and reached maximal values at 1 mM of the NO donor. A typical example depicting cell-cycle distribution after propidium iodide staining of RAW macrophages in response to NO is given in Figure 1B . It is important for calculating cell-cycle distribution that cell death—i.e., the sub-G1 population—was eliminated under these conditions.

NO modulates expression of cell-cycle regulators
Accumulation of the tumor suppressor p53 is known as a negative regulator of the cell cycle and an established target of NO. Because NO-evoked p53 accumulation is established [7 , 29 ], we sought to investigate its impact on cell-cycle progression. Western blot analysis displayed none or very little p53 in controls or in response to 100 µM GSNO (Fig. 2A ). However, p53 accumulated dose-dependently after adding 200, 500, or 1000 µM GSNO for 4 h.



View larger version (32K):
[in this window]
[in a new window]
  		Figure 2. NO-evoked accumulation of p53 and p21WAF1/CIP1. Macrophages were incubated for 4 and 8 h with increasing concentrations of GSNO (100–1000 µM) prior to Western blot analysis to detect (A) p53 or (B) p21WAF1/CIP1. Experimental details are described in Materials and Methods. This figure is representative of three similar experiments. Detection of ß-tubulin serves as a protein-loading control.

 
Because p53 is known to drive expression of the cell-cycle inhibitor p21WAF1/CIP1, we became interested in measuring the protein appearance of p21WAF1/CIP1 in response to GSNO, which provoked p21WAF1/CIP1 expression after an 8-h incubation period at concentrations between 100 and 1000 µM (Fig. 2B) . The response was weak at 100 µM and 200 µM, became significant at 500 µM, and was even stronger at 1000 µM GSNO. Controls revealed a faint immunoreactivity of p21WAF1/CIP1 only. Equal protein loading was confirmed by ß-tubulin staining (Fig. 2A and 2B , tops).

Antisense elimination of p21WAF1/CIP1 and p53 in affecting cell cycle and apoptosis
To verify the role of p21WAF1/CIP1 in affecting RAW 264.7 macrophage apoptosis, we transfected antisense p21WAF1/CIP1-encoding plasmids stably. Following single-clone selection and expansion, we determined the ability of various clones to express p21WAF1/CIP1 at the protein level in response to 1 mM GSNO (Fig. 3A ). As expected, p21WAF1/CIP1-antisense transfectants showed a variable degree of p21WAF1/CIP1 immunoreactivity. Clone 1 (Rp21-asn-1) suppressed the p21WAF1/CIP1 signal completely, clone 5 (Rp21-asn-5) attenuated p21WAF1/CIP1 expression partially, whereas clone 9 (Rp21-asn-9) was the least active in opposing p21WAF1/CIP1 accumulation. Therefore, clone 1 (Rp21-asn-1) was used in further examinations. In clone 1, the p21WAF1/CIP1 signal, following GSNO addition, was completely suppressed compared with parent macrophages (RAW 264.7) or a vector control (Rvector control). Equal protein loading was confirmed by ß-tubulin staining (Fig. 3A , top).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 3. NO-evoked accumulation of p21WAF1/CIP1 and p53 in antisense p21WAF1/CIP1- or antisense p53-expressing macrophages. Parent macrophages (RAW 264.7), antisense p21WAF1/CIP1-expressing cell clones 1, 5, and 9 (Rp21-asn), antisense p53-expressing cells (Rp{Delta}53-asn-11), or appropriate vector transfectants (Rvector control) were exposed to 1 mM GSNO. Western blot analysis was used to detect (A) p21WAF1/CIP1 after 8 h or (B) p53 after a 4-h incubation period. Experimental details are described in Materials and Methods. This figure is representative of three similar experiments. Detection of ß-tubulin serves as a protein-loading control.

 
To appreciate the potential impact of p53 in affecting p21WAF1/CIP1 accumulation, we examined the appearance of the cell-cycle inhibitor in previously characterized antisense p53-containing cells: Rp{triangleup}53-asn-11 [27 ]. The ability to express p21WAF1/CIP1 following GSNO utilization was abrogated significantly in cells that lost the competence to upregulate p53 (Rp{triangleup}53-asn-11).

Sustained accumulation of p53 in Rp{triangleup}53-asn-11 cells as a result of NO usage was verified by Western blot analysis (Fig. 3B) . Whereas p53 accumulated in parent macrophages or vector control-transfected RAW cells during a 4-h incubation period with 1 mM GSNO, the response was attenuated largely in p53-antisense transfectants, which is in line with our previous studies [27 ]. Investigating Rp21-asn-1 clones in parallel revealed that the p53 response remained intact, although p21WAF1/CIP1 accumulation was restrained. Obviously, p53 accumulation is upstream of the p21WAF1/CIP1 response in murine macrophages.

Considering the dual role of p53 in affecting cell-cycle regulatory components or eliciting apoptosis, we designed experiments to elucidate the impact of p21WAF1/CIP1 and p53 on cell death. For these studies, we analyzed the amount of apoptotic cells—i.e., cells in the sub-G1 phase—after adding 200–1000 µM GSNO for 24 h by comparing the behavior of parent macrophages, and antisense p21WAF1/CIP1- and antisense p53-cells (Fig. 4 ). In parent macrophages, we noticed apoptosis and/or necrosis in response to 500 µM GSNO with ~25% cells in the sub-G1 peak. The proapoptotic response of NO is confirmed by cytochrome c release, caspase activation, or DNA fragmentation, as shown in earlier studies [7 , 30 ]. In response to 1 mM GSNO, cells in the sub-G1 peak increased to roughly 50%, and the NO donor at 200 µM was noneffective. Compared with parent macrophages, Rp21-asn-1 cells responded with increased apoptosis at individual concentrations of the NO-releasing compound. This implied a cell-protective impact of p21WAF1/CIP1 and revealed sensitization of macrophages toward NO, with p21WAF1/CIP1 being eliminated.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 4. NO-mediated apoptosis in parent macrophages, antisense p21WAF1/CIP1-, or antisense p53-expressing macrophages. RAW 264.7 macrophages (RAW 264.7), antisense p21WAF1/CIP1- (Rp21-asn-1), or antisense p53-expressing clones (Rp{Delta}53-asn-11) were treated for 24 h with 200, 500, or 1000 µM GSNO, or remained as controls. Propidium iodide staining in combination with flow cytometry was used to analyze apoptotic cells in the sub-G1 phase of the cell cycle as described in Materials and Methods. Mean values ± SD of at least three determinations are presented.

 
In Rp21-asn-1 cells, 200 µM GSNO evoked ~20% apoptosis, 500 µM GSNO promoted between 50% and 60% apoptosis, and 1 mM GSNO led to apoptosis of roughly 85%. These values were significantly higher compared with corresponding levels determined in parent cells.

Performing similar experiments in Rp{triangleup}53-asn-11 cells unveiled a different situation. In those cells, apoptosis, in response to all GSNO concentrations, was largely attenuated. The amount of cells in the sub-G1 phase was consistently below 20%, thus indicating efficient termination of apoptosis, which is in line with our previous work [27 ]. After having established a modulatory role of p21WAF1/CIP1 with the implication that in the absence of p21WAF1/CIP1 the cells have to default to death, we went on and assessed cell-cycle distribution in those cells compared with parent macrophages (Fig. 5 ). Obviously, the cell-cycle arrest that was noticed in response to GSNO in RAW macrophages (Fig. 1A) was eliminated with p21WAF1/CIP1 or p53 being neutralized by antisense technology.



View larger version (40K):
[in this window]
[in a new window]
  		Figure 5. Cell-cycle distribution under the influence of NO in antisense p21WAF1/CIP1- or antisense p53-expressing macrophages. Antisense p21WAF1/CIP1- (Rp21-asn-1) or antisense p53-expressing clones (Rp{Delta}53-asn-11) of RAW 264.7 macrophages were treated for 24 h with 200, 500, or 1000 µM GSNO. Controls remained untreated. Propidium iodide staining in combination with flow cytometry was used to analyze cell-cycle distribution as described in Materials and Methods. Mean values ± SD of at least three determinations are shown.

 
With a functional p21WAF1/CIP1 or p53 response toward GSNO being offset, NO no longer combat cell-cycle distribution. The amount of cells in the G1, S, or G2/M phase prevailed for all doses of GSNO, with no variance between Rp21-asn-1 vs. Rp{triangleup}53-asn-11 cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
NO is known to affect cell-cycle progression and/or to initiate apoptosis, which is in line with its antiproliferative and/or cytotoxic properties [3 , 6 , 31 ]. Apoptosis, as a result of NO formation, is, at least in murine RAW 264.7 macrophages, closely associated with accumulation of the tumor suppressor p53 [7 ]. NO-evoked stabilization of p53 may signal transcriptional upregulation of the cell-cycle inhibitor p21WAF1/CIP1 or may promote apoptosis directly. Here, we demonstrate that NO causes a G1 cell-cycle arrest in tight correlation with p53 expression and subsequent upregulation of p21WAF1/CIP1. Elimination of p21WAF1/CIP1 by antisense technology sensitized macrophages toward NO-mediated apoptosis, and cell death and cell-cycle inhibition were largely attenuated with a p53 response being exterminated.

Induction of p21WAF1/CIP1 following NO exposure is appreciated for various cell systems in close association with attenuated cell-cycle progression [32 ]. In line, we noticed accumulation of p21WAF1/CIP1 and p53 under the influence of GSNO, which represents the most physiological NO donor. Because it is known that p21WAF1/CIP1 expression occurs in a p53-dependent and -independent manner, it was our intention to establish any correlation for RAW macrophages under the influence of NO. This seemed of particular interest because p53-independent regulation of p21WAF1/CIP1 is established for smooth muscle cells [32 ]. As a result of our Western blot analysis in antisense p21WAF1/CIP1- and antisense p53-transfected macrophages, we provide evidence that accumulation of p53 is a prerequisite for eliciting p21WAF1/CIP1 expression in RAW cells. In contrast, elimination of p21WAF1/CIP1 left p53 accumulation intact, which allows p53 to be positioned upstream of the cell-cycle regulator p21WAF1/CIP1. Arresting cells in G1 are related most probably to inhibition of cyclin/cyclin-dependent kinase activity, which is required for progression of cells through the G1 restriction point. Expression of p21WAF1/CIP1 by NO can be rationalized via p53 accumulation and transactivation of the p21WAF1/CIP1 promoter, which represents a classic target for p53-elicited transcriptional regulation, i.e., gene activation [15 16 17 ]. A p53-dependent step is proven in antisense p53-transfected macrophages and is further implied by a fast p53 vs. delayed p21WAF1/CIP1 response after NO exposure.

Having established that elimination of p21WAF1/CIP1 sensitizes macrophages toward NO-evoked apoptosis, it is in line with the notion that acute renal failure increases in null p21WAF1/CIP1 mice following cisplatin administration. Megyesi and collaborators [18 ] recognized that kidney cells in null p21WAF1/CIP1 mice progressed into the S phases following cellular damage, and a G1 cell-cycle arrest was noticed in wild-type littermates. Furthermore, certain tumor cells can be sensitized toward apoptosis by microtubule inhibitors under p21WAF1/CIP1 deficiency [19 ]. Moreover, it has been demonstrated that induction of p21WAF1/CIP1 prevents apoptosis following UV irradiation or treatment with an RNA polymerase II inhibitor, whereas loss of p21WAF1/CIP1 results in increased sensitivity to cell killing by ionizing radiation [20 , 21 ]. Based on our studies in macrophages, a similar scenario can be proposed for the response toward NO formation, which may promote p21WAF1/CIP1 expression that in turn blocks cell-cycle progression. The failure to upregulate p21WAF1/CIP1 converts a cell-cycle arrest signal into a proapoptotic death-promoting activity. Obviously, both signals demand accumulation of p53. In analogy to other systems, p21WAF1/CIP1 expression and a block in cell-cycle progression may provide time to repair the damage or otherwise exit into apoptosis in case of severe injury. It is interesting that cells deleted for p21WAF1/CIP1 or p53 show an increased G1 population (50–55% compared with 40% in RAW controls) under resting conditions. The reason is unclear presently, but a similar phenomenon has been described for glioma cells, where p21WAF1/CIP1-antisense transfection led to an attenuated cell-cycle progression [33 ]. Further experiments will be necessary to evaluate the underlying mechanism.

During severe cellular damage, the cleavage of p21WAF1/CIP1 by caspases that become activated during progression of apoptosis contributes to the initiation of a feed-forward amplification loop by eliminating a critical checkpoint target for cell-cycle arrest [34 ]. Expression of p21WAF1/CIP1 may prevent initiation of apoptosis at relatively low concentrations of NO—i.e., NO being released from 200 µM GSNO—thus acting as a safeguard to attenuate NO toxicity. However, at high concentrations of NO, the activation of proapoptotic signaling systems such as p53 accumulation, cytochrome c relocation, and caspase activation may override protective barriers, setting into motion NO-mediated apoptotic cell death [35 , 36 ]. It is interesting that elimination of p21WAF1/CIP1 sensitized RAW cells at higher doses of NO as well, which may suggest that the balance between anti- and proapoptotic signaling systems is effective still. In contrast, the action of p53 is strictly proapoptotic in macrophages, because elimination of the tumor suppressor hinders the onset of cell death. Obviously, a functional p53 response is required to complete the apoptotic program in murine macrophages. In line are our observations that the negative impact of NO on the cell cycle is abrogated in antisense p21WAF1/CIP1- and antisense p53-transfected cells.

In conclusion, our data suggest that the expression of p21WAF1/CIP1 modulates the apoptotic response of macrophages during NO exposure with the implication that the absence of p21WAF1/CIP1 increases the sensitivity to the potential toxic mediator NO. Further, we sum up that NO-evoked p53 accumulation is an essential component to complete apoptosis and to interfere with cell-cycle progression by provoking expression of p21WAF1/CIP1.


   ACKNOWLEDGEMENTS
 
We are grateful to Deutsche Forschungsgemeinschaft and Deutsche Krebshilfe for financial support. F. Y. is a recipient of a DAAD-K.C. Wong fellowship. F. Y. and A. v. K. contributed equally to this work.

Received March 19, 2000; revised July 5, 2000; accepted July 6, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. MacMicking, J., Xie, Q. W., Nathan, C. (1997) Nitric oxide and macrophage function Annu. Rev. Immunol. 15,323-350[Medline]
  2. Nathan, C. (1992) Nitric oxide as a secretory product of mammalian cells FASEB J 6,3051-3064[Abstract]
  3. Nüssler, A. K., Billiar, T. R. (1993) Inflammation, immunoregulation, and inducible nitric oxide synthase J. Leukoc. Biol. 54,171-178[Abstract]
  4. Moncada, S., Palmer, R. M., Higgs, E. A. (1991) Nitric oxide: physiology, pathophysiology, and pharmacology Pharmacol. Rev. 43,109-142[Medline]
  5. Sarih, M., Souvannavong, V., Adam, A. (1993) Nitric oxide synthase induces macrophage death by apoptosis Biochem. Biophys. Res. Commun. 191,503-508[Medline]
  6. Albina, J. E., Cui, S., Mateo, R. B., Reichner, J. S. (1993) Nitric oxide-mediated apoptosis in murine peritoneal macrophages J. Immunol. 150,5080-5085[Abstract]
  7. Messmer, U. K., Ankarcrona, M., Nicotera, P., Brune, B. (1994) p53 expression in nitric oxide-induced apoptosis FEBS Lett 355,23-26[Medline]
  8. Mannick, J. B., Miao, X. Q., Stamler, J. S. (1997) Nitric oxide inhibits Fas-induced apoptosis J. Biol. Chem. 272,24125-24128[Abstract/Free Full Text]
  9. Dimmeler, S., Haendeler, J., Nehls, M., Zeiher, A. M. (1997) Suppression of apoptosis by nitric oxide via inhibition of interleukin-1ß-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases J. Exp. Med. 185,601-607[Abstract/Free Full Text]
  10. Peunova, N., Enikolopov, G. (1995) Nitric oxide triggers a switch to growth arrest during differentiation of neuronal cells Nature 375,68-73[Medline]
  11. Sarkar, R., Gordon, D., Stanley, J. C., Webb, R. C. (1997) Cell cycle effects of nitric oxide on vascular smooth muscle cells Am. J. Physiol. 272,H1810-H1818[Abstract/Free Full Text]
  12. Agarwal, M. L., Taylor, W. R., Chernov, M. V., Chernova, O. B., Stark, G. R. (1998) The p53 network J. Biol. Chem. 273,1-4[Free Full Text]
  13. Weinert, T. (1998) DNA damage checkpoints update: getting molecular Curr. Opin. Genet. Dev. 8,185-193[Medline]
  14. Elledge, S. J. (1996) Cell cycle checkpoints: preventing an identity crisis Science 274,1664-1672[Abstract/Free Full Text]
  15. Gartel, A. L., Tyner, A. L. (1999) Transcriptional regulation of the p21(WAF1/CIP1) gene Exp. Cell Res. 246,280-289[Medline]
  16. Ewen, M. E., Miller, S. J. (1996) p53 and translational control Biochim. Biophys. Acta 1242,181-184[Medline]
  17. Ho, Y. S., Wang, Y. J., Lin, J. K. (1996) Induction of p53 and p21/WAF1/CIP1 expression by nitric oxide and their association with apoptosis in human cancer cells Mol. Carcinog. 16,20-31[Medline]
  18. Megyesi, J., Safirstein, R. L., Price, P. M. (1998) Induction of p21WAF1/CIP1/SDI1 in kidney tubule cells affects the course of cisplatin-induced acute renal failure J. Clin. Invest. 101,777-782[Medline]
  19. Stewart, Z. A., Mays, D., Pietenpol, J. A. (1999) Defective G1-S cell cycle checkpoint function sensitizes cells to microtubule inhibitor-induced apoptosis Cancer Res 59,3831-3837[Abstract/Free Full Text]
  20. Wouters, B. G., Giaccia, A. J., Denko, N. C., Brown, J. M. (1997) Loss of p21Waf1/Cip1 sensitizes tumors to radiation by an apoptosis- independent mechanism Cancer Res 57,4703-4706[Abstract/Free Full Text]
  21. Bissonnette, N., Hunting, D. J. (1998) p21-induced cycle arrest in G1 protects cells from apoptosis induced by UV-irradiation or RNA polymerase II blockage Oncogene 16,3461-3469[Medline]
  22. Shi-Qiong Xu, W. S. E-D. (2000) p21WAF1/CIP1 inhibits initiator caspase cleavage by TRAIL death receptor DR4 Biochem. Biophys. Res. Commun. 269,179-190[Medline]
  23. Esposito, F., Cuccovillo, F., Vanoni, M., Cimino, F., Anderson, C. W., Appella, E., Russo, T. (1997) Redox-mediated regulation of p21(waf1/cip1) expression involves a post-transcriptional mechanism and activation of the mitogen-activated protein kinase pathway Eur. J. Biochem. 245,730-737[Medline]
  24. Russo, T., Zambrano, N., Esposito, F., Ammendola, R., Cimino, F., Fiscella, M., Jackman, J., O’Connor, P. M., Anderson, C. W., Appella, E. (1995) A p53-independent pathway for activation of WAF1/CIP1 expression following oxidative stress J. Biol. Chem. 270,29386-29391[Abstract/Free Full Text]
  25. Huppi, K., Siwarski, D., Dosik, J., Michieli, P., Chedid, M., Reed, S., Mock, B., Givol, D., Mushinski, J. F. (1994) Molecular cloning, sequencing, chromosomal localization and expression of mouse p21 (Waf1) Oncogene 9,3017-3020[Medline]
  26. Arakawa, T., Laneuville, O., Miller, C. O., Lakkides, K. M., Wingerd, B. A., DeWitt, D. L., Smith, W. L. (1996) Prostanoid receptors of murine NIH 3T3 and RAW 264.7 cells. Structure and expression of the murine prostaglandin EP4 receptor gene J. Biol. Chem. 271,29569-29575[Abstract/Free Full Text]
  27. Messmer, U. K., Brüne, B. (1996) Nitric-oxide induced apoptosis: p53-dependent and p53-independent signalling pathways Biochem. J. 319,299-305
  28. Hart, T. W. (1985) Some observations concerning the S-nitroso and S-phenylsulphonyl derivates of L-cysteine and glutathione Tetrahedron Lett 26,2013-2016
  29. Glockzin, S., von Knethen, A., Scheffner, M., Brune, B. (1999) Activation of the cell death program by nitric oxide involves inhibition of the proteasome J. Biol. Chem. 274,19581-19586[Abstract/Free Full Text]
  30. Brockhaus, F., Brune, B. (1999) p53 accumulation in apoptotic macrophages is an energy demanding process that precedes cytochrome c release in response to nitric oxide Oncogene 18,6403-6410[Medline]
  31. Murad, F. (1997) What are the molecular mechanisms for the antiproliferative effects of nitric oxide and cGMP in vascular smooth muscle? Circulation 95,1101-1103[Free Full Text]
  32. Ishida, A., Sasaguri, T., Kosaka, C., Nojima, H., Ogata, J. (1997) Induction of the cyclin-dependent kinase inhibitor p21(Sdi1/Cip1/Waf1) by nitric oxide-generating vasodilator in vascular smooth muscle cells J. Biol. Chem. 272,10050-10057[Abstract/Free Full Text]
  33. Besson, A., Young, V. W. (2000) Involvement of p21(Waf1/Cip1) in protein kinase C alpha-induced cell cycle progression Mol. Cell. Biol. 20,4580-4590[Abstract/Free Full Text]
  34. Gervais, J. L., 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]
  35. Albina, J. E., Reichner, J. S. (1998) Role of nitric oxide in mediation of macrophage cytotoxicity and apoptosis Cancer Metastasis Rev. 17,39-53[Medline]
  36. Ushmorov, A., Ratter, F., Lehmann, V., Droge, W., Schirrmacher, V., Umansky, V. (1999) Nitric-oxide-induced apoptosis in human leukemic lines requires mitochondrial lipid degradation and cytochrome C release Blood 93,2342-2352[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
I. V. Smirnova, M. Kajstura, T. Sawamura, and M. S. Goligorsky
Asymmetric dimethylarginine upregulates LOX-1 in activated macrophages: role in foam cell formation
Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H782 - H790.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Hortelano, P. G. Traves, M. Zeini, A. M. Alvarez, and L. Bosca
Sustained Nitric Oxide Delivery Delays Nitric Oxide-Dependent Apoptosis in Macrophages: Contribution to the Physiological Function of Activated Macrophages
J. Immunol., December 1, 2003; 171(11): 6059 - 6064.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
A. L. Gartel and A. L. Tyner
The Role of the Cyclin-dependent Kinase Inhibitor p21 in Apoptosis
Mol. Cancer Ther., June 1, 2002; 1(8): 639 - 649.
[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 Yang, F.
Right arrow Articles by Brüne, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, F.
Right arrow Articles by Brüne, B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS