Suppression of arthritis by forced expression of cyclin-dependent kinase inhibitor p21Cip1 gene into the joints
Yoshinori Nonomura,
Hitoshi Kohsaka,
Kimio Nasu,
Yoshio Terada1,,
Masa-aki Ikeda2, and
Nobuyuki Miyasaka
Departments of Bioregulatory Medicine and Rheumatology,
1 Homeostasis Medicine and Nephrology, and
2 Craniofacial Molecular Embryology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
Correspondence to:
H. Kohsaka
 |
Abstract
|
---|
Rheumatoid synovial fibroblasts (RSF) express cyclin-dependent kinase (CDK) inhibitors p16INK4a and p21Cip1 when they are growth-inhibited in vitro. The induction of p16INK4a is characteristic of RSF and intra-articular p16INK4a gene therapy has been shown to suppress adjuvant arthritis (AA) of rats. The other inducible CDK inhibitor, p21Cip1, has multiple functions depending on the cell type. They include inhibition of CDK as well as promotion of active CDK complex formation and induction of apoptosis. This study is to discern the biological effects of p21Cip1 gene transfer into RSF and its therapeutic effects on AA. A recombinant adenovirus containing a human p21Cip1 gene and control adenoviruses were prepared. RSF infected with these viruses were examined for their cell growth. Apoptotic cell death was evaluated by nuclear staining and DNA fragmentation analysis. In vivogene therapy of rat AA was carried out by intra-articular injection of the viruses. Severity of the arthritis was clinically scored. The treated joints were examined histologically and proliferating cell nuclear antigens (PCNA) were detected immunohistochemically. The adenoviral p21Cip1 gene transfer inhibited growth of RSF without inducing apoptosis. p21Cip1 gene therapy suppressed AA clinically and histologically. The effects were comparable to p16INK4a gene therapy. PCNA expression was reduced in the p21Cip1-treated joints. The adenoviral gene transfer of p21Cip1 ameliorated rat AA. The effect was attributable to inhibition of proliferation. Because p21Cip1 is induced more easily by many chemicals than p16INK4a, it also appears to be a feasible target in developing anti-rheumatic drugs.
Keywords: adjuvant arthritis, apoptosis, cyclin-dependent kinase inhibitor, p21Cip1, rheumatoid arthritis
 |
Introduction
|
---|
Rheumatoid arthritis (RA) is characterized by chronic inflammation at multiple joints. T and B lymphocytes and macrophages infiltrate into the affected synovial tissues, and promote release of a high concentration of proinflammatory cytokines, including tumor necrosis factor (TNF)-
and IL-1 (1). In response to these cytokines, synovial fibroblasts proliferate vigorously and form pannus tissues, which destroy the cartilage and bone of the joints. In clinics, various drugs are used to control RA. Representative are conventional disease-modifying anti-rheumatic drugs including hydroxychloroquine, D-penicillamine, gold, sulfasalazine as well as immunosuppressive methotrexate. Although they do not have distinct target molecules known to be responsible for their anti-rheumatic effects, they appear to down-modulate the inflammatory processes (2). In contrast, newly developed drugs, such as leflunomide, cyclosporin A and FK506, inhibit intracellular molecules essential for activation of the immunocompetent cells involved in the rheumatoid synovitis. TNF-
antagonists (remicade and infliximab) neutralize the key inflammatory mediator released by the synovial cells. Many patients respond well to these drugs, but some are resistant or become so after some period of disease amelioration. Moreover, few have been proven effective in halting the disease progression in the long term. We assume that suppression of one inflammatory mediator might activate other mediators because of redundancy of the inflammatory pathways. Thus, suppression of the synovial cell proliferation itself seems to be the most effective therapeutic way to intervene in the joint destruction.
In general, cell cycle progression of eukaryotic cells is regulated largely by kinase activity of cyclin/cyclin-dependent kinase (CDK) complexes. There are several subtypes of cyclins and CDK, which undergo cyclic activation to promote cell cycle progression (3). The kinase activity is inhibited by a group of intranuclear proteins termed CDK inhibitors (CDKI). Up to now, two CDKI families have been identified (3). They are the INK4 family, which consists of p16INK4a, p15INK4b, p18INK4c and p19INK4d, and the Cip/Kip family, which consists of p21Cip1, p27Kip1 and p57Kip2. Previously, we investigated expression of cell-cycle proteins in rheumatoid synovial fibroblasts (RSF) (4). We found that p16INK4a and p21Cip1 expression was readily induced in vitro in RSF derived from joints with active synovitis. This induction was observed when growth of RSF was artificially inhibited. Of note, the induction of p16INK4a was characteristic of RSF, but not of other types of fibroblasts. Subsequently, using adjuvant arthritis (AA) of rats, an animal model of RA, we showed that adenoviral transfer of the p16INK4a gene to the synovial tissues suppressed pathology of the arthritis (4). This was the first evidence showing that cell-cycle control of synovial cells is effective for arthritis treatment.
The p16INK4a protein binds specifically to CDK4 and CDK6, and inhibits formation of the CDK/cyclin complexes (3). Physiologically, it is expressed by replicative senescent cells and by terminally differentiated cells (5). Forced expression of the p16INK4a gene induces cell-cycle arrest at G1 phase and phenotypic changes characteristic of senescent cells (6).
The p21Cip1 protein, being the other one of the two inducible CDKI in RSF, inhibits a wide variety of cyclin/CDK complexes (3). Forced expression of p21Cip1 also arrests the cell cycle of normal and tumor cells at the G1 phase (7). In fibroblasts, the expression level of p21Cip1 goes up with an increase of cell division and decreases when the cells approach senescence (8). In contrast, the p16INK4a protein level gradually rises and stays high in the senescent cells. Thus, p16INK4a gene expression appears to have a more direct impact in senescence induction (5). The biological function of p21Cip1 is more complex. In certain settings, p21Cip1 promotes formation of active kinase complexes by cyclin and CDK, forwarding rather than halting the cell cycle (9). Whereas the N-terminal domain interacts with CDK/cyclin complexes, the C-terminal domain binds to and inhibits proliferation cell nuclear antigen (PCNA), a subunit of DNA polymerase
, which is essential for DNA replication and repair (10). Also, the expression of p21Cip1 enhances NF-
B-dependent gene expression by inhibiting cyclin E/CDK2, which binds to complexes of p300/CBP co-activators and NF-
B (11). In this regard, NF-
B is a critical transcription factor involved in rheumatoid synovitis (12). Whereas proinflammatory cytokines such as IL-1 and TNF-
activate NF-
B in rheumatoid synovial tissues, the production of these cytokines depends on NF-
B (13,14). Moreover, forced expression of p21Cip1 induced apoptotic cell death of some cell lines (1518).
Thus, p21Cip1 ectopically expressed in the inflamed synovial tissues may exert differential effects than p16INK4a on the pathology of the arthritis. In the present study, we first transferred the p21Cip1 gene adenovirally into RSF. Subsequently, the gene was transferred in vivo to synovial tissues of rats with AA, which had been treated successfully with p16INK4a gene therapy. The results revealed that p21Cip1 is also a potentially ideal target for development of gene therapy as well as anti-rheumatic drugs.
 |
Methods
|
---|
Recombinant adenoviruses
A recombinant replication-defective adenovirus, AxCAp21, containing a CAG promoter and a human p21Cip1 gene was prepared (19). An AxCALacZ adenovirus containing a bacterial lacZ gene was a gift from Dr Saito (20). An Ax1w1 adenovirus without insertion was purchased from the Riken Gene Bank (Saitama, Japan). Previous studies showed that the product of the p21Cip1 gene in the AxCAp21 virus can inhibit CDK activity and thus keep pRB protein underphosphorylated (21).
Cell culture
Synovial tissues were obtained from five patients, under their consent, who underwent synovectomy or total knee joint replacement surgery for active rheumatoid synovitis at Nippon Medical School Hospital, Tokyo Metropolitan Bokuto or Huchu Hospital. They fulfilled the American College of Rheumatology criteria for classification of RA (22). RSF were isolated and cultured as described elsewhere (4). The cells at passages 36 were used for experiments.
Cell proliferation assay
In vitro adenoviral infection was performed as described previously (19). Cell growth was assessed by incorporation of [3H]thymidine. RSF were plated in 96-well plates, incubated in 10% FBS for 24 h and then infected with various concentrations of the adenoviruses for 1 h. Then the cells were incubated for 48 h. For the last 24 h, the cells were pulsed with [3H]thymidine (Amersham, Little Chalfont, UK) and then incorporated radioactivities counted.
Apoptosis assays
A half million RSF were infected with 5x107 p.f.u. of the AxCAp21 or Ax1w1 adenoviruses at 100 m.o.i. To induce apoptosis, the same number of cells were treated with 50 µM N-acetylsphingosine (Wako Pure Chemical, Osaka, Japan) in RPMI 1640 medium supplemented with 40 ng/ml platelet-derived growth factor (PDGF; Genzyme, Cambridge, MA) (23). After 4 days, the treated cells were isolated with trypsinEDTA solution (Immunobiology Laboratories, Gunma, Japan), fixed with 1% glutaraldehyde in PBS and stained with Hoechst 33258 (Molecular Probes, Eugene, OR). To quantify the apoptotic cells, 500 nuclei were visually examined. Total cellular DNA was extracted from the treated cells and their fragmentation was analyzed with 2% agarose gel DNA electrophoresis.
Gene therapy of AA
Six-week-old male Lewis rats were immunized with 1 mg of Mycobacterium butyricum emulsified in 100 µl of mineral oil. Intra-articular gene transfer to the knee joints was carried out by injecting 1x107 p.f.u. of the adenoviruses in 50 µl of saline. During the course of the disease, severity of the arthritis was clinically scored (24). Width of the knee joints at the lower edge of the patella was measured by a micrometer. At the end of the clinical observation, the joints were fixed for histological and immunohistochemical analyses. Injections of saline alone served as a control. All procedures in these animal experiments were reviewed and approved the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (approval no. 990107), and carried out under the control of the guidelines for animal experiments.
Histological analysis
The knee joints were embedded in paraffin wax after 10% PBSformalin fixation and decalcification. Their sections (5 µm) were stained with hematoxylin & eosin. Thickness of the synovial tissues and cartilage was measured at the position close to the synovial attachment to the tibial head. Mononuclear cells in 1.8x108 m2 of the synovial tissues were enumerated at the same position. Severity of the pannus invasion was scored as was performed previously (4).
Immunohistochemistry
For immunohistochemical analyses, the fixed sections (5 µm) were deparaffinized and treated by two rounds of microwave heating for 5 min in 10 µM sodium citrate (pH 6). They were incubated with 0.3% H2O2, with 10% normal goat serum in PBS and with an anti-PCNA mAb (PC10; Santa Cruz Biotechnology, Santa Cruz, CA) or a control mouse IgG antibody for 1 h. This antibody reacts with a PCNA peptide chain distinct from the binding site for p21Cip1, ruling out the possibility that binding of p21Cip1 to PCNA interferes with the antibody reactivity (25,26). They were subsequently incubated with a biotinylated goat anti-mouse IgG antibody (AP181B; Chemicon, Temecula, CA) and with horseradish peroxidase-labeled streptavidin (Southern Biotechnology Associates, Birmingham, AL). Bound antibodies were visualized with 0.02% 3,3'-diaminobenzidine tetrahydrochloride. The sections were counterstained with hematoxylin. The percentages of the PCNA+ cells in total cells [PCNA labeling indices (LI)] were calculated by examining 200 synovial cells (27).
Statistics
Statistical analyses were carried out with StatView-5.0J software (SAS, Cary, NC). The [3H]thymidine uptake of the cultured RSF, as well as knee width, thickness of the cartilage and synovial membranes and PCNA LI of the AxCAp21-treated and AxCALacZ-treated joints were compared with a paired t-test. The scores of pannus invasion were compared with a MannWhitney U-test. The percentages of the apoptotic RSF infected with the adenoviruses were compared with a t-test.
 |
Results
|
---|
Growth inhibition of RSF by the p21Cip1 gene transfer
Using recombinant adenoviruses, the effect of the ectopic expression of p21Cip1 on proliferation of RSF was evaluated. RSF were cultured to allow logarithmic growth with media containing 10% FBS. They were infected with the AxCAp21 adenoviruses containing a human p21Cip1 gene or with the Ax1w1 adenoviruses containing no inserted gene. Western blot analysis of the total cell lysates of the infected cells showed that the p21Cip1 protein was expressed specifically by the AxCAp21-infected cells but not by the Ax1w1-infected cells (data not shown). Proliferation of the cells was evaluated 24 h after the infection. Compared to the proliferation of the Ax1w1-infected RSF, that of the AxCAp21-infected RSF was significantly suppressed by the viruses. The suppressive effect depended on the titer of the viruses (Fig. 1
).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 1. Growth inhibition of the RSF by p21Cip1 gene transfer. RSF infected with the indicated m.o.i. of the Ax1w1 or AxCAp21 adenoviruses were stimulated with 10% FBS. [3H]Thymidine incorporation by the stimulated RSF is shown relative to that of control RSF infected with Ax1w1. At 100 m.o.i. infection, the difference was statistically significant (P < 0.01).
|
|
Apoptotic cell death of RSF with the overexpressed p21Cip1 gene
Growth inhibition observed in the AxCAp21-infected RSF could be due to apoptosis induced by the ectopic p21Cip1 expression. Thus, RSF infected with the AxCAp21 adenoviruses and those infected with the Ax1w1 adenoviruses were examined for apoptotic cell death.
As was reported previously, RSF undergo apoptotic cell death when they are treated with N-acetylsphingosine (23). The apoptotic RSF displayed characteristic morphological changes such as nuclear condensation and fragmentation when they were stained with Hoechst 33258 (Fig. 2A
). The RSF treated with a growth-inhibitory concentration (100 m.o.i.) of AxCAp21 or those with the same dose of Ax1w1 were also stained 4 days after the infection (Fig. 2B and C
). They were compared with RSF cultured without the viruses (Fig. 2D
). The percentages of apoptotic cells that showed characteristic nuclear changes were evaluated in three different samples for each type of the infection. The frequencies of the apoptotic cells in the non-infected, AxCAp21-infected and Ax1w1-infected RSF were not significantly different; the means ± SEM were 0.52 ± 0.45, 0.69 ± 0.60 and 0.60 ± 0.67% respectively.

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 2. Apoptosis assays of RSF with forced p21Cip1 expression. (AD) Hoechst 33258 staining of the nuclei. RSF treated with N-acetylsphingosine displayed nuclear condensation and fragmentation characteristic of the apoptotic cells (A). The AxCAp21-treated RSF (B) and Ax1w1-treated RSF (C) showed no signs of apoptosis compared to the non-treated RSF (D). (Original magnification x400.) See text for quantitation of the apoptotic cells. (E) Agarose gel electrophoresis of cellular DNA. Total cellular DNA of non-treated RSF (lane 2), RSF 2 days (Lane 3) and 4 days (lane 4) after the AxCAp21 infection, and RSF 2 days (lane 5) and 4 days (Lane 6) after the Ax1w1 infection were analyzed. The DNA of UV-treated HL-60 cells was fractionated to show typical nucleosomal DNA ladder (lane 7). Lane 1: DNA mol. wt marker ( X174 HaeIII digests).
|
|
Nuclear DNA of RSF infected with the AxCAp21 or control viruses was fractionated with agarose gel electrophoresis. Whereas DNA of the apoptotic HL-60 cells had typical nucleosomal DNA ladders, the AxCAp21-infected or Ax1w1-infected RSF had no fragmented DNA (Fig. 2E
). These results argue that the p21Cip1 overexpression did not induce apoptotic cell death of RSF.
Treatment of rat AA with adenoviral p21Cip1 gene transfer
Six rats were immunized with M. butyricum to induce AA. They were treated with injection of AxCAp21 into the right knees and that of AxCALacZ into the left knees of the same animals. The gene transfer was carried out once (7 days after the immunization) or 3 times (8, 15 and 22 days after the immunization). Swelling of the knee joints was monitored during the disease course. The arthritis developed in all rats ~10 days after the immunization. Without treatment, the knee joints width became maximal ~20 days after the immunization and decreased afterwards. The knee joints treated with the control adenoviruses followed this course (Fig. 3
). In comparison, the gene p21Cip1 transfer significantly suppressed swelling of the knees whether it was carried out once or 3 times (Fig. 3
, P < 0.05). The therapeutic effect lasted throughout the experiment to a statistically significant extent when the gene transfers were repeated 3 times with an interval of 1 week between transfers. Although the gene transfer carried out once also suppressed the joint swelling effectively, the effect became less clear after 2 weeks from the gene transfer.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3. Effect of p21Cip1 gene transfer on the joint swelling of rats with AA. The knee joints of rats with AA were treated with intra-articular injection of the AxCAp21 (filled diamonds) or AxCALacZ (open diamonds) adenoviruses. The points and bars represent the mean ± SEM of six rats. Arrows represent the timing of the gene transfer. The gene transfer was carried out 3 times (A) or once (B). Spontaneous decrease of the joint around day 22 seemed to be attributed to fibrosis of the joints. The asterisks represent statistically significant differences (P < 0.05) between the two groups.
|
|
To evaluate the effects of adenoviruses per se, the AxCALacZ viruses was injected into the right knee joints of normal rats. The same volume of saline was injected into the other knees. Either experiment induced no obvious joint swelling, and no difference in the width of the right and left knees was observed.
Effects of p21Cip1 gene transfer on the histopathology of rat AA
The joints of the rats treated 3 times with the gene transfer were histologically examined 1 week after the last treatment. The synovial tissues of the knee joints from normal rats had one or two layers of synovial lining cells that were supported by loose fatty connective tissues (Fig. 4A
). The arthritic joints treated with AxCALacZ or with saline had marked synovial thickening, which was accompanied by mononuclear cell infiltration. Also, destructive pannus tissues developed and invaded the adjacent bones (Fig. 4B and C
). The cartilage of the affected joints was degenerated. In contrast, synovial thickening and mononuclear cell infiltration were remarkably suppressed in the synovial tissues of the AxCAp21-treated joints (Fig. 4D
). Bone destruction by the pannus tissues was suppressed and cartilage was well preserved. The joints of normal rats which were treated with the control AxCALacZ viruses showed minimal and focal mononuclear cell infiltration, and slight stratification of the synovial lining cells (Fig. 4E
). No changes were observed in the joints treated with saline (Fig. 4F
).

View larger version (132K):
[in this window]
[in a new window]
|
Fig. 4. Effect of p21Cip1 gene therapy on histopathology of AA. The arthritic joints treated 3 times with gene transfer were histologically examined 4 weeks after the immunization. The synovial tissue sections around the patellar ligaments were stained with hematoxylin & eosin. (A) The normal joint, (B) the saline-treated joint, (C) the AxCALacZ-treated joint, (D) the AxCAp21-treated joint, (E) the AxCALacZ-treated normal joint and (F) the saline-treated normal joint. p, patella; s, synovial tissue; f, femur; t, subpatellar tendon.
|
|
Synovial thickness and cartilage thickness were measured, and infiltrating mononuclear cells were enumerated (4). Pannus invasion was scored for the severity. All of these measurements of the AxCAp21-treated and control LacZ-treated joints showed that p21Cip1 gene transfer significantly ameliorated the arthritis (Fig. 5
).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 5. Histological measurements of the treated knee joints. The joints treated with AxCAp21 (p21Cip1) and with AxCALacZ (LacZ) were examined for synovial thickness (A), mononuclear cell infiltration (B), pannus invasion (C) and cartilage thickness (D) by microscopy. The points and bars represent the mean ± SEM derived from six rats (A, B and D). The points in (C) represent the histological scores of the individual rats. The differences of all measurements were statistically significant.
|
|
Injection of the AxCALacZ virus in the normal joints did not change synovial or cartilage thickness, or induce pannus formation. However, it induced a marginal but significant increase of the mononuclear cell infiltration; the means ± SEM were 3.7 ± 6.7 and 173 ± 140/mm2 in the saline- and the AxCALacZ-treated joints respectively. Since the mean number of infiltrating mononuclear cells in the residual synovitis was 643 ± 516/mm2, the adenovirus appeared to contribute minimally to the residual inflammation, but not to the synovial thickening or the destruction of the bone and cartilage.
The cells in the S phase of cell cycle express PCNA. The arthritic synovial tissues were examined immunohistochemically for PCNA expression 1 week after the gene transfer. Synovial cells positive for PCNA staining were found more frequently in the synovial tissues of the AxCALacZ-treated joints than in those of the AxCAp21-treated joints (Fig. 6AD
). The PCNA LI, which reflect the frequency of the cells in the S phase, were calculated by examining three independent microscopic fields. They were significantly smaller in the AxCAp21-treated joints than in the AxCALacZ-treated joints (Fig. 6E
).

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 6. PCNA expression in the synovial tissues of the treated joints. The synovial tissues from the AxCAp21- (A and C) and AxCALacZ-(B and D) treated joints were stained with an anti-PCNA mAb (A and B) or with normal mouse IgG (C and D) as a control. Filled arrowheads and open arrowheads represent PCNA+ and PCNA nuclei respectively. PCNA LI were calculated, and are shown as columns and bars, representing the mean ± SEM (E). The difference was statistically significant (P < 0.01).
|
|
 |
Discussion
|
---|
Our studies have shown that forced expression of the p21Cip1 gene inhibits cell growth of RSF without inducing their apoptotic cell death. In vivo transfer of the p21Cip1 gene into the synovial tissues suppressed the pathology of AA. These in vivo and in vitro effects were comparable to those of the p16INK4a gene transfer. Reduced frequency of the PCNA-expressing cells in the p21Cip1-treated synovial tissues showed that the cell cycling of the synovial cells was indeed inhibited in vivo.
As was reviewed before, both p16INK4a and p21Cip1 are associated with replicative senescence. However, their expression is differentially regulated. p21Cip1 inhibits a wider variety of CDKs than p16INK4a. Moreover, their other biological effects than inhibition of kinase activity are distinct (9,10,28). Disruption of the p16INK4a gene, but not that of the p21Cip1 gene, resulted in frequent tumor development in a murine model (29). Nevertheless, the p16INK4a and p21Cip1 gene therapy ameliorated AA equally. They inhibited synovial thickening, mononuclear cell infiltration, pannus formation and cartilage degeneration. Thus, cell cycle control of the synovial cells by the senescence-related CDKI is effective to suppress pathology of the arthritis.
Generally, p21Cip1 keeps cells from undergoing the apoptosis when the cells respond to various apoptosis-inducing stimuli (3032). However, some immortalized cell lines died with apoptosis when they overexpressed the p21Cip1 gene (1518). The molecular events underlying the p21Cip1-induced apoptosis remain to be elucidated. In the rheumatoid synovial tissues, apoptotic cell death is observed commonly. However, when massive apoptosis of the synovial cells was induced artificially in animal models of RA, it ameliorated the pathology of the arthritis (3335). Thus, p21Cip1 induction might have exerted therapeutic effects, at least partially, through induction of apoptosis.
In our experiments, the gene transfer was performed once or 3 times with an interval of 1 week between transfers. Repeated gene transfers were required to maintain the statistically significant therapeutic effects. This agrees with the observation that ectopic p21Cip1 gene expression after the in vivo gene transfer was detectable only for 23 weeks (data not shown). Thus, anti-arthritic effects required sustained expression of p21Cip1. It was reported that induction of the p21Cip1 gene in human fibrosarcoma cells affected expression of many genes (36). Although the adenoviral p21Cip1 gene transfer must have altered the gene expression by the synovial cells, the changes did not provide prolonged anti-rheumatic effects.
By p21Cip1 transfer, mononuclear cell infiltration was also ameliorated. Lymphoid cells are generally resistant to the adenoviral infection. Indeed, in our experimental procedures, the recombinant adenoviruses delivered the exogene mainly to the synovial lining cells, and to some of the fibroblast-like and macrophage-like cells at the sublining and stromal area, but not to lymphoid cells (4). Thus the reduction of the mononuclear cells should not be due to direct inhibition of their proliferation, but probably to overall suppression of the synovial inflammation.
It should be noted that the gene transfer protocol in the present study was not necessarily optimized. Adenoviral gene transfer was applied because it introduces exogenes to the synovial cells with high efficacy (37). The aim of the present studies was to investigate the therapeutic effect of the p21Cip1 induction in the inflamed synovial tissues. For actual application to human RA, the gene delivery method should be improved. Injection of the adenoviruses into joints could induce inflammatory responses (37). Indeed, we observed residual synovitis in the AxCAp21-treated joints and minimal mononuclear cell infiltration in the joints of normal rats treated with the control adenoviruses. Thus, the residual synovitis in AxCAp21-treated joints might be partly attributable to the inflammatory effects of the adenoviruses. Although the therapeutic effects of CDKI gene transfer surpassed the inflammatory effects of the adenoviruses, it is desirable to use less inflammatory variants of the adenoviruses (38).
Little is understood about regulation of the p16INK4a gene expression. Inactivation of retinoblastoma gene product (pRB) by viral gene products promotes transcription of p16INK4a (39). However, responsive promoter elements for this induction are still unclear (39). A few methods that induce p16INK4a gene expression in pre-senescent cells include
-irradiation, bleomycin and actinomycin D treatment of certain cell lines (5,40). In contrast, multiple compounds have been identified to induce p21Cip1 in many cell lines (4145). The p21Cip1 gene is also induced by serum, growth factors and IL-6 in some cell lines (46,47). Thus, development of pharmaceuticals to induce p21Cip1 expression in vivo in the synovial tissues might be more feasible. However, unlike p16INK4a, p21Cip1 is readily induced in the fibroblasts of non-synovial origin (4,5,8). Since generalized p21Cip1 induction will probably impair homeostasis of the body, the induction should be confined within the inflamed synovial tissues by some means.
The gene therapy reported here aims at transforming synovial cells per se and is thus distinct from the previous trials, which were mostly to produce anti-inflammatory molecules in the joints (4854). It was shown that the intra-articular delivery of the anti-inflammatory cytokine and cytokine antagonist genes could exert their effects on non-treated joints (5456). Such a remote effect was not observed in our experiments (data not shown). However, this was expected because the products of the p21Cip1 gene should not be released from the virus-infected cells.
In theory, gene delivery of other cell-cycle regulators could be likewise effective in treating the arthritis. Nevertheless, as for the development of new anti-rheumatic drugs, p21Cip1 and p16INK4a appear to be attractive target candidates because they are inducible in RSF.
 |
Acknowledgments
|
---|
We thank Drs H. Nakamura, K. Uchida and J. Hasegawa for providing synovial samples, Dr S. Endo for his assistance in the animal facility, and Drs K. Hirokawa, N. Mizushima and K. Taniguchi for their technical advice. This study was supported by a grant from the Ministry of Health and Welfare, Japan and by Naito Foundation.
 |
Abbreviations
|
---|
AA adjuvant arthritis |
CDK cyclin-dependent kinase |
CDKI cyclin-dependent kinase inhibitor |
LI labeling indices |
PCNA proliferating cell nuclear antigen |
PDGF platelet-derived growth factor |
RA rheumatoid arthritis |
RSF rheumatoid synovial fibroblast |
TNF tumor necrosis factor |
 |
Notes
|
---|
Transmitting editor: T Sasazuki
Received 18 October 2000,
accepted 19 February 2001.
 |
References
|
---|
-
Hale, L. and Haynes, B. 1997. Pathology of rheumatoid arthritis and associated disorders. In Koopman, W., ed., Arthritis and Allied Conditions: A Textbook of Rheumatology, 13th edn, p. 993. Williams & Wilkins, Baltimore, MD.
-
Conaghan, P. G., Lehmann, T. and Brooks, P. 1997. Disease-modifying antirheumatic drugs. Curr. Opin. Rheumatol. 9:183.[Medline]
-
Sherr, C. J. and Roberts, J. M. 1999. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13:1501.[Free Full Text]
-
Taniguchi, K., Kohsaka, H., Inoue, N., Terada, Y., Ito, H., Hirokawa, K. and Miyasaka, N. 1999. Induction of the p16INK4a senescence gene as a new therapeutic strategy for the treatment of rheumatoid arthritis. Nat. Med. 5:760.[ISI][Medline]
-
Alcorta, D. A., Xiong, Y., Phelps, D., Hannon, G., Beach, D. and Barrett, J. C. 1996. Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proc. Natl Acad. Sci. USA 93:13742.[Abstract/Free Full Text]
-
Dai, C. Y. and Enders, G. H. 2000. p16INK4a can initiate an autonomous senescence program. Oncogene 19:1613.[ISI][Medline]
-
Dimri, G. P., Nakanishi, M., Desprez, P. Y., Smith, J. R. and Campisi, J. 1996. Inhibition of E2F activity by the cyclin-dependent protein kinase inhibitor p21 in cells expressing or lacking a functional retinoblastoma protein. Mol. Cell. Biol. 16:2987.[Abstract]
-
Tahara, H., Sato, E., Noda, A. and Ide, T. 1995. Increase in expression level of p21sdi1/cip1/waf1 with increasing division age in both normal and SV40-transformed human fibroblasts. Oncogene 10:835.[ISI][Medline]
-
LaBaer, J., Garrett, M. D., Stevenson, L. F., Slingerland, J. M., Sandhu, C., Chou, H. S., Fattaey, A. and Harlow, E. 1997. New functional activities for the p21 family of CDK inhibitors. Genes Dev. 11:847.[Abstract]
-
Li, R., Waga, S., Hannon, G. J., Beach, D. and Stillman, B. 1994. Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature 371:534.[ISI][Medline]
-
Perkins, N. D., Felzien, L. K., Betts, J. C., Leung, K., Beach, D. H. and Nabel, G. J. 1997. Regulation of NF-
B by cyclin-dependent kinases associated with the p300 coactivator. Science 275:523.[Abstract/Free Full Text]
-
Miagkov, A. V., Kovalenko, D. V., Brown, C. E., Didsbury, J. R., Cogswell, J. P., Stimpson, S. A., Baldwin, A. S. and Makarov, S. S. 1998. NF-
B activation provides the potential link between inflammation and hyperplasia in the arthritic joint. Proc. Natl Acad. Sci. USA 95:13859.[Abstract/Free Full Text]
-
Aupperle, K. R., Bennett, B. L., Boyle, D. L., Tak, P. P., Manning, A. M. and Firestein, G. S. 1999. NF-
B regulation by I
B kinase in primary fibroblast-like synoviocytes. J. Immunol. 163:427.[Abstract/Free Full Text]
-
Bondeson, J., Foxwell, B., Brennan, F. and Feldmann, M. 1999. Defining therapeutic targets by using adenovirus: blocking NF-
B inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators. Proc. Natl Acad. Sci. USA 96:5668.[Abstract/Free Full Text]
-
Tsao, Y. P., Huang, S. J., Chang, J. L., Hsieh, J. T., Pong, R. C. and Chen, S. L. 1999. Adenovirus-mediated p21(WAF1/SDII/CIP1) gene transfer induces apoptosis of human cervical cancer cell lines. J. Virol. 73:4983.[Abstract/Free Full Text]
-
Matsushita, H., Morishita, R., Kida, I., Aoki, M., Hayashi, S., Tomita, N., Yamamoto, K., Moriguchi, A., Noda, A., Kaneda, Y., Higaki, J. and Ogihara, T. 1998. Inhibition of growth of human vascular smooth muscle cells by overexpression of p21 gene through induction of apoptosis. Hypertension 31:493.[Abstract/Free Full Text]
-
Kondo, Y., Kondo, S., Liu, J., Haqqi, T., Barnett, G. H. and Barna, B. P. 1997. Involvement of p53 and WAF1/CIP1 in
-irradiation-induced apoptosis of retinoblastoma cells. Exp. Cell Res. 236:51.[ISI][Medline]
-
Sheikh, M. S., Rochefort, H. and Garcia, M. 1995. Overexpression of p21WAF1/CIP1 induces growth arrest, giant cell formation and apoptosis in human breast carcinoma cell lines. Oncogene 11:1899.[ISI][Medline]
-
Terada, Y., Inoshita, S., Nakashima, O., Yamada, T., Kuwahara, M., Sasaki, S. and Marumo, F. 1998. Lovastatin inhibits mesangial cell proliferation via p27Kip1. J. Am. Soc. Nephrol. 9:2235.[Abstract]
-
Kanegae, Y., Lee, G., Sato, Y., Tanaka, M., Nakai, M., Sakaki, T., Sugano, S. and Saito, I. 1995. Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase. Nucleic Acids Res. 23:3816.[Abstract]
-
Terada, Y., Yamada, T., Nakashima, O., Tamamori, M., Ito, H., Sasaki, S. and Marumo, F. 1997. Overexpression of cell cycle inhibitors (p16INK4 and p21Cip1) and cyclin D1 using adenovirus vectors regulates proliferation of rat mesangial cells. J. Am. Soc. Nephrol. 8:51.[Abstract]
-
Arnett, F. C., Edworthy, S. M., Bloch, D. A., McShane, D. J., Fries, J. F., Cooper, N. S., Healey, L. A., Kaplan, S. R., Liang, M. H., Luthra, H. S., et al. 1988. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31:315.[ISI][Medline]
-
Mizushima, N., Kohsaka, H. and Miyasaka, N. 1998. Ceramide, a mediator of interleukin 1, tumour necrosis factor
, as well as Fas receptor signalling, induces apoptosis of rheumatoid arthritis synovial cells. Ann. Rheum. Dis. 57:495.[Abstract/Free Full Text]
-
Eden, W. and Josee, P. A. 1996. Adjuvant arthritis in rat. In Coligan, E., Kruisbeek, A., Margulies, D., Shevach, E., Strober, W., eds, Current Protocols in Immunology, Suppl. 19, p. 15.4. Wiley, New York.
-
Roos, G., Landberg, G., Huff, J. P., Houghten, R., Takasaki, Y. and Tan, E. M. 1993. Analysis of the epitopes of proliferating cell nuclear antigen recognized by monoclonal antibodies. Lab. Invest. 68:204.[ISI][Medline]
-
Chen, J., Peters, R., Saha, P., Lee, P., Theodoras, A., Pagano, M., Wagner, G. and Dutta, A. 1996. A 39 amino acid fragment of the cell cycle regulator p21 is sufficient to bind PCNA and partially inhibit DNA replication in vivo. Nucleic Acids Res. 24:1727.[Abstract/Free Full Text]
-
Galand, P. and Degraef, C. 1989. Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues. Cell Tiss. Kinet. 22:383.[ISI][Medline]
-
Xiong, Y., Hannon, G. J., Zhang, H., Casso, D., Kobayashi, R. and Beach, D. 1993. p21 is a universal inhibitor of cyclin kinases. Nature 366:701.[ISI][Medline]
-
Serrano, M., Lee, H., Chin, L., Cordon-Cardo, C., Beach, D. and DePinho, R. A. 1996. Role of the INK4a locus in tumor suppression and cell mortality. Cell 85:27.[ISI][Medline]
-
Waldman, T., Lengauer, C., Kinzler, K. W. and Vogelstein, B. 1996. Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21. Nature 381:713.[ISI][Medline]
-
Bissonnette, N. and 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.[ISI][Medline]
-
Lu, Y., Yamagishi, N., Yagi, T. and Takebe, H. 1998. Mutated p21WAF1/CIP1/SDI1 lacking CDK-inhibitory activity fails to prevent apoptosis in human colorectal carcinoma cells. Oncogene 16:705.[ISI][Medline]
-
Firestein, G. S., Yeo, M. and Zvaifler, N. J. 1995. Apoptosis in rheumatoid arthritis synovium. J. Clin. Invest. 96:1631.[ISI][Medline]
-
Sakai, K., Matsuno, H., Morita, I., Nezuka, T., Tsuji, H., Shirai, T., Yonehara, S., Hasunuma, T. and Nishioka, K. 1998. Potential withdrawal of rheumatoid synovium by the induction of apoptosis using a novel in vivo model of rheumatoid arthritis. Arthritis Rheum. 41:1251.[ISI][Medline]
-
Zhang, H., Yang, Y., Horton, J. L., Samoilova, E. B., Judge, T. A., Turka, L. A., Wilson, J. M. and Chen, Y. 1997. Amelioration of collagen-induced arthritis by CD95 (Apo-1/Fas)-ligand gene transfer. J. Clin. Invest. 100:1951.[Abstract/Free Full Text]
-
Chang, B. D., Watanabe, K., Broude, E. V., Fang, J., Poole, J. C., Kalinichenko, T. V. and Roninson, I. B. 2000. Effects of p21Waf1/Cip1/Sdi1 on cellular gene expression: Implications for carcinogenesis, senescence, and age-related diseases. Proc. Natl Acad. Sci. USA 97:4291.[Abstract/Free Full Text]
-
Nita, I., Ghivizzani, S. C., Galea-Lauri, J., Bandara, G., Georgescu, H. I., Robbins, P. D. and Evans, C. H. 1996. Direct gene delivery to synovium. An evaluation of potential vectors in vitro and in vivo. Arthritis Rheum. 39:820.[ISI][Medline]
-
Steinwaerder, D. S., Carlson, C. A. and Lieber, A. 1999. Generation of adenovirus vectors devoid of all viral genes by recombination between inverted repeats. J. Virol. 73:9303.[Abstract/Free Full Text]
-
Hara, E., Smith, R., Parry, D., Tahara, H., Stone, S. and Peters, G. 1996. Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence. Mol. Cell. Biol. 16:859.[Abstract]
-
Robles, S. J. and Adami, G. R. 1998. Agents that cause DNA double strand breaks lead to p16INK4a enrichment and the premature senescence of normal fibroblasts. Oncogene 16:1113.[ISI][Medline]
-
Steinman, R. A., Hoffman, B., Iro, A., Guillouf, C., Liebermann, D. A. and el-Houseini, M. E. 1994. Induction of p21 (WAF-1/CIP1) during differentiation. Oncogene 9:3389.[ISI][Medline]
-
Jiang, H., Lin, J., Su, Z. Z., Collart, F. R., Huberman, E. and Fisher, P. B. 1994. Induction of differentiation in human promyelocytic HL-60 leukemia cells activates p21, WAF1/CIP1, expression in the absence of p53. Oncogene 9:3397.[ISI][Medline]
-
Barboule, N., Chadebech, P., Baldin, V., Vidal, S. and Valette, A. 1997. Involvement of p21 in mitotic exit after paclitaxel treatment in MCF-7 breast adenocarcinoma cell line. Oncogene 15:2867.[ISI][Medline]
-
Sheikh, M. S., Li, X. S., Chen, J. C., Shao, Z. M., Ordonez, J. V. and Fontana, J. A. 1994. Mechanisms of regulation of WAF1/Cip1 gene expression in human breast carcinoma: role of p53-dependent and independent signal transduction pathways. Oncogene 9:3407.[ISI][Medline]
-
Gorospe, M., Wang, X. and Holbrook, N. J. 1999. Functional role of p21 during the cellular response to stress. Gene Express 7:377.[ISI]
-
Gartel, A. L. and Tyner, A. L. 1999. Transcriptional regulation of the p21(WAF1/CIP1) gene. Exp. Cell Res. 246:280.[ISI][Medline]
-
Bellido, T., O'Brien, C. A., Roberson, P. K. and Manolagas, S. C. 1998. Transcriptional activation of the p21WAF1,CIP1,SDI1 gene by interleukin-6 type cytokines. A prerequisite for their pro-differentiating and anti-apoptotic effects on human osteoblastic cells. J. Biol. Chem. 273:21137.[Abstract/Free Full Text]
-
Pap, T., Gay, R. E. and Gay, S. 2000. Gene transfer: from concept to therapy. Curr. Opin. Rheumatol. 12:205.[ISI][Medline]
-
Otani, K., Nita, I., Macaulay, W., Georgescu, H. I., Robbins, P. D. and Evans, C. H. 1996. Suppression of antigen-induced arthritis in rabbits by ex vivo gene therapy. J. Immunol. 156:3558.[Abstract]
-
Makarov, S. S., Olsen, J. C., Johnston, W. N., Anderle, S. K., Brown, R. R., Baldwin, A. S., Jr, Haskill, J. S. and Schwab, J. H. 1996. Suppression of experimental arthritis by gene transfer of interleukin 1 receptor antagonist cDNA. Proc. Natl Acad. Sci. USA 93:402.[Abstract/Free Full Text]
-
Lubberts, E., Joosten, L. A., van Den Bersselaar, L., Helsen, M. M., Bakker, A. C., van Meurs, J. B., Graham, F. L., Richards, C. D. and van Den Berg, W. B. 1999. Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction. J. Immunol. 163:4546.[Abstract/Free Full Text]
-
Boyle, D. L., Nguyen, K. H., Zhuang, S., Shi, Y., McCormack, J. E., Chada, S. and Firestein, G. S. 1999. Intra-articular IL-4 gene therapy in arthritis: anti-inflammatory effect and enhanced Th2 activity. Gene Therapy 6:1911.[ISI][Medline]
-
Oligino, T., Ghivizzani, S., Wolfe, D., Lechman, E., Krisky, D., Mi, Z., Evans, C., Robbins, P. and Glorioso, J. 1999. Intra-articular delivery of a herpes simplex virus IL-1Ra gene vector reduces inflammation in a rabbit model of arthritis. Gene Therapy 6:1713.[ISI][Medline]
-
Whalen, J. D., Lechman, E. L., Carlos, C. A., Weiss, K., Kovesdi, I., Glorioso, J. C., Robbins, P. D. and Evans, C. H. 1999. Adenoviral transfer of the viral IL-10 gene periarticularly to mouse paws suppresses development of collagen-induced arthritis in both injected and uninjected paws. J. Immunol. 162:3625.[Abstract/Free Full Text]
-
Ghivizzani, S. C., Lechman, E. R., Kang, R., Tio, C., Kolls, J., Evans, C. H. and Robbins, P. D. 1998. Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor alpha soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects. Proc. Natl Acad. Sci. USA 95:4613.[Abstract/Free Full Text]
-
Lechman, E. R., Jaffurs, D., Ghivizzani, S. C., Gambotto, A., Kovesdi, I., Mi, Z., Evans, C. H. and Robbins, P. D. 1999. Direct adenoviral gene transfer of viral IL-10 to rabbit knees with experimental arthritis ameliorates disease in both injected and contralateral control knees. J. Immunol. 163:2202.[Abstract/Free Full Text]