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The Journal of Immunology, 2000, 165: 1882-1888.
Copyright © 00 by The American Association of Immunologists

In Vitro and In Vivo Transfection of p21 Gene Enhances Cyclosporin A-Mediated Inhibition of Lymphocyte Proliferation

Ashwani K. Khanna1 and Jeffrey D. Hosenpud

The Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, WI 53226


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cyclosporine has potent antiproliferative properties, some of which may be via the induction of the cyclin inhibitor p21. In this study, we describe the effects of in vitro and in vivo transfection of p21 in lymphoid and nonlymphoid cells. For in vitro studies, p21 sense plasmid DNA was transfected in A-549 cells (lung adenocarcinoma cell line) and Jurkat cells (human lymphoid cell line). This in vitro transfection of p21 resulted in the inhibition of spontaneous and mitogen-induced cellular proliferation ([3H]thymidine uptake) and also augmented the antiproliferative effects of cyclosporine. In vivo transfection of p21 was accomplished in mice via the i.m. injection of p21 sense plasmid DNA complexed with cationic lipids. As was the case in the cell lines, p21 mRNA was augmented in heart, lung, liver, and spleen 7 days after i.m. injection of p21 sense plasmid DNA. The mitogen (anti-CD3)-induced proliferation of splenocytes from p21-overexpressing mice was significantly decreased, and again this effect was augmented by cotreatment with cyclosporine. These novel findings demonstrate the potential of targeting the cell cycle directly to inhibit alloimmune activation in organ transplantation. This may serve as an alternate strategy to induce immunosuppression, perhaps with less toxicity than that which is seen with conventional immunosuppressive agents.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The current strategy for immunosuppression in organ transplantation is based on the primary treatment with either cyclosporine or tacrolimus combined with other antiproliferative agents and corticosteroids. Cyclosporine and tacrolimus, in addition to their effect on inhibiting IL-2, induce the expression of TGF-ß (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). This effect may be partially responsible for the antiproliferative effects of cyclosporine as well as responsible for the majority of the fibrogenic-related toxic effects of this agent, most importantly the nephrotoxicity.

The antiproliferative effects of TGF-ß are believed to be via induction of the cell cyclin inhibitor p21 (12, 13, 14). We have previously demonstrated that cyclosporine induces p21 via the induction of TGF-ß (15). The "potency" of a given cyclin inhibitor such as p21 to inhibit transplant-relevant lymphoid cell mitogenic responses is unknown. Furthermore, if p21 is immunosuppressive and given that cyclosporine’s effects on p21 require TGF-ß, it is conceivable that equivalent immunosuppression with lower toxicity could be achieved by directly inducing p21 and using either no or lessor amounts of cyclosporin A (CsA)2 (resulting in less fibrogenic TGF-ß). Accordingly, this study was designed to investigate some of the transplant-relevant biologic effects of p21 transfection both in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
To achieve p21 transfection, we constructed eukaryotic expression vectors with p21 in the sense orientation and transfected A-549 cells (human adenocarcinoma cell line) and Jurkat cells (human lymphoid cell line) in vitro. An increase in p21 mRNA and protein was determined by RT-PCR and Western blot analysis, respectively. Cell proliferation at rest and following nonspecific mitogen stimulation was performed using [3H]thymidine uptake with and without cyclosporine. To transfect p21 gene in vivo, we injected mice with p21 sense plasmid DNA complexed with cationic lipid i.m. and investigated mRNA coding for p21 at 7 days in various tissues using RT-PCR. Ex vivo splenocyte proliferation was determined following anti-CD3 stimulation in the presence or absence of cyclosporine using [3H]thymidine incorporation as above. Details of the methods are described below.

Plasmids

We constructed plasmids expressing sense and antisense p21. The p21 gene was cloned in TOPO (Invitrogen, Carlsbad, CA) cloning. Isolated bacterial colonies containing this fragment were grown, plasmid DNA was isolated and digested with EcoRI, and the insert was sequenced using an automated sequencer. The resultant sequence was run with the BLAST program (www.ncbi.nlm.nih.gov/blast), and the analysis confirmed it to be the p21 gene. This cloned 1.7-kb p21 gene (Fig. 1GoA) was then ligated into the ZeoCassette vectors (Invitrogen, pcDNA3.1/Zeo+/-), which are 5-kb mammalian expression vectors (Fig. 1GoB) driven by the CMV promoter and contain the Zeocin (an antibiotic)-resistant gene. These vectors allowed us to ligate the cloned p21 gene in both sense and antisense orientations. DNA from purified p21 gene, and Zeo+ and Zeo- expression vectors were prepared. DNA isolated from p21 gene, Zeo- and Zeo+ expression vectors were digested with XbaI and KpnI, and the 1.7-kb and 5-kb size fragments were obtained. DNA represented by these bands was eluted from the agarose gels using gel elute columns that simultaneously remove ethidium bromide from DNA. The authenticity of these was confirmed by agarose gel electrophoresis. Purified DNA from the p21 gene was ligated to the expression vector using a ligation protocol. The ligation reaction was transformed into Escherichia coli, isolated colonies were selected, and plasmid DNA was isolated. The plasmid DNA was linearized by StuI to confirm the insert in the expression vector representing a 6.7-kb band. The presence of the p21 insert was confirmed by KpnI and XbaI restriction endonucleases for Zeo+ and Zeo- expression vectors; results are shown in Fig. 1GoC. The electrophoretic analysis shows the plasmids containing inserts (6.7 kb) and after digestion demonstrates the empty vector (5 kb) and p21 gene insert (1.7 kb). The orientations of the p21 inserts were confirmed by sequencing these inserts in an automated sequencer.



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  		FIGURE 1. Construction of p21 sense and antisense plasmid DNA. Genomic DNA was amplified using p21-specific primers (A, A) and cloned into TOPO cloning vector (Invitrogen) (A, B) and authenticated by EcoRI digestion (A, C). The cloned p21 gene was digested and ligated into Zeo+ and Zeo- eukaryotic expression vectors (B), and the authenticity of the ligated p21 in the sense direction was confirmed by digestion with Kpn and XbaI restriction nucleases (C).

 
In vitro transfection of p21

p21 sense plasmid DNA was transfected into A-549 cells or Jurkat T cells, selected using Zeocin resistance, and propagated. The increased or de- creased levels of p21 mRNA are determined by RT-PCR. Untreated cells and cells transfected with empty vector plasmid DNA were used as controls.

In vivo transfection of p21

Plasmid DNA was isolated from competent E. coli cells transformed with either the empty pcDNA3.1/Zeo vector (Invitrogen) or the vector containing the full-length p21 gene in the sense direction. Groups of mice (four each) were noninjected, injected with empty vector, or injected with p21 gene (100 µg) complexed with lipofectamine. Mice were sacrificed after 7 days. In a separate experiment, groups of mice (eight each) were either untreated or injected with p21 sense plasmid DNA to study the effect of p21 transfection on CsA-mediated inhibition of splenocyte proliferation. The expression of p21 mRNA was studied in heart, kidney, liver, and spleen. A part of the spleen was used to study the spontaneous and anti-CD3-induced ex vivo proliferation of splenocytes. RNA from spleen and other tissues was isolated and reverse transcribed to cDNA and amplified for p21 and ß-actin genes using specific primers. Controls included mice injected with empty vector (tissue mRNA levels and resting and mitogen-stimulated splenocyte proliferation) and noninjected mice (tissue mRNA levels and mitogen-stimulated splenocyte proliferation with increasing doses of cyclosporine).

p21 mRNA levels

The modulation of p21 gene expression in these and different cell lines was studied by RT- PCR as previously described (15). Oligonucleotide primers used were as follows: p21, coding, 5'-AGG ATC CAT GTC AGA ACC GGC TGG-3'; noncoding, 5'-CAG GAT CCT GTG GGC GGA TTA GGG-3' (16); ß-actin, coding, 5'-TGA CGG GGT CAC CCA CAC TGT GAA CAT CTA-3'; noncoding, 5'-CTT GAA GCA TTT GCG GTG GAC GAT GGA GGG-3' (17). The PCR products were resolved using agarose gel electrophoresis containing ethidium bromide and photographed under UV light.

p21 protein expression

p21 protein expression was studied by Western blot analysis as described (15). Briefly, cell lysates were prepared using boiling lysis buffer (10 mM Tris-HCl, pH 7.4, containing 1% SDS, 1.0 mM sodium vanadate). The protein in each sample was quantified using Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA). Identical amounts of proteins (10 µg) were electrophoretically resolved in 12% polyacrylamide gels under reducing conditions and transferred to nitrocellulose paper. Expression was detected using 1:500 diluted p21 mAb (Transduction Laboratories, Louisville, KY) and visualized by chemiluminescence technique (Pierce, Rockford, IL).

In vitro and ex vivo cell proliferation studies

Cell proliferation was determined using [3H]thymidine incorporation as previously described (3). All proliferation assays of each experiment were performed in triplicate. A total of three individual experiments investigating proliferation of unaltered and p21-augmented A-549 cells in unstimulated cells in the absence or presence of increasing concentrations of cyclosporine and expressed as percent inhibition (ratio between cyclosporine-treated and untreated cells) were performed. A total of three individual experiments investigating the proliferation of unaltered and p21-augmented Jurkat cells were performed in unstimulated cells and cells activated with PMA and ionomycin. Briefly, 200,000 cells were added to each well of a round-bottom 96-well plate. PMA (10 ng/ml) and ionomycin (400 ng/ml) was added to the wells; controls were without PMA/ionomycin. Splenocytes were prepared as previously described (4). Briefly, spleens were removed aseptically from mice and were crushed and passed through a fine mesh, separated over a Ficoll-Hypaque gradient, and resuspended in RPMI 1640 medium containing 5% FBS at a concentration of 1 x 106/ml. A total of 200,000 cells were plated into each well of a 96-well round-bottom plate. The cells were cultured for 64 h at 37°C in a 95% air and CO2-enriched environment. The cultures were pulsed with [3H]thymidine (1 µCi/well) for the last 16 h of incubation, cells were harvested, and radioactivity was counted using a scintillation counter. [3H]Thymidine uptake was expressed as the mean cpm of triplicate samples. The magnitude of splenocyte proliferation from unaltered and p21-augmented mice was investigated at rest, following mitogen stimulation (anti-CD3 mAb, 100 ng/ml), and with increasing concentrations of cyclosporine.

Data analysis

Differences between groups were determined using two-tailed unpaired t test with significance considered present at p < 0.05. The results are expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vitro transfection of p21

Our initial efforts focussed on attempts to overexpress p21 in vitro. We initially used A-549 cells, as the biologic and antiproliferative effects of CsA are well described in this cell line. We then repeated the majority of these studies in a more relevant Jurkat lymphoid cell line. Fig. 2Go demonstrates the effects of p21 gene transfection in A-549 cells. In Fig. 2GoA, the mRNA levels coding for p21 are shown in control cells, cells transfected with antisense p21, and cells transfected with sense p21 gene. This is a representative gel of a total of three transfection experiments. Fig. 2GoB demonstrates p21 protein expression in control cells (lanes 1 and 2) and p21 gene-transfected cells (lanes 3 and 4), demonstrating the substantial increase in p21 protein expression in these cells following transfection. Fig. 2GoC relates to our primary hypothesis that the direct up-regulation of p21 would synergize with agents that indirectly up-regulated p21, specifically cyclosporine, which through TGF-ß stimulates p21. Unmodified A-549 cells show between a 15% and 40% inhibition of proliferation in the presence of CsA, depending on dose. In p21-transfected cells, an equivalent level of inhibition was obtained using half of the cyclosporine dose (n = 3 experiments). These data confirm the ability to transfect cells in vitro with p21 and obtain a measurable increase in p21 mRNA and protein, which results in a biologic effect.



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  		FIGURE 2. The overexpression of p21 in A-549 cells and its effect on proliferation and the inhibitory effects of cyclosporine. A-549 cells were transfected with p21 sense or antisense plasmid DNA. RNA from these cells were prepared and reverse transcribed to cDNA using PCR amplified for p21 mRNA. The ethidium bromide-stained agarose gel is shown, and the expression of p21 mRNA in untreated A-549 cells, transfected with p21 antisense plasmid DNA, and transfected with p21 sense plasmid DNA is shown in lanes 1, 2, and 3, respectively. The lysates from these cells were also prepared, and p21 protein was studied by Western blot analysis, representative results are shown in B. The expression of p21 protein from untreated cells is shown in lanes 1 and 2 and from p21 sense plasmid DNA-transfected cells in lanes 3 and 4, showing an increase in p21 protein expression. The effect of p21 transfection was studied by the inhibition of proliferation by CsA. As shown in C, an equivalent amount of inhibition of proliferation (mean ± SEM from three consecutive experiments) was observed with 250 ng/ml in p21-overexpressing cells as compared with 500 ng/ml of CsA in untreated A-549 cells.

 
Fig. 3Go presents data on transfected Jurkat cells, which is a more relevant lymphoid cell line. Fig. 3GoA demonstrates a representative gel showing the up-regulation of p21 mRNA in p21-transfected cells (lane 2) compared with unmodified cells (lane 1). As previously described, ß-actin is used as a control housekeeping gene. Fig. 3GoB demonstrates p21 protein expression in these cells. Lane 1 is p21 protein from unmodified cells, lane 2 from Jurkat cells transfected with the empty vector, and lanes 3–5 Jurkat cells transfected with the p21 gene. Fig. 3GoC demonstrates thymidine uptake as a measure of cell proliferation at rest and following nonspecific mitogen (ionomycin/PMA) stimulation. The p21-transfected Jurkat cells are able to be stimulated to proliferate, but the response is blunted compared with unmodified cells (n = 3 experiments). The results obtained with control and p21-overexpressing Jurkat cells activated in the presence of CsA (100 ng/ml) are also shown. These results demonstrate that p21 transfection increases the inhibitory effects of CsA. These data confirm the ability to transfect cells with p21 from an appropriate lymphoid lineage and that this transfection has the anticipated biologic effects.



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  		FIGURE 3. The effect of in vitro transfection of p21 in Jurkat T cells. Jurkat cells were transfected with p21 sense plasmid DNA. RNA from these cells were prepared and reverse transcribed to cDNA using PCR amplified for p21 mRNA. The ethidium bromide-stained agarose gel shows the expression of p21 mRNA in untreated Jurkat cells and transfected with p21 sense plasmid DNA. The transfection of p21 is demonstrated by the overexpression of p21 mRNA (A, lane 2) as compared with untransfected cells (lane 1); the expression of the house keeping gene ß-actin is also shown. The lysates from these cells were also prepared, and p21 protein was studied by Western blot analysis. p21 protein was increased in p21-overexpressing cells (B, lanes 3, 4, and 5) as compared with untransfected cells(B, lane 1) or cells transfected with empty vector (B, lane 2). The effect of p21 transfection was studied by the spontaneous and activated Jurkat T cells and quantified by [3H]thymidine uptake assay. The results (mean ± SEM, n = 3) are shown in C, demonstrating a reduced proliferation in p21-overexpressing Jurkat cells.

 
In vivo transfection of p21

Fig. 4Go demonstrates the data generated from in vivo transfection of p21 in mice sacrificed 7 days after transfection. A representative gel showing mRNA coding for p21 in heart, spleen, kidney, and liver, respectively, from an untreated animal, a mouse injected with empty vector, and a mouse injected with p21 sense plasmid DNA is presented in Fig. 4GoA. No constitutive p21 gene expression could be demonstrated in either of the control groups. The transfected group all demonstrated p21 mRNA in the four tissues studied. We also studied the expression of p21 in spleens of control and p21-overexpressing mice by Western blot analysis; the results (Fig. 4GoA) demonstrate that the expression of p21 was observed only in mice injected with p21 sense plasmid DNA, not in control mice.



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  		FIGURE 4. The in vivo overexpression of p21 in mice and its effect on splenocyte proliferation. Three groups of mice were used: untreated, i.m. injection of empty vector, and i.m. injection of p21 sense plasmid DNA. The expression of p21 was observed only in the third group (A); the expression of housekeeping gene ß-actin is also shown. The expression of p21, studied by Western blot analysis, is also shown, which was observed only in spleens of mice transfected with the p21 gene. The in vivo effect of p21 transfection was studied by ex vivo proliferation of splenocytes. The proliferation was also quantified by [3H]thymidine uptake assay, and data is presented as cpm (mean ± SEM, n = 4); there was no difference in the anti-CD3-activated splenocytes of mice (untreated vs injected with empty vector plasmid DNA). A significantly decreased inhibition of anti-CD3-induced proliferation can be seen in p21-overexpressing mice (B). The effect of CsA on the anti-CD3-induced proliferation of splenocytes was also studied by [3H]thymidine uptake assay, and data is presented as cpm (mean ± SEM, n = 8) as shown in C; significantly more inhibition by CsA was observed in p21-overexpressing mice as compared with the control mice.

 
Fig. 4GoB demonstrates anti-CD3-induced splenocyte proliferation from control (untreated mice and mice injected with empty vector DNA) and p21-overexpressing mice. We did not observe any difference in the proliferation of the splenocytes from these compared with control vector-injected mice; 41,957 ± 2,959 vs 38,061 ± 1,762, p = 0.34 (mean ± SEM); however, the proliferation of splenocytes from p21-augmented mice was significantly reduced (10,495 ± 4,471). Fig. 4GoC demonstrates data from another set of experiments relating again to our primary hypothesis that the direct up-regulation of p21 would synergize with agents that indirectly up-regulated p21. In these experiments, increasing doses of cyclosporine are added ex vivo to mitogen (anti-CD3)-stimulated splenocytes from mice transfected with p21 (n = 8) vs controls (nontransfected mice, n = 8). At every cyclosporine dose, splenocyte proliferation in p21-augmented animals was lower than controls. These data demonstrate that p21 can be transfected into animals in vivo and that this transfection has the anticipated biological effects on proliferating cells. Moreover, this biological effect is maintained when lymphoid cells are induced to proliferate as they would with Ag stimulation (allogeneic response).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The two mainstay immunosuppressive agents used in clinical transplantation today are cyclosporine and tacrolimus. Both are felt to inhibit T cell activation by inhibiting IL-2 production via the inhibition of calcineurin (18, 19). We and others have demonstrated that both cyclosporine and tacrolimus induce TGF-ß, which has both potent antiproliferative as well as fibrogenic effects (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). The significance of these latter effects on the immunosuppressive properties of these agents is still under active investigation; however, our own studies have suggested that TGF-ß appears to mimic much of the in vivo effects of cyclosporine in a mouse model (2). We have most recently demonstrated that cyclosporine induces p21, and this induction requires the augmentation of TGF-ß (15). Moreover, others have demonstrated that the antiproliferative effects of TGF-ß are likely mediated by its induction of p21 (12, 13, 14). Accordingly, our working hypothesis was that the direct augmentation of p21 in lymphoid tissue could produce immunosuppression while bypassing the potential fibrogenic side effects of TGF-ß. Furthermore, should p21 induction alone be insufficient to induce clinical immunosuppression, one would theoretically be able to use less cyclosporine or tacrolimus in the presence of p21 augmentation, hence reducing fibrogenic side effects (nephrotoxicity, gum disease, etc.).

The data presented here represent the first steps toward testing this hypothesis. We have demonstrated the ability to transfect the p21 gene into nonlymphoid and lymphoid cells and have shown that this transfection produces increased levels of p21 protein, which is biologically active. We have additionally demonstrated that p21 transfection can be accomplished in vivo, again with biologic effects. Finally, we have demonstrated that, as we anticipated, the effects of the immunosuppressive agent cyclosporine and direct p21 augmentation appear to be additive in inhibiting lymphocyte proliferation. What is yet to be accomplished is the targeted delivery of the p21 gene to lymphoid cells, specifically T cells, as well as the demonstration that this approach is effective in a true allogeneic model.

p21 is one of the most potent regulators of the cell cycle and is known to inhibit cell proliferation by two independent and functionally different ways. It binds to cyclin-dependent kinase (Cdk)-2 and also binds and inhibits proliferating cell nuclear Ag, which is an auxiliary protein in DNA polymerase needed for DNA synthesis and nucleotide excise and repair and has six binding sites for p21 (20). p21 has been shown to control proliferation in cancer and noncancerous cells. Fukui et al. (21) transfected the p21 gene into rat aortic smooth muscle cells, which resulted in their inhibition of migration and proliferation. Ihling et al. (22) analyzed the expression of p21 in human atherosclerotic plaques by immunochemistry and observed that the cells expressing p21 lacked proliferating activity. Brugarolas et al. (23) demonstrated that loss of p21 in mouse embryo fibroblasts results in their increased proliferation, and Brown et al. (24) found that cells lacking p21 could not arrest their cell cycle caused by the DNA damage. We focused on p21 as one of the most potent cyclin inhibitors. Hengst et al. (25), using a number of different techniques including analytical ultracentrifugation of purified p21/cyclin A/Cdk2 complexes, demonstrated that a single p21 molecule is sufficient for kinase inhibition and that p21-saturated complexes contain only one stably bound inhibitor molecule. Even phosphorylated forms of p21 remained efficient inhibitors of Cdk activities. Other cyclin inhibitors, such as p53 and p27, though possibly not as potent as p21, may be potential targets should this immunosuppressive approach be found promising.

A number of studies have demonstrated that during the process of T cell activation a number of cyclins and Cdks are activated. Ajchenbaum et al. (26) demonstrated that during human T cell activation, D-type cyclin mRNA and protein levels were increased. Nagasawa et al. (27) observed that the activity of Cdk6 increased very early after activation of T cells. Shipman et al. (28) demonstrated that cyclin mRNA and protein accumulation occurred during IL-2-induced proliferation of a murine T lymphocytic cell line. These findings were extended by Nourse et al. (29), who observed that during T cell proliferation Ag-receptor and IL-2 signaling result in the increased expression of cyclins and activation of cyclin E/Cdk2 complexes. Most recently, Kaplan et al. (30), using Stat6-deficient mice, showed that these proteins control IL-4-induced proliferation of activated T cells by regulating cell-cycle inhibitors. Therefore, this strategy of inhibiting the alloimmune response by counteracting activated Cdks by a potent cyclin inhibitor may provide the desired immunosuppression in organ transplantation.

Our method of using plasmid DNA to obtain in vitro and in vivo transfection of p21 is based on the data supporting the efficacy of i.m. injection of plasmid DNA for a number of genes (31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41). Piccirillo and Prud’homme (42) injected naked plasmid DNA expression vectors encoding either TGF-ß1 (pVR-TGF-ß1) or an IL-4-IgG1 chimeric protein (pVR-IL-4-IgG1) i.m., which resulted in production of TGF-ß1 or IL-4-IgG1, respectively, and protection from myelin basic protein-induced experimental allergic encephalomyelitis. Chun et al. (43) compared the modulatory effect of the cytokine IL-10 expressed via viral vector or plasmid DNA on viral Ag-induced cutaneous inflammatory lesions and observed that modulatory effects achieved by plasmid DNA-expressing IL-10 were delayed in onset and milder in effect but were far more persistent than those achieved by viral vectors. Chen et al. (44) demonstrated that intratumoral and i.v. delivery of cationic liposomes complexed with angiostatin and endostatin plasmids inhibited angiogenesis and breast cancer in a nude mouse model, compared with either empty vector or untreated vectors. The authors also emphasized the significance of nonviral delivery of the desired genes. Similar results were obtained by Blezinger et al. (45), who observed that a single i.m. injection of the endostatin gene resulted in the expression of the gene in the muscle, and the product was secreted into the blood stream up to 2 wk, resulting in the systemic inhibition of tumor growth. In another study, Blezinger et al. (46) tested the effect of intratracheal administration of IL-12 plasmid DNA complexes in a mouse lung cancer model. Allila et al (47) demonstrated that i.m. injection of human insulin-like growth factor-I plasmid complexed with polyvinylpyrrolidone resulted in a localized and sustained level of biological active human insulin-like growth factor-I. Anwer et al. (48) also reported that the i.m. injection of a single dose of plasmid DNA encoding human growth hormone complexed with polyvinylpyrrolidone caused a sustained levels of human growth hormone in muscle, which were 10- to 15-fold higher than observed with human growth hormone complexed with polyvinylpyrrolidone formulated in saline. The expression persisted for 21 days. Wolff et al. (49) also studied the effect of direct gene transfer into muscle in vivo and noted the desired expression for at least 2 mo. Earlier, Budker et al. (50) have demonstrated the ability of nonviral plasmid DNA to achieve a high level of expression in the muscles of adult animals larger than mice. Based on their studies, Davis et al. (51) concluded that plasmid DNA provide a simple, safe, and viable alternative for gene therapy involving muscle tissue.

As noted above, we obtained a systemic overexpression of the p21 molecule to demonstrate its efficacy. The targeted transfection of p21 in immune cells should be a more precise way to obtain inhibition of alloimmune activation in transplant recipients. One method might be the transfection with p21 ligated to an expression vector containing T lymphocyte cell-surface receptor CD3 promoter. We have begun efforts in this area based on the work of others using targeted expression other molecules. Wu et al. (52) using the liver-specific gene transthretin promoter generated p21 transfection specifically in hepatocytes of mice and demonstrated the role p21 in normal development and regeneration of liver. Vacik et al. (53) also demonstrated the ability of plasmid DNA to target specific organs or tissues. They employed the promoter from the smooth muscle {gamma}-actin gene, whose expression is limited to smooth muscle cells, and created a series of reporter plasmids that were expressed selectively in smooth muscle cells. The nuclear import of the smooth muscle {gamma}-actin promoter-containing plasmids was achieved when the smooth muscle-specific transcription factor serum response factor was expressed in stably transfected CV1 cells, which supported the nuclear import of plasmids. These nuclear targeting sequences were also able to increase gene expression in liposome- and polycation-transfected nondividing cells in a cell-specific manner, similar to their nuclear import activity. These results provided credence for the development of cell-specific nonviral vectors for any desired cell type.

In summary, the currently available and most widely used immunosuppressive agents appear to ultimately induce the cyclin inhibitor p21. If this is a major mechanism for their immunosuppressive effects, the direct manipulation of p21 may provide more specific and less toxic immunosuppression. The data in this report demonstrates the potential feasibility of this approach and represents the first attempt at directly manipulating the cell cycle to achieve immune suppression.


   Acknowledgments
 
We thank Matthew Plummer for his excellent technical assistance.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Ashwani Khanna, The Cardiovascular Research Center, Room H4125, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. Back

2 Abbreviations used in this paper: CsA, cyclosporin A; Cdk, cyclin-dependent kinase. Back

Received for publication December 15, 1999. Accepted for publication June 2, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Khanna, A., V. Cairns, J. D. Hosenpud. 1999. Tacrolimus induces increased expression of transforming growth factor-ß in lymphoid and non-lymphoid cells. Transplantation 67:614.[Medline]
  2. Khanna, A., V. Cairns, C. G. Becker, J. D. Hosenpud. 1999. Transforming growth factor-ß (TGF-ß) mimics and anti-TGF-ß antibody abrogates the in-vivo effects of cyclosporine: demonstration of a direct role of TGF-ß in immunosuppression and nephrotoxicity of CsA. Transplantation 67:882.[Medline]
  3. Khanna, A., V. Cairns, C. G. Becker, J. D. Hosenpud. 1998. TGF-ß: a link between immunosuppression, nephrotoxicity and CsA. Transplant. Proc. 30:944.[Medline]
  4. Khanna, A., S. Kapur, V. Sharma, B. Li, M. Suthanthiran. 1997. In-vivo hyperexpression of transforming growth factor-ß1 in mice: stimulation with cyclosporine (CsA). Transplantation 63:1037.[Medline]
  5. Khanna, A., B. Li, P. Li, M. Suthanthiran. 1994. Regulation of transforming growth factor-ß1 (TGF-ß1) expression with a novel TGF-ß1 complementary DNA. Biochem. Biophys. Res. Commun. 204:1061.[Medline]
  6. Khanna, A. K, B. Li, K. H. Stenzel, M. Suthanthiran. 1994. Regulation of new DNA synthesis in mammalian cells by cyclosporine: demonstration of a transforming growth factor-ß dependent mechanism of inhibition of cell growth. Transplantation 57:577.[Medline]
  7. Wolf, G., F. Thaiss, R. A. Stahl. 1995. Cyclosporine stimulates expression of transforming growth factor-ß in renal cells: possible mechanism of cyclosporines’s antiproliferative effects. Transplantation 60:237.[Medline]
  8. Ahuja, S. S., S. Shrivastav, D. Danielpour, J. Balow, D. T. Boumpas. 1995. Regulation of transforming growth factor-ß1 and its receptors by cyclosporine in human T cells. Transplantation 60:718.[Medline]
  9. Shehata, M., J. H. Cope, T. S. Johonson, A. T. Raftery, A. M. el Nahas. 1995. Cyclosporine enhances the expression of TGF-ß in the juxtaglomerular cells in the rat kidney. Kidney Int. 48:1487.[Medline]
  10. Shihab, F. S., T. F. Andoh, A. M. Tanner, N. A. Noble, W. A. Border, N. Franceschini, W. A. Bannet. 1996. Role of transforming growth factor-ß1 in experimental chronic cyclosporine nephropathy. Kidney Int. 49:1141.[Medline]
  11. Pankewycz, O. G., L. Miao, R. Isaacs, J. Guan, T. Pruett, G. Haussmann, C. Sturgill. 1996. Increased renal tubular expression of transforming growth factor ß in human allografts correlates with cyclosporine toxicity. Kidney Int. 50:1634.[Medline]
  12. Datto, M. B., Y. Yu, X.-P. Wang. 1995. Functional analysis of the transforming growth factor-ß responsive elements in the WAF/Cip1/p21 promoter. J. Biol. Chem. 270:28623.[Abstract/Free Full Text]
  13. Li, C. Y., L. Suardet, J. B. Little. 1995. Potential role of WAF1/Cip1/p21 as a mediator of TGF-ß cytoinhibitory effect. J. Biol. Chem. 270:4971.[Abstract/Free Full Text]
  14. Elbendary, A., A. Berchuck, P. Davis, L. Havrilesky, R. C. Bast, J. D. Iglehart, and J. R.Marks, J. R. 1994. Transforming growth factor ß1 can induce CIP1/WAF1 expression independent of the p53 pathway in ovarian cancer cells. Cell Growth Differ. 5:1301.
  15. Khanna, A. K., J. D. Hosenpud. 1999. Cyclosporine induces the expression of the cyclin inhibitor p21. Transplantation 67:1262.[Medline]
  16. Xiang, Y., G. J. Hannon, H. Zhang, D. Casso, R. Kobayashi, D. Beach. 1993. p21 is a universal inhibitor of cyclin kinases. Nature 366:701.[Medline]
  17. Ponte, P., S. Y. Ng, J. Engel, P. Gunning, L. Kedes. 1984. Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human ß-actin cDNA. Nucleic Acids Res. 12:1687.[Abstract/Free Full Text]
  18. Shreiber, S., G. Crabtree. 1992. The mechanism of action of cyclosporine A and FK-506. Immunol. Today 13:136.[Medline]
  19. Sigal, N. H., F. J. Dumont. 1992. Cyclosporine A, FK-506, and rapamycin: pharmacological probes of lymphocyte signal transduction. Annu. Rev. Immunol. 10:519.[Medline]
  20. Waga, S., G. J. Hannon, D. Beach, E. Stillman. 1994. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interacting with PCNA. Nature 369:574.[Medline]
  21. Fukui, R., N. Shibata, E. Kohbayashi, M. Amakawa, D. Furutama, M. Hoshiga, N. Negoro, T. Nakaouji, T. M. Ii, T. Ishihara, N. Ohsawa. 1997. Inhibition of smooth muscle cell migration by the p21 cyclin-dependent kinase inhibitor (Cip1). Atherosclerosis 132:53.[Medline]
  22. Ihling, C., G. Menzel, E. Wellens, J. S. Monting, H. E. Schaefer, A. M. Zeiher. 1997. Topographical association between the cyclin-dependent kinase inhibitor p21, p53 accumulation, and cellular proliferation in himan atherosclerotic tissue. Arteriscler. Thromb. Vasc. Biol. 17:2218.[Abstract/Free Full Text]
  23. Brugarolas, J., C. Chandrasekaran, J. L. Gordon, D. Beach, T. Jacks, J. G. Hannon. 1995. Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature 377:552.[Medline]
  24. Brown, J. P. W. Wei, J. M. Sedivy. 1997. Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science 277:831.[Abstract/Free Full Text]
  25. Hengst, L., U. Gopfert, H. A. Lashuel, S. I. Reed. 1998. Complete inhibition of Cdk/cyclin by one molecule of p21(Cip1). Genes Dev. 12:3882.[Abstract/Free Full Text]
  26. Ajchenbaum, F., K. Ando, J. A. Decaprio, J. D. Griffin. 1993. Independent regulation of human D-type cyclin gene expression during G1 phase in primary human T lymphocytes. J. Biol. Chem. 268:4113.[Abstract/Free Full Text]
  27. Nagasawa, M., I. Melamed, A. Kupfer, E. W. Gelfand, J. J. Lucas. 1997. Rapid nuclear translocation and increased activity of cyclin-dependent kinase 6 after T cell activation. J. Immunol. 158:5146.[Abstract]
  28. Shipman, P. M., D. E. Sabath, A. H. Fischer, P. G. Comber, K. Sullivan, E. M. Tan, M. B. Prystowsky. 1988. Cyclin mRNA and protein expression in recombinant interleukin 2-stimulated cloned murine T lymphocytes. J. Cell Biochem. 38:189.[Medline]
  29. Nourse, J., E. Firpo, W. M. Flanagan, S. Coats, K. S., M. H. Poyak, J. Lee, J. Massague. G. R. Crabtree, J. M. Roberts. 1994. Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature 372:570.[Medline]
  30. Kaplan, M. H., C. Daniel, U. Schindler, M. J. Grusby. 1998. Stat proteins control lymphocyte proliferation by regulating p27Kip1 expression. Mol. Cell Biol. 18:1996.[Abstract/Free Full Text]
  31. Ishii, K. J, W. R. Weiss, M. Ichino, D. Verthelyi, D. M. Klinman. 1999. Activity and safety of DNA plasmids encoding IL-4 and IFN-{gamma}. Gene Ther. 6:237.[Medline]
  32. Nakanishi, M., G. R. Adami, R. S. Robetorye, A. Noda, S. F. Venable, D. Dimitrov, O. M. Pereira-Smith, J. R. Smith. 1995. Exit from G0 and entry into the cell cycle of cells expressing p21 antisense RNA. Proc. Natl. Acad. Sci. USA 92:4352.[Abstract/Free Full Text]
  33. Croxford, J. L., K. Triantaphyllopoulos, O. L. Pudhajcer, M. Feldmann, D. Baker, Y. Chernajovsky. 1998. Cytokine gene therapy in experimental allergic encephalomyelitis by injection of plasmid DNA-cationic liposome complex into the central nervous system. J. Immunol. 160:5181.[Abstract/Free Full Text]
  34. Danko, I., J. A. Wolffe. 1994. Direct gene transfer into muscle. Vaccine 12:1499.[Medline]
  35. Danko, I., J. D. Fritz, S. Jiao, K. Hogan, J. S. Latendresse, I. Bock, J. A. Wolffe. 1994. Pharmacological enhancement of in vivo foreign gene expression in muscle. Gene Ther. 1:114.[Medline]
  36. Nitta, Y., F. Tashiro, M. Tokul, A. Shimada, I. Takel, K. Tabayashi, J.-I. Miyazaki. 1998. Systemic delivery of interleukin 10 by intramuscular injection of expression plasmid DNA prevents autoimmune diabetes in nonobese diabetic mice. Hum. Gene Ther. 9:1701.[Medline]
  37. Danko, I., P. Willuams, H. Herweijer, G. Zhang, J. S. Latendresse, I. Bock, J. A. Wolffe. 1997. High expression of naked plasmid DNA in muscles of young rodents. Hum. Mol. Genet. 6:1435.[Abstract/Free Full Text]
  38. Daheshia, M., N. Kuklin, E. Manickan, S. Chun, B. T. Rouse. 1998. Immune induction and modulation by topical ocular administration of plasmid DNA encoding antigens and cytokines. Vaccine 16:1103.[Medline]
  39. Song, X., M. Gu, W. W. Jin, D. M. Klinman, S. M. Wahl. 1998. Plasmid DNA encoding transforming growth factor-ß1 suppresses chronic disease in a streptococcal cell wall-induced arthritis model. J. Clin. Invest. 101:2615.[Medline]
  40. Kuklin, N. A., M. Daheshia, S. Chun, B. T. Rouse. 1998. Immunomodulation of mucosal gene transfer using TGF-ß DNA. J. Clin. Invest. 102:438.[Medline]
  41. Nomura, T., K. Yasuda, T. Yamada, S. Okamoto, R. I. Mahato, Y. Watanabe, Y. Takakura, M. Hashida. 1999. Gene expression and antitumor effects following direct interferon (IFN)-{gamma} gene transfer with naked plasmid DNA and DC-chol liposome complexes in mice. Gene Ther. 6:121.[Medline]
  42. Piccirillo, C. A., G. J. Prud’homme. 1999. Prevention of experimental allergic encephalomyelitis by intramuscular gene transfer with cytokine-encoding plasmid vectors. Hum. Gene Ther. 10:1915.[Medline]
  43. Chun, S., M. Daheshia, S. Lee, B. T. Rouse. 1999. Immune modulation by IL-10 gene transfer via viral vector and plasmid DNA: implication for gene therapy. Cell Immunol. 194:194.[Medline]
  44. Chen, Q. R., D. Kumar, S. A. Stass, A. J. Mixson. 1999. Liposomes complexed to plasmids encoding angiostatin and endostatin inhibit breast cancer in nude mice. Cancer Res. 59:3308.[Abstract/Free Full Text]
  45. Blezinger, P. J., M. Wang, A. Gondo, D. Quezada, M. Mehrens, A. French, S. Singhal, A. Sullivan, R. Ralston Rolland, W. Min. 1999. Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene. Nat. Biotechnol. 17:343.[Medline]
  46. Blezinger, P., B. D. Freimark, M. Matar, E. Wilson, A. Singhal. W. Min, J. L. Nordstrom, F. Pericle. 1999. Intratracheal administration of interleukin 12 plasmid-cationic lipid complexes inhibits murine lung metastases. Hum. Gene Ther. 10:723.[Medline]
  47. Alila, H., M. Coleman, H. Nitta, M. French, K. Anwer, Q. Liu, T. Meyer, J. Wang, R. Mumper, D. Oubari, S. Long, J. Nordstrom, A. Rolland. 1997. Expression of biologically active human insulin-like growth factor-I following intramuscular injection of a formulated plasmid in rats. Hum. Gene Ther. 8:1785.[Medline]
  48. Anwer, K., M. Shi, M. F. French, S. R. Muller, W. Chen, Q. Liu, B. L. Proctor, J. Wang, R. J. Mumper, A. Singhal, A. P. Rolland, H. W. Alila. 1998. Systemic effect of human growth hormone after intramuscular injection of a single dose of a muscle-specific gene medicine. Hum. Gene Ther. 9:659.[Medline]
  49. Wolff, J. A., R. W. Malone, P. Williams, W. Chong, G. Acsadi, A. Jani, P. L. Felgner. 1990. Direct gene transfer into mouse muscle in vivo. Science 247:1465.[Abstract/Free Full Text]
  50. Budker, V., G. Zhang, I. Danko, P. Williams, J. Wolff. 1998. The efficient expression of intravascularly delivered DNA in rat muscle. Gene Ther. 5:272.[Medline]
  51. Davis, H. L., B. A. Demeneix, B. Quantin, J. Coulombe, R. G. Whalen. 1993. Plasmid DNA is superior to viral vectors for direct gene transfer into adult mouse skeletal muscle. Hum. Gene Ther. 4:733.[Medline]
  52. Wu, H., L. Krall, J. Grisham, Y. Xiang, T. Van Dyke. 1996. Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, post natal liver development and regeneration. Genes Dev. 10:245.[Abstract/Free Full Text]
  53. Vacik, J., B. Dean, W. Zimmer, D. Dean. 1999. Cell-specific nuclear import of plasmid DNA. Gene Ther. 6:1006.[Medline]



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