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

Abstract

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.

Recombinant p21 Protein Inhibits Lymphocyte Proliferation and Transcription Factors

“…We have demonstrated previously that anti-inflammatory/immunosuppressive drugs, e.g., cyclosporine (CsA) 2 , tacrolimus (TAC), and sirolimus (SRL) (11)(12)(13), induce the expression of cyclin inhibitor p21. Also, in vitro and in vivo overexpression of p21 in lymphocytes results in decreased response to mitogenic stimuli and greater sensitivity to the effects of CsA (14) and prolongation of graft survival in a rat heart transplant model (15). These studies demonstrated an indirect effect of p21 on various biological effects; however, a purified recombinant protein can accomplish the direct effect.…”

“…Briefly, p21 cDNA cloned into Topo vector (14) was excised and overexpressed in plasmid pET-3d and named as pET-p21, which was transformed into Escherichia coli strain BL21 (DE3). The transformed cells were grown in Luria-Bertani culture medium at 37°C overnight.…”

p21CIP1/WAF1 Controls Proliferation of Activated/Memory T Cells and Affects Homeostasis and Memory T Cell Responses

“…Although previous studies showed that p21 has a role in various T cell proliferation systems, the conditions that render T cell proliferation p21 dependent remained undefined (4,8,14,28,29). In this study, we explored the role of p21 in T cell proliferation of activated/memory T cells and reached the following conclusions: 1) p21 does not affect naive T cell proliferation, but exerts its control on activated/memory T cells; 2) p21 does not affect AICD, but regulates proliferation of memory T cells that survive AICD; 3) p21 regulates responses of in vivo generated memory T cells; and 4) p21 deficiency increases CD4…”

“…These findings suggest that p21 control of cycling may depend on T cell activation status. Other studies showed inhibition of T cell proliferation after transfection with p21 (28) or direct uptake of recombinant p21 protein (29), although T cell status is not discussed.…”

T-lymphocyte Inactivation and Anti-atopic Effects of Diarylheptanoid Hirsutenone Isolated from Alnus japonica

“…T 세포의 분화 및 증식은 효과적인 면역반응을 위해 요구되며 따라서 TCR 복합체를 통한 자극유입 시 cell cycling에 진입하게 된다 (30). 이 때 calcineurin inhibitor인 cyclosporin A 처리 시 p21 유전자 발현증가에 의해 세포주기 단속의 결과 보다 효과적 으로 T 세포 활성을 억제하는 것으로 보고되었다 (31) (Fig. 2H).…”