Cyclin kinase inhibitor as a viable and potential alternate strategy for immunosuppression
in organ transplantation:
The success of a graft depends on the control
of the immune response of the recipient. Donor antigens interact with T cell receptors of the recipient and the activation
of T lymphocytes is initiated. This activation is one of the events leading to allograft rejection. Current immunosuppression
strategies involve inhibition of lymphocyte activation. During T cell activation and proliferation, cyclins and cyclin-dependent
kinases (CdKs) are activated and the expression of cytokines like IL-2, TNF-a, and IFN-g
is increased. Therefore, cyclin inhibitors like p21 could inhibit T cell activation and inflammation, based on our preliminary
studies; this may in part be due to induction of immune tolerance. The following section discusses the mechanisms and modes
of the inhibitory effects of p21 specifically of T lymphocytes and its relevance to organ transplantation.
Problems Associated with Current Immunosuppressive Drugs:
Nephrotoxicity: The introduction of CsA as an immunosuppressive agent in solid organ transplantation
lead to a marked increase in allograft survival. However, long-term treatment with CsA is associated with nephrotoxicity.
Nephrotoxicity is characterized by vascular injury and fibrosis and renal failure. Initially, it was believed that TAC
might have less nephrotoxicity; however, clinical and experimental studies demonstrated nephrotoxicity with tacrolimus. While
it was once believed that SRL did not cause significant nephrotoxicity, long-term clinical studies using SRL have demonstrated
that SRL can also be nephrotoxic. In addition, prolonged use of SRL is associated with increases in serum cholesterol and
triglycerides, thrombocytopenia and leucopenia, increased incidences of lymphocele formation, and the delayed healing of wounds.
Viral infections: Viral infections are a leading cause of morbidity and mortality in organ transplant
recipients. These viruses include Cytomegalovirus (CMV), Polyoma Virus, Epstein Bar Virus (EBV)
and, more recently, West Nile virus. The use of more potent immunosuppressive agents is, in part, considered to the cause
of this increased incidence of viral infections.
also increases risk in malignancy in transplant recipients. The chronic use of immunosuppressive agents to prevent allograft
rejection increases the long-term risk of malignancy to approximately 100 times that of the general population. Based on studies,
patients receiving SRL-based therapy without CsA or SRL maintenance therapy after early CsA withdrawal still experience malignancies,
but lower rates of malignancy in the first 2 years after renal transplantation.
Effect of Cellular Localization and Function of p21: p21 protein has a transduction domain, GRKKRRQRRR
that permits it to be transported across cell membranes (1). Because of this domain, strategies such as electroporation, osmotic
shock or covalent linkage to viral proteins containing PTDs such as the HIV-TAT, VP22 or drosophila Antennapedia (Antp)
protein and HSV VP22 protein from HSV will not be required to permit delivery of p21 protein into cells.
Relative advantages of p21 as an immunosuppressive agent in organ transplantation
Three major negative side effects of prolonged treatment with CsA, TAC, and SRL are nephrotoxicity, viral
infections, and malignancies. Due to the significance of each effect, we believe that as our preliminary data indicates, p21
is as effective as CsA, TAC, or SRL in preventing allograft rejection, and then a significant reduction in any one of the
three major side effects would justify further development of p21 as a therapy. However, based on our preliminary observations
and those of others, we believe that p21 therapy could potentially result in a relative reduction in each of three major side
effects associated with CsA, TAC, and SRL. Nephrotoxicity: We believe it is unlikely that p21 will be nephrotoxic
when used as an immunosuppressant. The mechanism of nephrotoxicity by CsA, TAC, and SRL is attributed in part to their ability
to induce expression of TGF-b, which results in the expression of pro-fibrogenic genes such as
collagen and fibronectin, and consequently, vascular injury and fibrosis. Although p21 expression is also induced by TGF-beta,
p21 is primarily regulator of and is a mediator of TGF-b’s antiproliferative effects. Moreover,
p21-/- but not the wild type mice develop nephrotoxicity. Thus, our expectation is that p21 will provide the same benefits
of inhibiting graft-rejection as current therapies, without inducing nephrotoxic fibrosis and vascular injuries. Overexpression
of p21 completely protected cultured proximal tubules from cisplatin toxicity. The reno-protective effects of number of agents
including glucocorticoids are mediated by p21. The cell cycle-inhibitory drug roscovitine
protected kidney cells in vitro from cisplatin-induced apoptosis and in vivo models of mesangial proliferative
nephritis and collapsing glomerulopathy in mice without any signs of toxicity.
Malignancies and viral infections: Our hypothesis is that malignancies and viral Infections associated
with CsA, TAC, or SRL treatment are a result of the complex cascade of effects that these drugs induce that are propagated
through pleiotropic factors such as calcineurin and TGF-b. It is our expectation that by suppressing
lymphocyte proliferation using a more discretely acting inhibitor such as p21, there will be fewer side-effects resulting
from the beneficial effects of increasing graft survival.Malignancies: It is clear that, to varying degrees,
CsA, TAC and SRL are responsible for a higher rate of malignancies in patients receiving these drugs. However, it is not clear
how these drugs induce these higher rates. The precise mechanism of malignancy with immunosuppressive agents is not clear.
To the contrary, studies suggest a role for p21 in suppressing tumorigenesis (discussed below). While there
are indications that malignancies associated with CsA, TAC or SRL treatment may be propagated through TGF-b,
the evidence indicates this may not be a function of impaired immune surveillance. However, it instead may be mediated by
a cell-autonomous mechanism, independent of the host immune system, that acts through TGF-b.
In this regard, p21 may actually counter-act the effects of such mutagens. For example, some DNA-damaging agents
activate the p53-p21 pathway and cause G(1)-phase arrest. Deregulation of cyclin dependent kinases (Cdks) can result in uncontrolled
cellular proliferation, and deregulation of Cdks is associated with a majority of human malignancies. Since cyclin inhibitor
p21 prevents cells from entering the cell cycle, p21 may suppress malignancies. In fact, due to its antiproliferative properties,
p21 has shown potential therapeutic value as inhibitors of lung, pancreatic, bladder and renal cancers. Furthermore loss of
p21 expression has been shown to be associated with abnormal cellular proliferation, differentiation, increased mutation rates,
gene amplification and genomic instability. All these parameters are perquisite for abnormal cellular activation and cancer
progression. Therefore, we propose that the inhibitory effects of p21 on cell cycle entry will inhibit the development of
cancer,p21 and anti-viral immunity: Polyomavirus-associated nephropathy (PVAN) affects1-10% of kidney transplant
patients. While little is known about the exact mechanism of susceptibility, there is little doubt that PVAN is a function
of substantial immune suppression since most cases are reported to arise while patients are receiving combinations of powerful
immunosuppressants such as tacrolimus and/or mycophenolate mofetil, along with corticosteroids. Immunological control of polyomavirus
infections can be established in these patients by replacing, reducing, or eliminating components of these combinations, which
presumably allows limited restoration of immune system function. However, it is our conjecture that the more focused effects
of p21 may either alone or in combination with other drugs, have value in preventing graft rejection without compromising
immunity to viral infections as drastically as current combination treatments. p21 and virus replication: It
is well established that cellular immunity is critical to the control of infections, and there is a correlation between the
degree of suppression of the immune system in transplant patients and the susceptibility to infection with certain viruses.
In this regard, p21 may show no advantage over current therapies due to its immunosuppressive effects. However, p21 actually
plays suppressive role in the replicative cycle of a number of viruses, making the outcome of long-term p21
therapy difficult to predict. Cyclin dependent kinases are required for replication of many viruses in non-dividing cells
as well as in dividing cells. For example, viruses that cause nephritis such as HIV or polyoma apparently require Cdks for
replication or protein expression. Consistent with this requirement is the observation that pharmacological Cdk inhibitors
(PCIs) possess potent antiviral activity, and may ultimately be useful as antiviral agents. In fact, p21 has been implicated
as a mediator of the antiviral effects of cidofovir in treating solid tumors caused by human papillomaviruses. Thus,
considering the effects of p21 on cyclins, p21 may actually suppress or limit viral infections that would otherwise occur
in patients receiving immunosuppressive drugs such as CsA, TAC, and SRL Toxicity of cyclin inhibitors: Pharmacological
inhibitors of CDKs are currently being evaluated for therapeutic use against cancer, alopecia, neurodegenerative disorders
(e.g. Alzheimer's disease, amyotrophic lateral sclerosis and stroke), cardiovascular disorders (e.g. atherosclerosis and restenosis),
glomerulonephritis, viral infections (e.g. HCMV, HIV and HSV) and parasitic protozoa (Plasmodium sp. and Leishmania sp.).
These inhibitors have not been shown to be toxic and, on the contrary, are protective against the toxicity of drugs like cisplatin.
We propose studies to test the feasibility of using p21 protein
as a novel therapeutic to prevent organ transplant rejection. Our first aim consists of in vivo dose-ranging studies
in rats to determine the highest dose of recombinant p21 protein that inhibits of lymphocyte proliferation without causing
significant (if any) toxicity. The second aim is compare p21 and a widely used immunosuppressant, CsA, in rat heart and kidney
transplant models for efficacy and toxicity. We believe that two significant outcomes of p21 therapy will be: 1) a comparable
or superior allograft survival rate relative to CsA, and 2) a significant reduction or elimination of nephrotoxicity and other
side-effects relative to those that are observed when CsA, TAC, or SRL. The idea of develping
p21 as a viable potential and clinically reliable immunosuppressive drug is based on our following studies:
1. Khanna AK, Mehra MR. Targeted in vitro and in vivo gene transfer into T Lymphocytes: potential
of direct inhibition of allo-immune activation. BMC-IMMUNOLOGY 2006 Nov 10; 7:26-33
2. Khanna A, Plummer, M, Nilakantan V, and Pieper G. p21 protein inhibits lymphocyte activation
and transcription factors. J Immunology 2005; 174: 7610-7.
Khanna A Reciprocal role of cyclins and cyclin kinase inhibitor p21 on lymphocyte
proliferation, allo-immune activation and inflammation BMC-IMMUNOLOGY, 6: 22-30, 2005
4. Khanna AK Tacrolimus induces the Expression of the
Cyclin Inhibitor p21 in lymphoid and non-lymphoid cells Biochem Biophys Res Commun. 2003; 303:266-72
5. Khanna, A Cell cycle control
and immunosuppression in organ transplantation Transplantation Proc. 2001; 33: 2101-2106
6. Khanna A K and Hosenpud JD In vitro and
in vivo transfection of p21 gene enhances Cyclosporine mediated inhibition of lymphocyte proliferation. J Immunol. 2000; 165: 1882-8
7. Khanna AK and Hosenpud J.D Cyclosporine induces
the expression of the cyclin inhibitor p21. Transplantation 1999; 67: 1262-1268Our interest in developing p21 as an immunosuppressive therapeutic was enhanced by our discovery
that, remarkably, exogenously supplied recombinant p21 protein can pass directly into lymphocytes, enter their nuclei, and
interact with transcription factors that regulate immune response genes. This unusual property of p21 is apparently due to
the presence of a naturally occurring protein transduction domain (PTD) in p21 that resembles the well-characterized PTD of
the HIV Tat protein (1). Due to its unique properties, we believe that recombinantly expressed p21 may be suitable for use
as a therapeutic without the need for amino acid sequence modifications, unusual formulation methods, or gene-therapy approaches.