Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1995-07-24
2003-07-08
Wehbe′, Anne M. (Department: 1632)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C514S04400A, C435S320100, C435S325000, C435S242000, C530S350000, C424S093210
Reexamination Certificate
active
06590086
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of regulation of cell growth and proliferative diseases. More specifically, the present invention relates to mutants of the RB-1 and p53 genes and therapeutic uses of such mutants.
2. Description of the Related Art
The control of cell proliferation is a complex process which involves multiple interacting components. Whether a cell grows or not depends on the balance of the expression of negatively-acting and positively-acting growth regulatory genes. Negatively-acting growth regulatory genes are those that, when expressed in or provided to a cell, lead to suppression of cell growth. Positively-acting growth regulatory genes are those which, when expressed in or provided to a cell, stimulate its proliferation.
Recently, several negatively acting growth regulatory genes called tumor suppressor genes which have a negative effect on cell proliferation have been identified. These genes include, but are not limited to, the human retinoblastoma gene, RB-1, and the p53 gene. The absence or inactivation of some of these negative growth regulatory genes has been correlated with certain types of cancer.
The human retinoblastoma gene, RB-1, is the prototype of this class of tumor suppressor genes in which the absence of both alleles of the gene in a cell or the inhibition of the expression of the gene or its gene product will lead to neoplastic or abnormal cellular proliferation. At the molecular level, loss or inactivation of both alleles of RB-1 is involved in the clinical manifestation of tumors such as retinoblastoma and clinically related tumors, such as osteosarcomas, fibrosarcomas, soft tissue sarcomas and melanomas. In addition, loss of the function of RB-1 has also been associated with other types of primary cancer such as primary small cell lung carcinoma, bladder carcinoma, breast carcinomas, cervical carcinomas and prostate carcinomas.
The Tumor Suppressor Genes Rb and p53 Suppress the Proliferation of Cancerous and Noncancerous Cells
The reintroduction of a wild-type cDNA of RB-1 or p53 have been shown to partially restore normal growth regulation as the reintroduced genes induce growth arrest or retardation in many different tumor cell types. The designation of the Rb gene as a tumor suppression gene stemmed from the fact that inactivation of an allele of the Rb gene is a predisposition to the development of cancer. However, the growth suppression effect of the Rb gene is not restricted to tumor cells. Normal cells which have 2 copies can be growth arrested or retarded by the introduction of extra copies of the Rb gene under certain growth conditions. Likewise, the ability of a wild type p53 to suppress the growth of noncancerous cells is well documented. Thus, the step controlled by Rb and p53 may not directly affect the tumorigenic phenotype but rather affects the steps that control the growth of tumor and normal cells alike.
Mechanisms of Pathological Proliferation of Normal Cells
There is a wide variety of pathological cell proliferative conditions for which novel methods are needed to provide therapeutic benefits. These pathological conditions may occur in almost all cell types capable of abnormal cell proliferation. Among the cell types which exhibit pathological or abnormal growth are (1) fibroblasts, (2) vascular endothelial cells, and (3) epithelial cells. It can be seen from the above that methods are needed to treat local or disseminated pathological conditions in all or almost all organ and tissue systems of the individual.
For instance, in the eye alone, novel methods may be utilized to treat such a wide variety of pathologic disease states which are due to abnormal proliferation of normal, benign or malignant cells or tissues including, but not limited to, the following: fibroproliferative, vasoproliferative and/or neoplastic diseases of the eye: retinopathy of prematurity, proliferative vitreoretinopathy, proliferative diabetic retinopathy, capillary hemangioma, choroidal neovascular nets, subretinal neovascular nets, senile macular degeneration due to subretinal, neovascularization, corneal neovascularization, macular pucker due to epiretinal membrane proliferation, adult cataracts due to nuclear sclerosis, fibrous ingrowth following trauma or surgery, optic nerve gliomas, angiomatosis retinae, neovascular glaucoma, cavernous hemangioma, rubeosis iridis, sickle cell proliferative retinopathy, epithelial downgrowth after eye surgery or injury, after-cataract membrane, papilloma, retinal neovascularization in thalassemia, subretinal neovascularization due to pseudoxanthoma elasticum, and neurofibromatosis type I and II, retinoblastoma, uveal melanoma, and pseudotumor of the orbit. Other benign cell proliferative diseases for which the present invention is useful include, but are not limited to, psoriasis, ichthyosis, papillomas, basal cell carcinomas, squamous cell carcinoma, and Stevens-Johnson Syndrome.
It should be noted that in the case of normal cells, there are already two normal alleles each of the Rb gene and the P53 gene and yet these wild type proteins fail to prevent the cells from proliferating when the cells were provided growth factors, as for example in tissue culture growth conditions. The uncontrolled proliferation of otherwise resting cells in pathological conditions is also due to the exposure of the cells to growth factors induced by the pathological conditions (see below). For an example, uncontrolled proliferation of blood vessels in the eye in thalassemia can lead to detachment of the retina if the proliferation is not stopped. The introduction of extra copies of the Rb gene or the P53 gene into such cells may or may not be sufficient to suppress the cells from growing. In fact, the introduction of exogenous Rb gene into many tumor cells only retarded the growth rate of the cells instead of complete arrest. The previously reported cell lines such as Saos-2 and DU145 are good examples of this phenomenon. Many more copies of the gene may need to be introduced and it is difficult to control on the one hand the number of copies that need to be or could be introduced. On the other hand, growth factor exposure may lead to inactivation of the expressed Rb proteins.
Regulation of the Activity of Rb Protein by Phosphorylation
If the inactivation of growth suppressor genes is an essential step in the removal of the constraint on cell growth, there must be mechanisms whereby the activities of these gene products are regulated so that a cell may proliferate in a controlled manner. It is conceivable that the expression of a given growth suppressor gene may be regulated quantitatively or qualitatively. In the case of the Rb gene, the ratio of the steady state level of the protein to the cell volume is a constant throughout the cell cycle. This lack of variation of the Rb protein concentration suggests that there must be other mechanisms whereby a cell can regulate the activity of this protein. There are several lines of evidence to show that the activity of the Rb protein (pRb) is regulated by its phosphorylation state. Various growth stimulatory or inhibitory factors exert their effects by perturbing the phosphorylation of growth suppressor gene products such as the Rb protein. Evidence for a role of growth stimulatory factors in the induction of phosphorylation of the Rb proteins have come from the observation that the Rb proteins exist in the underphosphorylated forms in quiescent cells. In addition, when quiescent cells were stimulated to proliferate by exposure either to serum or to growth factors such as EGF together with insulin and transferrin, the predominant form of the Rb protein was underphosphorylated in G1 but became hyperphosphorylated as the cells enter the G1/S boundary and the cells. These data suggest that the Rb protein is a target of the signal transduction pathway induced by serum or growth factors. Finally, senescent human fibroblast cells incapable of responding to the proliferation stimulatory effects of growth factors al
Benjamin Aaron Adler
Research Development Foundation
Wehbe′ Anne M.
LandOfFree
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