Senscent cell-derived inhibitors of DNA synthesis

Details Senscent cell-derived inhibitors of DNA synthesis Senscent cell-derived inhibitors of DNA synthesis

1994-10-24

2002-04-16

Kunz, Gary L. (Department: 1647)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C514S002600, C514S012200, C530S350000

Reexamination Certificate

active

06372249

ABSTRACT:

FIELD OF THE INVENTION
The present invention is in the field of recombinant DNA technology. This invention is directed to a gene sequence and a protein that effects the ability of cells to become senescent. This invention was supported with Government funds. The Government has certain rights in this invention.
BACKGROUND OF THE INVENTION
Normal human diploid cells have a finite potential for proliferative growth (Hayflick, L. et al.,
Exp. Cell Res.
25:585 (1961); Hayflick, L.,
Exp. Cell Res.
37:614 (1965)). Indeed, under controlled conditions in vitro cultured human cells can maximally proliferate only to about 80 cumulative population doublings. The proliferative potential of such cells has been found to be a function of the number of cumulative population doublings which the cell has undergone (Hayflick, L. et al.,
Exp. Cell Res.
25: 585 (1961); Hayflick, L. et al.,
Exp. Cell Res.
37: 614 (1985)). This potential is also inversely proportional to the in vivo age of the cell donor (Martin, G. M. et al.,
Lab. Invest.
23:86 (1979); Goldstein, S. et al.,
Proc. Natl. Acad. Sci.
(U.S.A.) 64:155 (1969); Schneider, E. L.,
Proc. Natl. Acad. Sci.
(U.S.A.) 73:3584 (1976); LeGuilty, Y. et al.,
Gereontologia
19:303 (1973)).
Cells that have exhausted their potential for proliferative growth are said to have undergone “senescence.” Cellular senescence in vitro is exhibited by morphological changes and is accompanied by the failure of a cell to respond to exogenous growth factors. Cellular senescence, thus, represents a loss of the proliferative potential of the cell. Although a variety of theories have been proposed to explain the phenomenon of cellular senescence in vitro, experimental evidence suggests that the age-dependent loss of proliferative potential may be the function of a genetic program (Orgel, L. E.,
Proc. Natl. Acad. Sci.
(U.S.A.) 49:517 (1963); De Mars, R. et al.,
Human Genet.
16:87 (1972); M. Buchwald, Mutat. Res. 44:401 (1977); Martin, G. M. et al.,
Amer. J. Pathol.
74:137 (1974); Smith, J. R. et al.,
Mech. Age. Dev.
13:387 (1980); Kirkwood, T. B. L. et al.,
Theor. Biol.
53:481 (1975).
Cell fusion studies with human fibroblasts in vitro have demonstrated that the quiescent phenotype of cellular senescence is dominant over the proliferative phenotype (Pereira-Smith, O. M et al.,
Somat. Cell Genet.
8:731 (1982); Norwood, T. H. et al., Proc.
Natl. Acad. Sci.
(U.S.A.) 71:223 (1974); Stein, G. H. et al.,
Exp. Cell Res.
130:155 (1979)).
Insight into the phenomenon of senescence has been gained from studies in which senescent and young (i.e. non-senescent) cells have been fused to form heterodikaryons. In order to induce senescence in the “young” nucleus of the heterodikaryon (as determined by an inhibition in the synthesis of DNA), protein synthesis must occur in the senescent cell prior to fusion (Burmer, G. C. et al.,
J. Cell. Biol.
94:187 (1982); Drescher-Lincoln, C. K. et al.,
Exp. Cell Res.
144:455 (1983); Burner, G. C. et al.,
Exp. Cell Res.
145:708 (1983); Drescher-Lincoln, C. K. et al.,
Exp. Cell Res.
153:208 (1984).
Likewise, microinjection of senescent fibroblast mRNA into young fibroblasts has been found to inhibit the ability of the young cells to synthesize DNA (Lumpkin, C. K. et al.,
Science
232:393 (1986)). Researchers have identified unique mRNAs that are amplified in senescent cells in vitro (West, M. D. et al.,
Exp. Cell Res.
184:138 (1989); Giordano, T. et al.,
Exp. Cell Res.
185:399 (1989)).
The human diploid endothelial cell presents an alternative cell type for the study of cellular senescence because such cells mimic cellular senescence in vitro (Maciag, T. et al.,
J. Cell. Biol.
91:420 (1981); Gordon, P. B. et al.,
In Vitro
19:661 (1983); Johnson, A. et al.,
Mech Age. Dev.
18:1 (1982); Thornton, S. C. et al.,
Science
222:623 (1983); Van Hinsbergh, V. W. M. et al.,
Eur. J. Cell Biol.
42:101 (1986); Nichols, W. W. et al.,
J. Cell. Physiol.
132:453 (1987)).
In addition, the human endothelial cell is capable of expressing a variety of functional and reversible phenotypes. The endothelial cell exhibits several quiescent and non-terminal differentiation phenotypes (Folkman, J. et al.,
Nature
288:551 (1980); Maciag, T. et al.,
J. Cell Biol.
94:511 (1982); Madri. J. A. et al.,
J. Cell Biol.
97:153 (1983); Montesano, R.,
J. Cell Biol.
99:1706 (1984); Montesano, R. et al.,
J. Cell Physiol.
34:460 (1988)).
It has been suggested that the pathway of human cell differentiation in vitro involves the induction of cellular quiescence mediated by cytokines that inhibit growth factor-induced endothelial cell proliferation in vitro (Jay, M. et al.,
Science
228:882 (1985); Madri, J. A. et al.,
In Vitro
23:387 (1987); Kubota, Y. et al.,
J. Cell Biol.
107:1589 (1988); Ingber, D. E. et al.,
J. Cell Biol.
107:317 (1989)).
Inhibitors of endothelial cell proliferation also function as regulators of immediate-early transcriptional events induced during the endothelial cell differentiation in vitro, which involves formation of the capillary-like, tubular endothelial cell phenotype (Maciag, T., In: Imp. Adv. Oncol. (De Vita, V. T. et al., eds., J. B. Lippincott. Philadelphia, 42 (1990); Goldgaber, D. et al.,
Proc. Natl. Acad. Sci. (U.S.A.)
86:7606 (1990); Hla, T. et al.,
Biochem. Biophys. Res. Commun.
167:637 (1990)). The inhibitors of cell proliferation include:
1. Interleukin-1a (IL-1a) (Montesano, R. et al.,
J. Cell Biol.
99:1706 (1984); Montesano, R. et al.,
J. Cell Physiol.
122:424 (1985); Maciag, T. et al. (
Science
249:1570-1574 (1990));
2. Tumor necrosis factor (Frater-Schroder, M. et al.,
Proc. Natl. Acad. Sci. (U.S.A.)
84:5277 (1987); Sato, N. et al.,
J. Natl. Cancer Inst.
76:1113 (1986); Pber, J. P., Amer. J. Pathol. 133:426 (1988); Shimada, Y. et al.,
J. Cell Physiol.
142:31 (1990));
3. Transforming growth factor-&bgr; (Baird, A. et al.,
Biochem. Biophys. Res. Commun.
138:476 (1986); Mullew, G. et al.,
Proc. Natl. Acad. Sci.
(U.S.A.) 84:5600 (1987); Mairi, J. A. et al.,
J. Cell Biol.
106:1375 (1988));
4. Gamma-interferon (Friesel, R. et al.,
J. Cell Biol.
104:689 (1987); Tsuruoka, N. et al.,
Biochem. Biophys. Res. Commun.
155:429 (1988)) and
5. The tumor promoter, phorbol myristic acid (PMA) (Montesano, R. et al.,
Cell
42:469 (1985); Doctrow, S. R. et al.,
J. Cell Biol.
104:679 (1987); Montesano, R. et al.,
J. Cell. Physiol.
30 130:284 (1987); Hoshi, H. et al.,
FASAB J.
2:2797 (1988)).
The prospect of reversing senescence and restoring the proliferative potential of cells has implications in many fields of endeavor. Many of the diseases of old age are associated with the loss of this potential. Restoration of this ability would have far-reaching implications for the treatment of this disease, of other age-related disorders, and, of aging per se.
In addition, the restoration of proliferative potential of cultured cells has uses in medicine and in the pharmaceutical industry. The ability to immortalize nontransformed cells can be used to generate an endless supply of certain tissues and also of cellular products.
The significance of cellular senescence has accordingly been appreciated for several years (Smith, J. R., Cellular Ageing, In:
Monographs in Developmental Biology,
Sauer, H. W. (Ed.), S. Karger, New York, N.Y. 17:193-208 (1984); Smith, J. R. et al.
Exper. Gerontol.
24:377-381 (1989), herein incorporated by reference). Researchers have attempted to clone genes relevant to cellular senescence. A correlation between the existence of an inhibitor of DNA synthesis and the phenomenon of cellular senescence has been recognized (Spiering, A. I. et al,
Exper. Cell Res.
179:159-167 (1988); Pereira-Smith, O. M. et al.,
Exper. Cell Res.
160:297-306 (1985); Drescher-Lincoln, C. K. et al.,
Exper. Cell Res.
153:208-217 (1984); Drescher-Lincoln, C. K. et al.,
Exper. Cell Res.
144:455-462 (1983)). Moreover, the relative abundance of certain senescence-associated RNA molecules has been identified (Lumpkin, C. K. et al.,
Science
232:393-395 (1986)).
Several labora

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