Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-02-25
2001-09-25
Zitomer, Stephanie (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S091100, C435S091500, C435S320100, C435S219000, C436S548000, C436S501000, C530S350000, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06294332
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the length of telomeres and to their effect on proliferation and senescence in cells. More particularly, the present invention relates to hnRNP A1, UP1 or derivatives thereof to maintain or alter the length of telomeres in cells. The present invention also relates to methods and compositions for increasing or decreasing the proliferative capacity of cells and to delay or precipitate the onset of senescence. The invention further relates to pharmaceutical, therapeutic and diagnostic reagents which relate to telomere length and/or to the modulations thereof.
BACKGROUND OF THE INVENTION
Telomeres are the DNA structure at the ends of the chromosomes of eukaryotes, including human, and are comprised of variable lengths of double strander repeats terminating with single-stranded repeats originally identified in yeast and protozoa (Makarov et al., 1997, Cell 88:657-666).
Review articles concerning telomeres include Greider, 1996, Ann. Rev. Biochem. 65:337 and Zakian, 1995, Science 270:1601. Relevant articles on various aspects of telomeres include Cooke and Smith, 1986, Cold Spring Harbor Symp. Quant. Biol. 51:213; Morin, 1989, Cell 59:521; Blackburn et al., 1989, Genome 31:553; Szostak, 1989, Nature (London) 337:303; Gall, 1990, Nature (London) 344:108; Henderson et al., 1990, Biochemistry 9:732; Gottschling et al., 1990, Cell 630:751; Harrington et al., 1991, Nature (London) 353:451; Muller et al., 1991, Cell §67:815; Yu et al., 1991, Cell 67:823; Gray et al., 1991, Cell 67:807; de Lange, 1995, “Telomere Dynamics and Genome Instability in Human Cancer”, E. Blackburn and C. W. Greider (eds), in Telomeres, Cold Spring Harbor Laboratory Press, pp. 265-293; Rhyu, 1995, J. Natl. Cancer Inst 87:884; Greider and Harley, 1996, “Telomeres and Telomerase in Cell Senescence and Immortalization”, in Cellular Aging and Cell Death, Wiley-Liss, Inc., pp. 123-138. Other articles of some relevance include Lundblad et al., 1989, Cell 57:633 and Yu et al., 1990, Nature (London) 344:126.
Maintenar the integrity of teloees is essential for cell suival (Muller, 1938, The Collectirg Net 13:181-195; Sandell et al., 1993, Cell 75:729-739). The proliferative potential of cells has been correlated with alterations in the length of these tandemly repeated sequences (Zakian, 1989, Ann. Rev. Genet 23:579-604; Counter et al., 1992, EMBO J. 11:1921-1929).
The finite replicative capacity of normal human cells, e.g., fibroblasts, is characterized by a cessation of proliferation in spite of the presence of serum growth factors. This cessation of replication after a maximum of 50 to 100 population doublings in vitro is referred to as cellular senescence. See, Goldstein, 1990, Science 249:1129; Hayflick and Moorehead, 1961, Exp. Cell Res. 25.585; Hayflick, 1985, ibid., 37:614; Ohno, 1979, Mech. Aging. Dev. 11:179; Ham and McKeehan, 1979, “Media and Growth Requirements”, W. B. Jacoby and I. M. Pastan (eds), in Methods of Enzymology, Academic Press, NY, 58:44-93. The replicative life span of cells is inversely proportional to the in vivo age ofthe donor (Martin et al., 1979, Lab. Invest. 23:86; Goldstein et al. 1969, Proc. Natl. Acad. Sci. USA 64:155; Schneider and Mitsui, 1976, ibid, 73:3584) and is therefore suggested to reflect in vivo ageing on a cellular level.
Cellular immortalization (unlimited life span) may be thought of as an abnormal escape from cellular senescence (Shay et al., 1991, Emp. Cell Res. 196:33). Normal human somatic cells appear to be mortal, i.e., have finite replication potential. In contrast, the germ line and malignant tumor cells are immortal (have indefinite proliferative potential). Human cells cultures in vitro appear to require the aid of transforming oncoproteins to become immortal and even then the frequency of immortalization is 10
−6
to 10
−7
(Shay et al., 1989, Emp. Cell Res. 184:109). A variety of hypotheses have been advanced over the years to explain the causes of cellular senescence. One such hypothesis proposes that the loss of telomec DNA with age, eventually triggers cell cycle exit and cellular senescence (Zakian, 1989, Ann. Rev. Genet 23:579-604; Harley et al. 1990, Nature (London) 345:458-460; Hastie et al., 1990, Nature (London) 346:866-868; Allsopp et al., 1992, Proc, Natl. Acad. Sci. USA 89:10114-10118; Counter et al., 1992, EMBO J. 11:1921-1929).
Human primary fibroblasts in culture enter crisis after a precise number of cell division associated with gradual telomere shortening, at which point all the cells die (de Lange, 1994, Proc. Natl. Acad. Sci. USA 91:2882-2885). Mouse primary fibroblasts have longer and/or more stable telomeres and display a similar behavior when cultured in vitro (Prowse and Greider, 1994, Proc. Natl. Acad. Sci. USA, 92:4818-4822). However, after crisis, primary mouse cells in culture spontaneously immortalize with a frequency of 10
−6
, possibly because longer telomeres facilitate the growth of mutant cells (de Lange, 1994, Proc. Natl. Acad. Sci. USA 91:2882-2885).
It should be noted, as mentioned above, that other hypotheses have been advanced to explain senescence and that there is yet to be a consensus or a universally accepted hypothesis therefor. Previously, the causal relationship between telomeres and cancer/ageing/senescence had been built entirely on correlative studies.
Recent data has shown that telomeres play a direct role in cell senescence and transformation. Indeed, Wright et al., 1996, EMBO J. 15:1734-1741, using telomerase-negative cells which have limited life span in tissue culture, have shown that the introduction of oligonucleotides carrying telomeric repeats causes telomere elation and increases the proliferative capacity of these cells. Moreover, the authors state that “previous studies had shown a remarkable correlation between telomere length and cellular senescence. The present results provide the first experimental evidence for a true causal relationship between telomere length and a limited proliferative capacity”. Feng et al., 1995, Science 269:1236-1241 showed that human cell line (HeLa) transfected with an antsense telomere RNA, loose telomeric DNA and begin to die after 23-26 cell doublings. The author claim that “the results support the hypothesis that telomere loss leads to crisis and cell death once telomeres are shortened to a critical length”.
The postulated link between senescence/proliferation of cells and telomere length has led to therapeutic and diagnostic methods relating to telomere length or to telomerase, the ribonucleoprotein enzyme involved in the synthesis of telomeric DNA. PCT Publication No. 93/23572 describes oligonucleotide agents that either reduce the loss of telomeric sequence during passage of cells in vitro, or increase telomeric length of immortal cells in vitro. The same type of approach is also taught in PCT Publication No. 94/13383 and U.S. Pat. No. 5,484,508 which refer to methods and compositions for the determination of telomere length and telomerase activity, as well as to methods to inhibit telomerase activity in the treatment of proliferative diseases. Methods to increase or decrease the length of telomeres through an action on telomerase is also taught. The agents which are shown to reduce telomere loss of telomere length during proliferation are oligonucleotides which promote synthesis of DNA at the telomere ends, as well as telomerase.
PCT Publication No. 95/13383 discloses a method and compositions for increasing telomeric length in normal cells so as to increase the proliferative capacity of the cells and to delay the onset of cellular senescence. PCT Publication No. 96/10035 teaches that telomere length serves as a biomarker for cell turnover. Furthermore, it discloses that measuemert of telomere length can be used to diagnose and stage cancer and other diseases as well as cell senescence.
Heterogeneous nuclear ribonucleoprotein particles (hnRNP) proteins are abundant proteins mammalian cells, of which the A to U members have been best characterized due to their RNA binding properties (Dreyfuss
Clark & Elbing LLP
Kristina Bieker-Brady
Telogene Inc.
Wilder Cynthia B.
Zitomer Stephanie
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