Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-27
2002-03-19
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S004000
Reexamination Certificate
active
06358687
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 specifically, it concerns the ability of hnRNP A1 and its shortened derivative UP1 to alter the length of telomeres in cells. More precisely, the invention relates to the ability of A1/UP1 to bind telomerase RNA, to bind and to protect mammalian telomeric DNA, and to modulate telomere extension and replication. Finally, the present invention relates to agents which can interfere with the binding of A1/UP1 to telomeres and telomerase, and to the use of protection, extension and replication assays to measure the biological impact of these agents.
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-stranded repeats terminating with single-stranded G-rich repeats originally identified in yeast and protozoa (McElligot and Wellinger, 1997, EMBO J. 16:3705).
Review articles concerning telomeres include Greider, 1996, (Ann. Rev. Biochem. 65:337). Relevant articles on various aspects of telomeres include 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. Nati. 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. Thus, telomeres are involved in the maintenance of chromosome structure and function. Furthermore, it appears that loss of telomeric DNA activates cellular processes involved in the detection and control of DNA damage, and affects cellular proliferation and senescence.
Maintenance of the integrity of telomeres is essential for cell survival (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 (Counter et al., 1992, EMBO J. 11:1921-1929). In addition, maintenance of telomere length and the regulation thereof are essential, pluripotent cellular functions as they are involved in the transmission of genetic information to daughter cells, senescence, cell growth and cancer (Blackburn, 1992,
Annu. Rev. Biochem.
61:113-129).
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. The replicative life span of cells is inversely proportional to the in vivo age of the donor (Martin et al., 1979, Lab. Invest. 23:86) 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. 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, Exp. 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 telomeric DNA with age, eventually triggers cell cycle exit and cellular senescence (Harley et al. 1990, Nature (London) 345:458-460; 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. 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 elongation 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 a human cell line (HeLa) transfected with an antisense telomerase RNA, looses telomeric DNA and begins to die after 23-26 cell doublings. The authors 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 telomerase is part of a multi-component ribonucleoprotein complex. The RNA component of the human telomerase ribonucleoprotein has been identified. The catalytic protein subunit has recently been cloned (Nakamura et al., 1997, Science 277:955).
More recent advances have confirmed the role of telomeres in cell senescence. Overexpression of the catalytic protein component of telomerase can lead to telomere elongation and extension of the proliferative capacity of telomerase-negative fibroblasts in culture (Bodnar et al. 1998, Science 279:349). Overexpression of this protein also prevents the accelerated ageing of human fibroblasts derived from patients with Werner syndrome (Wyllie et al. 2000, Nat. Genet.). Mice and murine ES cells that do not express telomerase RNA show telomere shortening and become impaired in long-term viability (Lee et al. 1998, Nature 392:569; Niida et al. 1998, Nat. Genet. 19:203). Recent studies have also supported the role of telomeres in cellular transformation. The expression of a catalytically inactive form of telomerase or the inactivation of telomerase RNA in human immortal and cancer cell lines promotes telomere shortening, growth arrest and cell death (Hahn et al. 1999, Nat. Med. 5:1164; Herbert et al. 1999, Proc. Natl. Acad. Sci. USA 96:14276; Zhang et al. 1999, Genes Dev. 13:2388).
The length of telomeres and cell viability can also be affected by proteins that bind to vertebrate telomeres. TRF1 and TRF2 are proteins that bind to double-stranded telomeric repeats. Overexpression of TRF1 promotes telomere shortening (van Steensel and de Lange 1997, Nature 385:740). Expression of a dominant negative version of TRF2 promotes end-to-end fusion of chromosomes, an event which leads to p53-dependent cell death by apoptosis (van Steensel et al. 1998, Cell 92:401; Karlseder et al. 1999, Science 283:1321).
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 tel
Chabot Benoit
Wellinger Raymund
Merchant & Gould P.C.
Myers Carla J.
Telogene Inc.
LandOfFree
Methods for monitoring the binding of A1/UP1 to... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods for monitoring the binding of A1/UP1 to..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods for monitoring the binding of A1/UP1 to... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2873464