Senescence responsive transcriptional element

#This invention relates to the field of regulation of gene expression.

#Animal cells undergo replicative senescence--age-dependent decrements in function and integrity. One aspect of replicative senescence is that cells have a finite replicative life-span. After undergoing a limited number of mitotic divisions, they can no longer divide. Upon replicative senescence, cells stably arrest growth with a G1 DNA content, irreversibly losing the ability to enter S phase in response to physiologic mitogens. Another aspect of replicative senescence is that senescent cells acquire altered functions, resembling terminally differentiated cells. A third aspect of replicative senescence is that senescent cells acquire resistance to apoptotic cell death.

#Senescent cells exhibit altered gene expression compared with pre-senescent cells. In particular, several genes increase expression. For example, senescent human fibroblasts show a marked increase in interstitial collagenase mRNA and protein. Burke et al., Exp. Gerontol., (1994) 29:37-53. Senescent cells also exhibit increased expression of stromelysin, the adhesion molecule ICAM, and certain inflammatory mediators, such as IL-1. Other genes, such as certain mitogen inducible genes and IL-6, are repressed in senescent cells. Altered gene expression manifests itself at the tissue and organismic level.

#FIG. 1: Human interstitial collagenase is over-expressed in senescent human fibroblasts (WI-38) due to increased transcription initiation. The 3470 bp collagenase promoter driving the luciferase reporter (pGL-3470) was transfected into pre-senescent and senescent cells and reporter activity was measured 72 h later. Senescent cells expressed 15- to 20-fold more luciferase activity than pre-senescent cells. The promoter-less reporter vector (pGL3) was inactive in both cell types.

#FIG. 2: The region between -35 and -100 (1=start of transcription) accounts for the largest increase in promoter activity in senescent cells. Several 5' deletions in the collagenase promoter in pGL-3470 were made, and the deletion vectors were transfected into pre-senescent and senescent cells. Deletion up to -134 resulted in a 3-4 fold reduction in promoter activity in senescent cells; deletion up to -34 resulted in a 20- to 25-fold reduction in promoter activity in senescent cells. All deletion constructs showed 10- to 15-fold less activity in pre-senescent cells.

#FIG. 3: The promoter region -35 to -134 contains the major determinant of the enhanced transcription in senescent cells. Deletion of this region (PGL 522.DELTA.34/134 construct) resulted in a drastic decrease in the promoter activity in senescent cells.

#Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2d ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

#"Polynucleotide" refers to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids ("PNAs"), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. "Nucleic acid" typically refers to large polynucleotides. "Oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T."

#Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5'-end of the RNA transcript are referred to as "upstream sequences"; sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."