Abnormal expression of p27kip1 protein in levator ani muscle of aging women with pelvic floor disorders – a relationship to the cellular differentiation and degeneration

BACKGROUND: Pelvic floor disorders affect almost 50% of aging women. An important role in the pelvic floor support belongs to the levator ani muscle. The p27/kip1 (p27) protein, multifunctional cyclin-dependent kinase inhibitor, shows changing expression in differentiating skeletal muscle cells during development, and relatively high levels of p27 RNA were detected in the normal human skeletal muscles. METHODS: Biopsy samples of levator ani muscle were obtained from 22 symptomatic patients with stress urinary incontinence, pelvic organ prolapse, and overlaps (age range 38–74), and nine asymptomatic women (age 31–49). Cryostat sections were investigated for p27 protein expression and type I (slow twitch) and type II (fast twitch) fibers. RESULTS: All fibers exhibited strong plasma membrane (and nuclear) p27 protein expression. cytoplasmic p27 expression was virtually absent in asymptomatic women. In perimenopausal symptomatic patients (ages 38–55), muscle fibers showed hypertrophy and moderate cytoplasmic p27 staining accompanied by diminution of type II fibers. Older symptomatic patients (ages 57–74) showed cytoplasmic p27 overexpression accompanied by shrinking, cytoplasmic vacuolization and fragmentation of muscle cells. The plasma membrane and cytoplasmic p27 expression was not unique to the muscle cells. Under certain circumstances, it was also detected in other cell types (epithelium of ectocervix and luteal cells). CONCLUSIONS: This is the first report on the unusual (plasma membrane and cytoplasmic) expression of p27 protein in normal and abnormal human striated muscle cells in vivo. Our data indicate that pelvic floor disorders are in perimenopausal patients associated with an appearance of moderate cytoplasmic p27 expression, accompanying hypertrophy and transition of type II into type I fibers. The patients in advanced postmenopause show shrinking and fragmentation of muscle fibers associated with strong cytoplasmic p27 expression

Evidence that exposure of the telomere 3′ overhang sequence induces senescence

Normal human cells cease proliferation after a finite number of population doublings, a phenomenon termed replicative senescence. This process, first convincingly described by Hayflick and Moorhead [Hayflick, L. & Moorhead, P. S. (1961) Exp. Cell Res. 25, 595–621] for cultured human fibroblasts 40 years ago, is suggested to be a fundamental defense against cancer. Several events have been demonstrated to induce the senescent phenotype including telomere shortening, DNA damage, oxidative stress, and oncogenic stimulation. The molecular mechanisms underlying senescence are poorly understood. Here we report that a 1-week exposure to oligonucleotide homologous to the telomere 3′-overhang sequence TTAGGG (T-oligo) similarly specifically induces a senescent phenotype in cultured human fibroblasts, mimicking serial passage or ectopic expression of a dominant negative form of the telomeric repeat binding factor, TRF2(DN). We propose that exposure of the 3′ overhang due to telomere loop disruption may occur with critical telomere shortening or extensive acute DNA damage and that the exposed TTAGGG tandem repeat sequence then triggers DNA-damage responses. We further demonstrate that these responses can be induced by treatment with oligonucleotides homologous to the overhang in the absence of telomere disruption, a phenomenon of potential therapeutic importance

Telomeres Shorten during Aging of Human Fibroblasts

The terminus of a DNA helix has been called its Achilles' heel. Thus to prevent possible incomplete replication and instability of the termini of linear DNA, eukaryotic chromosomes end in characteristic repetitive DNA sequences within specialized structures called telomeres. In immortal cells, loss of telomeric DNA due to degradation or incomplete replication is apparently balanced by telomere elongation, which may involve de novo synthesis of additional repeats by novel DNA polymerase called telomerase. Such a polymerase has been recently detected in HeLa cells. It has been proposed that the finite doubling capacity of normal mammalian cells is due to a loss of telomeric DNA and eventual deletion of essential sequences. In yeast, the est1 mutation causes gradual loss of telomeric DNA and eventual cell death mimicking senescence in higher eukaryotic cells. Here, we show that the amount and length of telomeric DNA in human fibroblasts does in fact decrease as a function of serial passage during ageing in vitro and possibly in vivo. It is not known whether this loss of DNA has a causal role in senescence

Requirement for p27(KIP1) in Retinoblastoma Protein-Mediated Senescence

In vivo and in vitro evidence indicate that cells do not divide indefinitely but instead stop growing and undergo a process termed cellular proliferative senescence. Very little is known about how senescence occurs, but there are several indications that the retinoblastoma protein (pRb) is involved, the most striking being that reintroduction of RB into RB(−/−) tumor cell lines induces senescence. In investigating the mechanism by which pRb induces senescence, we have found that pRb causes a posttranscriptional accumulation of the cyclin-dependent kinase inhibitor p27(KIP1) that is accompanied by an increase in p27(KIP1) specifically bound to cyclin E and a concomitant decrease in cyclin E-associated kinase activity. In contrast, pRb-related proteins p107 and p130, which also decrease cyclin E-kinase activity, do not cause an accumulation of p27(KIP1) and induce senescence poorly. In addition, the use of pRb proteins mutated in the pocket domain demonstrates that pRb upregulation of p27(KIP1) and senescence induction do not require the interaction of pRb with E2F. Furthermore, ectopic expression of p21(CIP1) or p27(KIP1) induces senescence but not the morphology change associated with pRb-mediated senescence, uncoupling senescence from the morphological transformation. Finally, the ability of pRb to maintain cell cycle arrest and induce senescence is reversibly abrogated by ablation of p27(KIP1) expression. These findings suggest that prolonged cell cycle arrest through the persistent and specific inhibition of cdk2 activity by p27(KIP1) is critical for pRb-induced senescence