Invited Symposium: Molecular Mechanisms of Ageing
Senescent growth arrest is a common phenomena observed in a variety of human and animal somatic cells when they are put into culture, and thus the majority of these cells have a finite proliferative potentials (1).Currently, the causative mechanisms for growth arresting signal transduction in sensecent cell is unclear, butĘcandidates for the actual growth arresting factor has been discoverd. We and others isolated and determined the senescent cell inhibitors of cell growth (2,3). The effector molecules are the G1 Cyclin/Cdk inhibiting small proteins that overexpress in senescent cells. We here show the approaches to isolate senescent inhibitors that could be induced or activated during serial proliferation of normal cells. This strategy may also be applied for the isolation of the upstream regulators of the inhibitor expression in senescent cells. The identification of such regulatory factor would clarify the possible molecular link between the inhibitor expression and division counting "clock", e.g. telomerase shortening or decrease of genomic DNA methylations or any other unknown irreversible changes gradually progress in cell aging process.
Materials and Methods
Isolation of sensecent inhibitors by expression screening.
Fig.1:Cloning strategy for senescent inhibitor gene.
The percentage inhibiton of DNA synthesis was calculated by the formula [(% labeled nuclei in cells transfected with control plasmid - % labeled nuclei in cells transfected with sensecent cell-derived inhibitors (sdi) plasmid) / % labeled nuclei in cells transfected with control plasmid] X 100. In this way, DNA synthesis activities in b-gal positive cells were determined, and cDNA pools that showed growth inhibitory was further divied into individual plasmid. Finally, candidate cDNA clones (senescent inhibitor of DNA synthesis) were isolated, and their sequences were analyzed.
Northen blot and protein expression analyses.
Northern analysis was performed by standard methods using 1 micro g of polyA+ RNA isolated from young and senescent cells. b-actin or GAPDH expression was also examined as an internal controls for northern analysis. Furthermore, the correlation between p21(sdi1) expression and cell PDs in culture was also analyzed.
Microinjection and antisense expression.
Nuclear microinjection of the isolated plasmids into young cycling or sensecent cells were performed. The plasmid combinations were either pCMVb + pcDSRaD-sdi1(p21) ,or pCMVb + pcDSRaD (control).In each expreriment, 50 micro g/ml of each plasmid was injected. sdi-1(p21) antisense expression construct (pcDSRaD-anti-sdi1) was also microinjected into young and sensecent cells.
Results and Discussion
Cloning strategy for senescent cell inhibitors.
Fig. 2: Sense and anti-sense sdi gene transfections to young cycling cells.
(left) Sense- and antisense- sdi1 expression plasmid (pcDSRaD-sdi1 or pcDSRaD-anti-sdi1) and pCMVb were co-transfected into young cells and % inhibition of DNA synthesis was examined. Lanes 1: pcDSRaD-cat, 2: pcDSRaD-sdi1. 3: pcDSRaD-anti-sdi1, 4: pcDSRaD-sdi2, 5: pcDSRaD-anti-sdi2.(right) Autoradiograph of the sdi1 transfections. A: transfection of control plasmid, B: pcDSRaD-sdi1 transfection. Blue cells (b-gal positive) containing nuclear silver grain indicate DNA synthsis after transfection.
Fig. 2 shows innhibitory effects of sdi-1 and sdi-2 sense and anti-sense transfections to young cycling cells. Overexpression of sdi sequence resulted strong inhibition of young cell growth, whereas that of anti-sense expression showed no inhibition or enhancement. No inhibition was observed in pcDSRaD-cat transfection, indicating that inhibition by sdi was not caused by any full-length cDNA expressed at a high level: not from the overexpression artifacts in the cells. The same results were obtained by microinjection experiments.
Sdi1 expression during cellular senescence.
Changes in sdi1 mRNA expression during cellular senescence was examined by northern analysis. Using full-length cDNA as a probe, we observed the 2.1 kb sdi1 message increased about 20-fold during the in vitro life span of the cells (Fig. 3). While the increase was gradual, the major change in mRNA level occurred during the final few population doublings, correlating closely with expression of senescent phenotype and loss of proliferative potential.
Fig. 3: Sdi1 expression during cellular senescence.
(A) Northern anaysis of sdi1 expression in young and senescent cells. (B) Relative amount of sdi1 expression during in vitro life span.
Role of sdi1 in senescent growth arrest.
Fig. 4 summarizes current understanding of the role of sdi1 in senescent growth arrest. Sdi1 protein appeared to be identical to p21 Cyclin/Cdk inhibitor (4). It could act to inhibit G1 Cyclin/Cdk complexes, therby preventing phosphorylation of the retinoblastoma tumor supressor protein (RB1), which is known tobe hypophosphorylated in senescent cells. This in turn would result in sequestration of E2F transcription factors by hypophosphorylated RB1 and therefore lack of activation of many genes that act during Go/G1 to S transition (5). In fact, knock out experiments of p21 gene revealed that the cells lacking p21 bypassed senescence and continued to replicate (6). However, the cells underwent crisis after 20-30 PD extension, indicating that inactivation of p21 was not a sole requisite for cell immortalization.
Fig. 4.: Changes of cell cycle regulatory gene expression in growth stimulated senescent cells as compared to growth stimulated young prolifelation competent cells.
Signals that cause senescent growth arrest and senescent phenotype.
Changes in many gene expression during cellular aging process has been reported. Theses include extra matrix components (5), cell cycle reguratory genes (7), and transcription factors. p16(ink4), another Cdk inhibitor, is also upregulated in senescent cells (3,8). p21 and p16 could be a direct growth inhibitor whereas others might be a regulator ,or they might be expressed as a result of senescent phenotype.
Fig. 5.: Senescence signals in cell aging.
Identification of signal transducing proteins that induce or activate p21 or p16 might reveal possible link between actual growth arrest and gradual irreversible genome changes, shortening of telomere or decrease in DNA methylation, during cell aging.
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|Noda, A; (1998). Molecular Mechanisms of Cellular Aging and Senescent Growth Arrest.. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/higuchi/noda0326/index.html|
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