James R. Smith

Replicative senescence of human skin fibroblasts correlates with a loss of regulation and overexpression of collagenase activity

The atrophy of extracellular matrix is a common event during the aging of connective tissues. In this study, we tested the hypothesis that the altered ability of senescent cells to be modulated by serum growth factors correlated with a loss of regulation of collagenase synthesis. We examined the levels of immunoreactive procollagenase and collagenase inhibitor (the tissue inhibitor of metalloproteinases, TIMP) associated with young and senescent fibroblasts cultured in vitro. Young fibroblasts cultured in low (0.5%) concentrations of fetal bovine serum respond to increased (10%) serum by increasing levels of procollagenase and TIMP beginning 4.0 h after serum stimulation. In contrast, senescent fibroblasts constitutively produce relatively high levels of procollagenase even when cultured in low levels of serum and do not respond to serum stimulation by increasing procollagenase synthesis. In addition, senescent fibroblasts constitutively express a relatively small amount of TIMP which is not induced upon serum stimulation. This altered expression of collagenase and TIMP appears unique to the senescent phenotype and not merely a result of growth inhibition, since young cells growth arrested by density-dependent growth inhibition displayed a temporal pattern of procollagenase and TIMP expression upon serum stimulation similar to that of subconfluent young cultures. An assay of net collagenase activity revealed a greater than 20-fold elevation of activity in trypsin-activated extracts from senescent versus young fibroblasts when cultured in a low concentration of fetal bovine serum. These results demonstrate for the first time a direct correlation between alterations in the molecular pathways regulating connective tissue homeostasis and those of replicative senescence. The increased collagenolytic activity of senescent compared to young fibroblasts cultured in the presence of a low serum concentration suggests that aging fibroblasts may become increasingly fibroclastic causing many of the age-associated alterations in dermal collagen observed during aging in vivo.

Molecular Basis for Impaired Muscle Differentiation in Myotonic Dystrophy

ABSTRACT Differentiation of skeletal muscle is affected in myotonic dystrophy (DM) patients. Analysis of cultured myoblasts from DM patients shows that DM myoblasts lose the capability to withdraw from the cell cycle during differentiation. Our data demonstrate that the expression and activity of the proteins responsible for cell cycle withdrawal are altered in DM muscle cells. Skeletal muscle cells from DM patients fail to induce cytoplasmic levels of a CUG RNA binding protein, CUGBP1, while normal differentiated cells accumulate CUGBP1 in the cytoplasm. In cells from normal patients, CUGBP1 up-regulates p21 protein during differentiation. Several lines of evidence show that CUGBP1 induces the translation of p21 via binding to a GC-rich sequence located within the 5′ region of p21 mRNA. Failure of DM cells to accumulate CUGBP1 in the cytoplasm leads to a significant reduction of p21 and to alterations of other proteins responsible for the cell cycle withdrawal. The activity of cdk4 declines during differentiation of cells from control patients, while in DM cells cdk4 is highly active during all stages of differentiation. In addition, DM cells do not form Rb/E2F repressor complexes that are abundant in differentiated cells from normal patients. Our data provide evidence for an impaired cell cycle withdrawal in DM muscle cells and suggest that alterations in the activity of CUGBP1 causes disruption of p21-dependent control of cell cycle arrest.

Identification of the active region of the DNA synthesis inhibitory gene p21Sdi1/CIP1/WAF1.

The cloning of the negative growth regulatory gene, p21Sdi1, has led to the convergence of the fields of cellular senescence, cell cycle regulation and tumor suppression. This gene was first cloned as an inhibitor of DNA synthesis that was overexpressed in terminally non‐dividing senescent human fibroblasts (SD11) and later as a p53 transactivated gene (WAF1) and a Cdk‐interacting protein (CIP1, p21) that inhibited cyclin‐dependent kinase activity. To identify the active region(s) of p21Sdi1, cDNA constructs encoding various deleted forms of the protein were analyzed. Amino acids 22‐71 were found to be the minimal region required for DNA synthesis inhibition. Amino acids 49‐71 were involved in binding to Cdk2, and constructs deleted in this region expressed proteins that were unable to inhibit Cdk2 kinase activity in vitro. The latter stretch of amino acids shared sequence similarity with amino acids 60‐76 of the p27Kip1 protein, another Cdk inhibitor. Point mutations made in p21Sdi1 in this region confirmed that amino acids common to both proteins were involved in DNA synthesis inhibition. Additionally, a chimeric protein, in which amino acids 49‐65 of p21Sdi1 were substituted with amino acids 60‐76 of p27Kip1, had almost the same DNA synthesis inhibitory activity as the wild‐type protein. The results indicate that the region of sequence similarity between p21Sdi1 and p27Kip1 encodes an inhibitory motif characteristic of this family of Cdk inhibitors.

Identification of a Gene That Reverses the Immortal Phenotype of a Subset of Cells and Is a Member of a Novel Family of Transcription Factor-Like Genes

ABSTRACT Based on the dominance of cellular senescence over immortality, immortal human cell lines have been assigned to four complementation groups for indefinite division. Human chromosomes carrying senescence genes have been identified, including chromosome 4. We report the cloning and identification of a gene, mortality factor 4 (MORF 4), which induces a senescent-like phenotype in immortal cell lines assigned to complementation group B with concomitant changes in two markers for senescence. MORF 4 is a member of a novel family of genes with transcription factor-like motifs. We present here the sequences of the seven family members, their chromosomal locations, and a partial characterization of the three members that are expressed. Elucidation of the mechanism of action of these genes should enhance our understanding of growth regulation and cellular aging.

Isolation of a cDNA that hybrid selects antiproliferative mRNA from rat liver

Studies of chromosome loss in inherited cancers, of fusions between proliferating and quiescent cells, and of microinjection of RNA from quiescent cells into proliferation competent cells have all provided evidence for antiproliferative genes in mammalian cells. In this report, we describe a partial cDNA clone isolated on the basis of its preferential hybridization to RNA from normal versus regenerating rat liver. The corresponding mRNA, enriched by hybrid selection, was microinjected into normal human diploid fibroblasts in cell culture, resulting in a 53% decrease in the fraction of nuclei incorporating tritiated thymidine. This mRNA is 2 kb in size and is expressed in eight tissues examined.

Competition of CUGBP1 and calreticulin for the regulation of p21 translation determines cell fate

Induction of p21 in senescent human fibroblasts plays a key role in the inactivation of cyclin‐dependent kinases and the resulting irreversible growth arrest in the early stages of cell senescence. We found that RNA‐binding proteins are critical regulators of p21 during senescence. Two RNA‐binding proteins, CUGBP1 and calreticulin (CRT), interact with the same nucleotide sequences within the 5′ region of p21 mRNA, but have opposite effects on the translation of p21 mRNA. CUGBP1 increases translation of p21 mRNA, whereas CRT blocks translation of p21 via stabilization of a stem–loop structure within the 5′ region of the p21 mRNA. CUGBP1 and CRT compete for binding to p21 mRNA and thereby the regulation of p21 translation. In senescent fibroblasts, CUGBP1 displaces CRT from the p21 mRNA and releases CRT‐dependent repression of p21 translation leading to growth arrest and development of a senescent phenotype. These data present evidence that competition between RNA‐binding proteins for the regulation of p21 translation determines cell fate.

Fibronectin expression increases during in vitro cellular senescence: Correlation with increased cell area

Several changes in the functional characteristics of fibronectin have been noted as cells become senescent in culture. In this report we show that steady state levels of both fibronectin mRNA and protein increase significantly during the process of cellular aging. The greatest change in the proportion of cells expressing high levels of fibronectin occurs near the end of a cultures proliferative potential. The proportion of cells unable to synthesize DNA has previously been shown to follow a similar pattern. We also found that increasing cell size correlates closely with higher levels of fibronectin expression. Thus, there is a clear correlation between increased fibronectin mRNA content and in vitro cellular senescence. It remains to be determined whether the change in fibronectin production is a contributing cause or a result of in vitro cellular senescence.

Senescent and quiescent cell inhibitors of DNA synthesis: Membrane-associated proteins☆

Cytoplasts derived from senescent and quiescent human diploid cells inhibit DNA synthesis initiation when fused with cells capable of proliferation. When the cytoplasts were subjected to a variety of conditions (trypsin and cycloheximide treatment and growth on fibronectin), this inhibitory activity was lost, suggesting that the inhibitors involved were proteins associated with the surface membranes of the cells. We have studied the quiescent cell inhibitor in greater detail and determined that surface membrane-enriched preparations isolated from quiescent cells and proteins extracted from these membrane preparations have DNA synthesis-inhibitory activity.

MRG15 regulates embryonic development and cell proliferation

ABSTRACT MRG15 is a highly conserved protein, and orthologs exist in organisms from yeast to humans. MRG15 associates with at least two nucleoprotein complexes that include histone acetyltransferases and/or histone deacetylases, suggesting it is involved in chromatin remodeling. To study the role of MRG15 in vivo, we generated knockout mice and determined that the phenotype is embryonic lethal, with embryos and the few stillborn pups exhibiting developmental delay. Immunohistochemical analysis indicates that apoptosis in Mrg15 − / − embryos is not increased compared with wild-type littermates. However, the number of proliferating cells is significantly reduced in various tissues of the smaller null embryos compared with control littermates. Cell proliferation defects are also observed in Mrg15 − / − mouse embryonic fibroblasts. The hearts of the Mrg15 − / − embryos exhibit some features of hypertrophic cardiomyopathy. The increase in size of the cardiomyocytes is most likely a response to decreased growth of the cells. Mrg15 − / − embryos appeared pale, and microarray analysis revealed that α-globin gene expression was decreased in null versus wild-type embryos. We determined by chromatin immunoprecipitation that MRG15 was recruited to the α-globin promoter during dimethyl sulfoxide-induced mouse erythroleukemia cell differentiation. These findings demonstrate that MRG15 has an essential role in embryonic development via chromatin remodeling and transcriptional regulation.

The Genetics of Cellular Senescence

the reversible quiescent state that some young cells undergo in the absence of growth factors. The role of replicative senescence in aging of the organism remains controversial, but correlative evidence suggests that it is an in vitro manifestation of an in vivo phenomenon. First, the replicative capacity of cells cultured from old donors is less than that of young donors, suggesting that cells keep track of the number of cell doublings that they have undergone in vivo. Second, the replicative capacity of cells is roughly proportional to the maximum life span of the animals from which they originated. A third line of evidence comes from the study of diseases, such as Werner syndrome (WS), that mimic premature aging. Fibroblasts obtained from WS patients senesce much earlier than normal age-matched controls (Norwood et al. 1979). The WS gene (WRN) has recently been cloned and displays homology to some DNA helicases, although all mutations thus far identified map outside the helicase region, suggesting other possible functions for this protein (Yu et al. 1996). Two predominant models have been suggested to explain the loss of proliferation in senescence. The first proposes that cellular aging results from the accumulation of errors that occur, perhaps, as a result of either impaired DNA repair mechanisms or inadequate free radical‐scavenging abilities. Thus, the cell is seen as passive in the aging process, although it may be able to regulate the process by repairing or removing damaged cellular components. The second model proposes that cells age according to an intrinsic genetic program and, hence, suggests that cells are active participants in their own aging. The majority of the data described below implicate genetic factors in the activation of cellular senescence.