Olivia M. Pereira-Smith

A role for both RB and p53 in the regulation of human cellular senescence

We present evidence for the possible involvement of both the RB and p53 proteins in the regulation of cellular senescence. Human fibroblasts immortalized with an inducible SV40 T-antigen become senescent following the de-induction of T-antigen. Plasmids expressing an alternative source of intact T-antigen restore proliferation but T-antigen deletion mutants lacking either the RB or p53 binding domains are unable to do so. Similarly, combinations of adenovirus E1A + E1B or human papillomavirus E6 + E7 genes are able to replace T-antigen functions and permit cell proliferation, whereas the individual genes do not. These results are discussed in terms of a two-stage model for the escape from in vitro cellular senescence.

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.

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.

A gene involved in control of human cellular senescence on human chromosome 1q.

Normal cells in culture exhibit limited division potential and have been used as a model for cellular senescence. In contrast, tumor-derived or carcinogen- or virus-transformed cells are capable of indefinite division. Fusion of normal human diploid fibroblasts with immortal human cells yielded hybrids having limited life spans, indicating that cellular senescence was dominant. Fusions of various immortal human cell lines with each other led to the identification of four complementation groups for indefinite division. The purpose of this study was to determine whether human chromosome 1 could complement the recessive immortal defect of human cell lines assigned to one of the four complementation groups. Using microcell fusion, we introduced a single normal human chromosome 1 into immortal human cell lines representing the complementation groups and determined that it caused loss of proliferative potential of an osteosarcoma-derived cell line (TE85), a cytomegalovirus-transformed lung fibroblast cell line (CMV-Mj-HEL-1), and a Ki-ras(+)-transformed derivative of TE85 (143B TK-), all of which were assigned to complementation group C. This chromosome 1 caused no change in proliferative potential of cell lines representing the other complementation groups. A derivative of human chromosome 1 that had lost most of the q arm by spontaneous deletion was unable to induce senescence in any of the immortal cell lines. This finding indicates that the q arm of human chromosome 1 carries a gene or set of genes which is altered in the cell lines assigned to complementation group C and is involved in the control of cellular senescence.

Conservation of the MORF4 related gene family: identification of a new chromo domain subfamily and novel protein motif

The seven member, human MORF4 related gene (MRG) family was recently identified based on the ability of Mortality factor on chromosome 4 (MORF4) to induce replicative senescence in immortal cell lines assigned to complementation group B (Bertram et al., 1999. Mol. Cell Biol. 19, 1479-1485). Initial computer based similarity searches identified human retinoblastoma binding protein 1 (RBP-1), Drosophila melanogaster male specific lethal-3 (Msl-3), S. pombe altered polarity-13 (Alp13) and S. cerevisiae Eaf3p, a component of the yeast NuA4 HAT complex (Galarneau et al., 2000. Mol. Cell 5, 927-937), as having similarity to the human MRG protein family. This suggested that the MRG family might be found in multiple species, and analysis of other homologs would provide functional and evolutionary insights into this gene family. Here, we report that MRG family members are present in twenty-three species based on molecular assays and sequence similarity searches. The new family members were divided into two groups based on similarity to the predominant human MRG family members, MRG15 and MRGX. The family members similar to MRG15 define a new, highly conserved subsection of the chromo domain superfamily. Additionally, conservation in the C-terminal two thirds of all the MRG family members and the Drosophila and human MSL-3 proteins defines a new protein domain, the MRG domain. These results indicate a highly conserved role for the MRG family in transcriptional regulation via chromatin remodeling by histone acetylation.

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 activates the B-myb promoter through formation of a nuclear complex with the retinoblastoma protein and the novel protein PAM14

The MORF4-RelatedGene on chromosome 15 (MRG15) is a member of a novel family of genes originally identified in studies to reveal cell senescence-inducing factors. MRG15 contains several predicted protein motifs, including a nuclear localization signal, a helix-loop-helix region, a leucine zipper, and a chromodomain. These motifs are commonly associated with transcription factors, suggesting that MRG15 may likewise function as a transcriptional regulator. To examine the potential function(s) of MRG15, we sought to identify cellular factors associated with thisMRG family member. In this regard, we have found that both the retinoblastoma tumor suppressor (Rb) and a novel nuclear protein PAM14 (Protein Associated with MRG,14 kDa) specifically associate with MRG15. We have further demonstrated that these interactions require the helix-loop-helix and leucine zipper domains of MRG15. Interestingly we have found all three proteins present in a multiprotein complex, suggesting that at least some of their functions may be interdependent. Although the functions of PAM14 have yet to be elucidated, Rb has several well characterized activities, including repression of E2F-activated promoters such as that of B-myb. Significantly we have demonstrated that MRG15 blocks the Rb-induced repression of this promoter, leading toB-myb promoter activation. Collectively these results suggest that MRG15 regulates transcription through interactions with a cellular protein complex containing Rb and PAM14.

Evidence That High Telomerase Activity May Induce a Senescent-like Growth Arrest in Human Fibroblasts

Expression of the catalytic subunit of human telomerase (hTERT), in normal human fibroblasts allows them to escape replicative senescence. However, we have observed that populations of hTERT-immortalized human fibroblasts contain 3–20% cells with a senescent morphology. To determine what causes the appearance of these senescent-like cells, we used flow cytometry to select them from the population and analyzed them for various senescence markers, telomere length, and telomerase activity. This subpopulation of cells had elevated levels of p21 and hypophosphorylated Rb, but telomere length was similar to that of the immortal cells in the culture that was sorted. Surprisingly, telomerase activity in the senescent-like cells was significantly elevated compared with immortal cells from the same population, suggesting that high telomerase activity may induce the senescent phenotype. Furthermore, transfection of normal fibroblasts with a hTERT-expressing plasmid that confers high telomerase activity led to the induction of p21, a higher percentage of SA-β-galactosidase-positive cells, and a greater number of cells entering growth arrest compared with controls. These results suggest that excessive telomerase activity may act as a hyperproliferative signal in cells and induce a senescent phenotype in a manner similar to that seen following overexpression of oncogenic Ras, Raf, and E2F1. Thus, there must be a critical threshold of telomerase activity that permits cell proliferation.

Identification of an alternatively spliced form of the Tat Interactive Protein (Tip60), Tip60(β)

Tip60 was originally isolated as a Tat interactive protein. It was subsequently shown that Tip60 had histone acetyltransferase (HAT) activity. In studies to understand gene-expression regulation that might involve HAT activity, we PCR-amplified Tip60 from a human heart marathon-ready cDNA library. As a result, we identified an alternatively spliced form of Tip60, Tip60beta (we refer to the previously cloned Tip60 as Tip60alpha). Tip60beta cDNA is slightly smaller than Tip60alpha, and sequencing indicates that there is a deletion of 156 bp in the coding region of the gene. The predicted Tip60beta protein therefore lacks 52 amino acids when compared with Tip60alpha. The Tip60alpha gene is encoded by 14 exons, and Tip60beta is an alternatively spliced form resulting from the exclusion of exon 5 during the splicing process. Exon 5 encodes a proline-rich region that is known to be important for protein-protein interaction. Tip60beta is expressed in a variety of human tissues and cell lines, and the protein is present in both the nucleus and cytoplasm in contrast to Tip60alpha, which is entirely nuclear. The results suggest that Tip60beta may have functions additional to those of Tip60alpha in cells and tissues.