Replicative senescence: implications for in vivo aging and tumor suppression.

Normal cells have limited proliferative potential in culture, a fact that has been the basis of their use as a model for replicative senescence for many years. Recent molecular analyses have identified numerous changes in gene expression that occur as cells become senescent, and the results indicate that multiple levels of control contribute to the irreversible growth arrest. These include repression of growth stimulatory genes, overexpression of growth inhibitory genes, and interference with downstream pathways. Studies with cell types other than fibroblasts will better define the role of cell senescence in the aging process and in tumorigenesis.

Evidence for the recessive nature of cellular immortality.

Fusion of immortal cell lines with normal human fibroblasts or certain other immortal cell lines yields hybrids having limited division potential. Cellular immortality was found to be a recessive phenotype in hybrids. It was also found that at least two separate events in the normal cell genome can result in immortality. In fusions involving certain immortal parent cells, these events can be complemented to result in hybrids with finite division capacity.

Existence of high abundance antiproliferative mRNA's in senescent human diploid fibroblasts.

Polyadenylated RNA isolated from senescent human diploid fibroblasts (HDF) inhibited DNA synthesis in proliferation-competent cells after microinjection, whereas polyadenylated RNA from young HDF had no inhibitory effect. Polyadenylated RNA from young cells made quiescent by removal of serum growth factors had a slight inhibitory effect on DNA synthesis. The abundance level of inhibitor messenger RNA (mRNA) from senescent cells was estimated at 0.8 and that of quiescent cells at 0.005 percent. These results demonstrate the existence of one or more antiproliferative mRNA's in nonproliferating normal human cells; these RNA's code for factors that either work antagonistically to initiators of DNA synthesis or regulate the expression of the initiators in some way. The abundance level of the inhibitory mRNA in senescent cells indicates the feasibility of developing a complementary DNA probe that will be useful in studying cell cycle control mechanisms.

Functional simian virus 40 T antigen is expressed in hybrid cells having finite proliferative potential.

Simian virus 40-transformed human cells fused with other independently derived simian virus 40-transformed cells and tumor-derived cells containing activated H-ras and N-ras oncogenes yielded hybrids capable of indefinite division. Fusions with various other immortal cells yielded hybrids that had limited division potential. T antigen expressed in limited-division hybrids was functional for the induction of cellular DNA synthesis.

Reversible cellular senescence: implications for immortalization of normal human diploid fibroblasts.

IMR-90 normal human diploid fibroblasts, transfected with a steroid inducible mouse mammary tumor virus-driven simian virus 40 T antigen, were carried through crisis to yield an immortal cell line. Growth was dependent on the presence of the inducer (dexamethasone) during both the extended precrisis life span of the cells and after immortalization. After dexamethasone removal, immortal cells divided once or twice and then accumulated in G1. These results are best explained by a two-stage model for cellular senescence. Mortality stage 1 (M1) causes a loss of mitogen responsiveness and arrest near the G1/S interface and can be bypassed or overcome by the cellular DNA synthesis-stimulating activity of T antigen. Mortality stage 2 (M2) is an independent mechanism that is responsible for the failure of cell division during crisis. The inactivation of M2 is a rare event, probably of mutational origin in human cells, independent of or only indirectly related to the expression of T antigen. Under this hypothesis, T-antigen-immortalized cells contain an active but bypassed M1 mechanism and an inactivated M2 mechanism. These cells are dependent on the continued expression of T antigen for the maintenance of immortality for the same reason that precrisis cells are dependent on T antigen for growth: both contain an active M1 mechanism.

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.

Mitogenic effects of the proto-oncogene and oncogene forms of c-H-ras DNA in human diploid fibroblasts.

Nuclear microinjection of c-H-ras DNA induced DNA synthesis in reversibly nonproliferating quiescent human cells. The proto-oncogene and oncogene forms were equally effective inducers. In contrast, c-H-ras DNA either alone or in combination with the adenovirus E1A gene did not cause terminally nondividing senescent cells to synthesize DNA.

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.

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.

Genetic and functional analyses exclude mortality factor 4 (MORF4) as a keratinocyte senescence gene.

Approximately 50% of immortal human keratinocyte lines show loss of heterozygosity of chromosome region 4q33-q34, and the reintroduction of chromosome 4 into one such line, BICR 6, causes proliferation arrest and features of replicative senescence. Recently, a candidate gene, mortality factor 4 (MORF4), was identified in this region and sequenced in 21 immortal keratinocyte lines. There were no mutations or deletions, and two of the seven lines that showed loss of heterozygosity at 4q33-q34 were heterozygous for MORF4 itself. Furthermore, the transfer of a chromosomal segment containing the entire MORF4 gene did not mimic the senescence effect of chromosome 4 in BICR 6. These results suggest that the inactivation of MORF4 is not required for human keratinocyte immortality.

Photodamaging effects of merocyanine 540 on neutrophils and HL-60 cells.

Merocyanine 540 (MC540) is a photosensitizing dye that has been used in several preclinical models and in a phase I clinical trial for the extracorporeal purging of tumor cells from autologous bone marrow grafts. The mechanism of the cytotoxic activity of MC540 is not yet fully understood, and the subcellular targets of MC540-mediated photodynamic damage remain to be identified. The human neutrophil provides an attractive model with which to study the effects of photoactivated MC540 on several well-defined cellular functions. As we report in this paper, simultaneous exposure of neutrophils to MC540 and light inhibited phagocytosis, random migration, chemotaxis, hydrogen peroxide production, and oxygen consumption. By contrast, the ability of neutrophils to kill engulfed bacteria and to produce superoxide radical was not compromised. Intracellular ATP levels and the activities of the cytosolic enzymes superoxide dismutase, catalase, and myeloperoxidase were only slightly reduced. Even in HL-60 leukemia cells, which bind more dye and are more readily killed by MC540-mediated photodynamic therapy than neutrophils, superoxide dismutase, catalase, and myeloperoxidase activities remained at normal or near-normal levels. These results are compatible with the view that plasma membrane components are primary targets of MC540-mediated photodynamic damage.

Plasma membrane properties regulating the sensitivity of leukemia, lymphoma, and solid tumor cells to merocyanine 540-sensitized photoirradiation.

Merocyanine 540 (MC 540) is a photosensitizing dye that has been used in a phase I clinical trial for the purging of leukemia and lymphoma cells from autologous bone marrow grafts. In this paper we examine the role of plasma membrane negative charge, plasma membrane fluidity, and plasma membrane hydrophobicity in the regulation of a cell's susceptibility to MC 540-sensitized photoirradiation. Among solid tumor cells, we found an inverse correlation between surface electronegativity, affinity for dye molecules, and susceptibility to MC 540-sensitized photoinactivation. That is, the least electronegative cells bound the highest amount of dye and were the most susceptible to dye-sensitized photoirradiation. By contrast, no such correlations were found among leukemia/lymphoma cells. This suggested that dye binding and susceptibility to MC 540-mediated photodynamic damages are regulated differently in hematopoietic/lymphopoietic and solid tumor cells.

Is there a role for physician involvement in introducing research to surrogate decision makers in the intensive care unit? (The Approach trial: a pilot mixed methods study).

PURPOSE: To assess the feasibility of conducting a randomized trial comparing two strategies [physician (MD) vs. non-physician (non-MD)] for approaching substitute decision makers (SDMs) for research and to evaluate SDMs' experiences in being approached for consent. METHODS: A pilot mixed methods study of first encounters with SDMs. RESULTS: Of 137 SDMs (162 eligibility events), 67 and 70 were randomized to MD and non-MD introductions, respectively. Eighty SDMs (98 events) provided consent and 21 SDMs (24 events) declined consent for studies, including 2 SDMs who provided and declined consent. We identified few missed introductions [4/52 (7.7 %)] and protocol violations [6/117 (5.1 %)], high comfort, satisfaction and acceptance scores and similar consent rates in both arms. SDMs provided consent significantly more often when a patient update was provided in the MD arm. Most SDMs (85.7 %) felt that physician involvement was inconsequential and preferred physician time to be dedicated to patient care; however, SDM experiences were closely related to their recall of being approached and recall was poor. SDMs highlighted 7 themes of importance to them in research surrogate decision-making. CONCLUSION: SDMs prioritized the personal attributes of the person approaching them over professional designation and preferred physician time to be dedicated to patient care. A mixed methods design evaluated intervention fidelity and provided the rationale for not proceeding to a larger trial, despite achieving all feasibility metrics in the pilot trial. TRIAL REGISTRATION NUMBER: NCT01232621.

Identification of an alternatively spliced form of the Tat interactive protein (Tip60), Tip60(beta).

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.

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.

Mouse and human chromosomal assignments of mortalin, a novel member of the murine hsp70 family of proteins.

Mortalin has been shown to exhibit differential distributions in cells with mortal and immortal phenotypes. In the present study, we report mot-2 cDNA cloning from RS-4 cells--an immortal clone from CD1-ICR mouse embryonic fibroblasts--and the chromosomal assignments of mortalin related genes to mouse chromosomes 18 and X by fluorescence in situ hybridization. Similar analysis assigned the gene to chromosome 5q31.1 in human.

Why are transformed cells immortal? Is the process reversible?

Normal cells have finite proliferative potential in culture. In contrast, cells derived from tumors immortalized by chemical carcinogens or viruses are able to divide indefinitely. A question of major importance is the mechanism that limits the proliferative potential of normal cells, and conversely, the process by which immortal cells have escaped irreversible growth cessation. To address this question we fused a number of different normal human fibroblast cell lines with various immortal human cell lines and determined the proliferative behavior of the resulting hybrids. In all cases the hybrids had a limited ability to proliferate in culture. These results suggested that the finite proliferative capacity of normal human cells was dominant and that immortal cells had acquired recessive changes in their genetic program, which allowed them to escape senescence. We were also able to assign approximately 30 immortal human cell lines to four complementation groups for indefinite division.

A long-lived human diploid fibroblast line for cellular aging studies: applications in cell hybridization.

We have isolated a diploid fibroblast culture from human fetal lung with an in vitro lifespan of about 100 population doublings. The culture grows very well at clonal densities and long-lived clones can be isolated for use in cellular aging studies. The longer in vitro lifespan of the culture has allowed us to isolate from it a clone, containing a dominant and recessive mutation, having significant remaining proliferative potential. The nature of the mutations will allow for hybrid selection, after fusion of the mutant clone with wild type human cells. The mass culture and clones derived from it provide a valuable resource for cell aging studies.

A general method for determining the replicative age of normal animal cell cultures.

The percentage of cells capable of forming colonies of at least a given size has been found to provide a reliable estimate of the remaining doubling potential of several human diploid fibroblast cell lines and for chick embryo fibroblasts. In five independently derived cell lines of human diploid fibroblasts we have found the same linear relationship between population doublings remaining and the percentage of cells able to form colonies of at least 16 cells. For chick embryo fibroblasts there is a linear relationship between the percentage of cells forming colonies of 64 or more cells and remaining proliferative potential. When human diploid fibroblasts were grown in medium containing hydrocortisone at 5 microgram/ml the increased in vitro lifespan was reflected in the colony size distribution long before the end of the in vitro lifespan.

Neurons in Alzheimer disease emerge from senescence.

A number of cell cycle markers are associated with the selective neuronal pathology found in Alzheimer disease. However, the significance of such cell cycle markers is clouded by duplicity of function in that many such proteins are also involved in apoptosis and/or DNA repair following oxidative damage. To clarify whether or not neurons in Alzheimer disease do in fact emerge from a quiescent status, with subsequent entry into the G1 phase of the cell cycle, in this study we focused on a family of MORF4-related proteins that are associated with emergence from senescence. Our results show that many neurons in vulnerable regions of Alzheimer disease brain, but not in control brain, have increased MORF4-related proteins indicating re-entry into the cell cycle. Immunoblot analysis showed a specific disease-related increase in a 52 kDa protein that is likely the human homologue of the MORF4-related transcription factor. The novel localization of such a transcriptional activating protein to selectively vulnerable neurons in Alzheimer disease provides compelling evidence for mitotic re-entry as part of the pathogenesis of neuronal dysfunction and death in Alzheimer disease.

Molecular genetic studies of cellular senescence.

The limited doubling potential of normal cells in culture was first proposed as a model for cellular aging by Hayflick in 1961. This phenomenon of in vitro cellular senescence is now well documented for a number of different normal human cell types. In an attempt to determine whether random events or programmed genetic processes were responsible for cellular aging, we performed a series of cell fusion studies. We determined that hybrids from fusion of normal with immortal human cells had limited proliferative potential, indicating that senescence is a dominant phenotype. We exploited the fact that immortality was recessive to assign a large number of different immortal human cell lines to four complementation groups for indefinite division. More recently, we have determined that the introduction of a single normal human chromosome 4 into HeLa (cervical carcinoma) cells by microcell fusion induced senescence in this immortal line. The results of these whole cell and microcell fusion studies support the hypotheses that propose senescence results from active, genetic mechanisms.