The cells stop dividing; the studies keep multiplying.
Exogenous expression of the ras oncogene triggers senescence in human fibroblasts. Staining with DAPI reveals the gradual appearance of senescence-associated heterochromatin foci, transcriptionally silent regions of DNA. Four days after cells are infected with a ras-containing retrovirus, nuclei also show a marked colocalization between heterochromatin protein 1ß and promyelocytic leukemia protein; ten days after infection, colocalization decreases. When aging and damaged cells undergo apoptosis or malignant transformation, their lives reach a dramatic dénouement: suicide or cancer. But when they undergo senescence, their destiny seems drab in comparison. They irreversibly exit the cell cycle, linger indefinitely, and die of undetermined causes. Senescent fibroblasts, in particular, “get big and flat, and they look ugly,” resembling fried eggs, notes Scott W. Lowe, a professor at Cold Spring Harbor Laboratory on Long Island.
For decades, senescent human cells were found only in culture. But since the 1990s, investigators have detected senescence in T lymphocytes extracted from HIV-infected people and in tumors exposed to cancer treatments. Persistent doubts about the phenomenon’s relevance in vivo should have softened. Yet the senescent cell remained a cytological ugly duckling. Not as scientifically glamorous, as revealing, or as well defined as the apoptotic or transformed cell, it received far less attention.
Lately, however, that ugly duckling has matured into a more formidable bird. Accumulating evidence suggests that even if few senescent cells normally exist in vivo, their secretions promote diseases and aging, says Felipe Sierra, director of the cell structure and function program at the US National Institute on Aging (NIA). As a result, he senses a modest resurgence in the senescence field after what he calls “a very dark period a few years ago.” Last January, an NIA-sponsored workshop explored possible interactions between senescent cells and the extracellular matrix. The meeting, held at the Buck Institute for Age Research in Novato, Calif., was intended as a wake-up call to its 18 participants to “start worrying” about the physiological effects of these interactions, says Sierra.
Another boost to senescence studies comes from new findings on intracellular mechanisms. Earlier research established that the p53 and Rb tumor-suppressor pathways are vital to the process, and the cyclin-dependent kinase inhibitors p16 and p21 also play roles. Yet senescence is far from fully characterized on the molecular level. One recent paper identifies proteins and mechanisms in a novel pathway, and another report implicates an enzyme not previously linked to senescence.
A deeper understanding of tumorigenesis is the likeliest outcome of these in vitro advances. René Bernards, a professor at the Netherlands Cancer Institute in Amsterdam, acknowledges that cell-culture senescence could be an experimental artifact. But Bernards, who conducts RNA interference screens for senescence-related genes, contends, “It’s a useful artifact because it involves many of the players that are normally deregulated in cancer.”
Links between senescence and the organism-wide aging process, on the other hand, are more tenuous and their therapeutic lessons more problematic. Efforts to reduce senescence in aging tissues “might end up promoting cancer,” cautions Peter D. Adams, a biologist at Fox Chase Cancer Center in Philadelphia.
Scientists originally induced senescence by serially passaging cells in culture. After a cell line has replicated several dozen times, telomeric erosion leads to mitotic arrest. In the past decade, studies have established that activated oncogenes, DNA damage, or oxidative stress can also trigger senescence. Different experimental protocols and culture conditions yield subtly different types of senescence. Moreover, its manifestations vary between cell types and even within a broad class of cells such as the fibroblasts.
Researchers agree nevertheless that all senescent cells probably undergo chromatin remodeling that permanently prevents their reentry into the mitotic cycle. Estela E. Medrano, a biology and dermatology professor at Baylor College of Medicine in Houston, studies histone acetylation and deacetylation, chromatin changes that respectively enhance and repress gene transcription. Her specialty is the melanocyte whose senescence might cause aging human skin to become mottled, and whose inability to senesce could foster melanomas.
Medrano postulates that senescence is mediated by a competition between histone acetyltransferases (HATs) and histone deacetylases (HDACs) to bind to promoters of cell-cycle genes. Excessive HAT or HDAC levels can each trigger senescence, she maintains. Her lab is investigating cellular complexes that sense these levels. “We want to know how and when the complex formations occur when the cells are aging in culture,” says Medrano.
In 2003, Lowe’s lab reported a striking chromatin development in some senescent fibroblasts. After DNA staining, their nuclei displayed many small, distinct spots containing heterochromatin, which is transcriptionally inactive. In contrast, DNA staining and heterochromatin markers were more uniformly distributed both in quiescent cells, which temporarily forgo mitosis under low-serum conditions, and in senescent fibroblasts that Lowe now hypothesizes lack a robust p16 response.
Formation of these small spots, which Lowe called senescence-associated heterochromatic foci (SAHF), was linked to repression of genes targeted by the transcription factor E2F; these genes encode mitogenic proteins. Lowe recognizes that SAHF might merely be a consequence, not a cause, of senescence. Yet he plans to explore “how these genes get silenced” and particularly how p16 and Rb contribute to the process.
Adams has already uncovered a pathway to SAHF that he says might operate parallel to the Rb pathway. By his own admission, Adams is not a senescence expert. But he recalls wondering, in reaction to Lowe’s paper, whether SAHF creation and exit from the cell cycle might be promoted by human homologs of certain yeast proteins; other researchers had found that the yeast proteins contribute to gene-silencing by helping form heterochromatin. Adams’ group subsequently uncovered several landmark events in a SAHF pathway. Participants include a histone H2A variant and nuclear bodies containing promyelocytic leukemia protein, a tumor suppressor.
Adams views his task now as filling in the gaps between these events. “It’s like digging a tunnel from England to France,” he explains. “You dig from England and you dig from France. And, hopefully at some point, you meet in the middle.” Lowe says that follow-up experiments should test whether manipulations to the molecules in Adams’ model would, in the long term, make cells resistant to senescence or, if not, would enable senescent cells to proliferate again.
Heterochromatin represses gene activity, but many genes in senescent cells actually display higher expression levels. The repression of repressors could account for some of this increased activity. Senescence experts insist, however, that euchromatin, which facilitates gene transcription, must also be involved. Data from a cDNA microarray study provide tentative support for this contention. Hong Zhang, a postdoc working with genetics professor Stanley N. Cohen at Stanford University School of Medicine, examined the gene-expression profiles of human fibroblasts and mammary epithelial cells. By filtering out genes upregulated in quiescence, the study identified transcriptional fingerprints unique to senescence, not those merely correlated with cell-cycle arrest. It also found that upregulated senescence-specific genes were physically clustered, an arrangement consistent with euchromatin formation.
“The clustering is sort of an in silico experiment,” notes Zhang. “You do it computationally, and it’s very exciting. But I think the first thing I need to do is to confirm it experimentally, to see whether there is a chromatin-structure alteration that occurs during senescence.” One possible approach, he adds, is a so-called “ChIP-Chip” experiment. Antibodies that bind, for example, to acetylated histones could be used to immunoprecipitate euchromatin-associated genomic regions, which then would be fragmented and identified on a genomic microarray.
Instead, Zhang has focused on smurf2, a ubiquitin ligase whose gene is upregulated in senescent cells. He and Cohen induced high levels of smurf2 expression in early-passage human fibroblasts, whose telomeres presumably were not exhausted. Showing no stress response or detectable DNA damage, the cells entered senescence if their p53 or Rb pathway was functioning. Intriguingly, the smurf2-induced senescence did not appear to depend on the enzyme’s ubiquitin ligase activity.
Protein upregulation, not as a cause but as an effect of senescence, is the bailiwick of Judith Campisi, a senior scientist at Lawrence Berkeley National Laboratory in Berkeley, Calif. A theory that she and others have touted is that senescent cells, far from being physiologically inert, secrete proteins that stimulate tissue aging and tumorigenesis. These secretions include degradative enzymes, inflammatory cytokines, and growth factors.
Campisi estimates that 30 to 40 proteins are involved.
In a study published in February, lab members irradiated human fibroblasts, causing them to senesce. The researchers injected these cells into mice together with immortal but nontumorigenic mouse mammary epithelial (MME) cells, and the murine cells formed malignant tumors. Further experiments suggested that this tumorigenic conversion was partly mediated by matrix metalloproteinase-3, an enzyme secreted by the senescent cells. In other experiments, the senescent fibroblasts were cultured with another nontumorigenic MME cell line. The MME cells formed abnormal alveolar structures and produced twofold less of a major milk protein.
Campisi draws two lessons from the study. The first is that senescent cells in vitro can disrupt a normal tissue’s function and structure, a process that she suggests might similarly occur during aging. The second is that, as experiments increasingly reveal which secreted factors yield particular outcomes, “we might be able to modify the senescent phenotype in a tissue-specific and situation-specific manner” so as “to intervene in an intelligent way.”
Chromatin remodeling is thought to mediate senescence in human melanocytes. According to one hypothesis, the histone acetyltransferase p300 is removed from promoters of certain cell-cycle regulatory genes, and histone deacetylases (HDACs) are added. The resulting repression of those genes leads to a halt in mitosis. Investigations of senescence in vivo are also continuing. Rita B. Effros, a pathology and laboratory medicine professor at the University of California, Los Angeles, has long taken a leading role in characterizing senescence in CD8+ T lymphocytes. In 1996, she and colleagues reported that, in HIV-infected people, some of these so-called cytotoxic or killer T cells displayed short telomeres and could no longer proliferate. To combat the virus, which infects CD4+ helper T cells, these HIV-targeted CD8+ cells presumably divided so much that they became senescent. A similar phenomenon occurs in elderly people harboring cytomegalovirus, another latent virus.
Senescent T cells, which resist apoptosis, accumulate over time. The immune response eventually suffers, according to Effros, possibly because the cells secrete certain cytokines or because their overwhelming presence depresses the generation of T cells that target other antigens. In a recent study, Effros’ lab inserted a gene encoding human telomerase into a culture of HIV-targeted killer T cells taken from people infected with the virus. This manipulation kept the cells from senescing but failed to improve their cytotoxic efficiency. Acknowledging the impracticality, if not danger, of gene therapy, Effros says she is collaborating with Geron Corporation, of Menlo Park, Calif., to test the effects of a telomerase-activating compound on immune cells. The goal, she adds, is a “pharmacologic way of manipulating telomerase that would selectively affect normal T cells and improve their function.”
While Effros is trying to prevent senescence, Igor B. Roninson, director of the cancer center at Ordway Research Institute in Albany, NY, hopes to impose a safe form of senescence on the wildly proliferating cells of malignant tumors. In the 1990s, Roninson’s lab discovered that various chemotherapeutic drugs could induce terminal proliferation arrest in different human tumor cell lines. This effect, which also occurs after radiation treatment, was later detected in breast carcinomas excised from patients who underwent chemotherapy.
Cancer treatments appear to cause cellular senescence, Roninson explains, by damaging DNA and thereby activating various signal-transduction pathways. He observes that the process is often not immediate; video microscopy indicates that some senescing cells first pass through a state called mitotic catastrophe. Induction of senescence is also not without its hazards. A cDNA microarray study by Roninson’s lab showed that senescent cancer cells upregulate genes that encode tumor-promoting, as well as tumor-suppressive, secreted factors.
Roninson’s newly formed company, Senex Biotechnology, also in Albany, is seeking drugs that stimulate the beneficial side of this process. One class of compounds would “induce senescence with minimal cytotoxicity and with preferential expression of growth-inhibitory genes over tumor-promoting genes,” he says. Retinoids belong to this class, but Roninson notes that their utility is limited because many tumor cells lose their retinoid receptors. Another class of compounds would prevent the induction of tumor-promoting genes in cells that have senesced as a result of other treatments. Roninson reports that such activity is faintly displayed by nonsteroidal anti-inflammatory drugs that inhibit the transcription factor NF-ĸB. Senex is developing more efficient compounds, he adds.
Some researchers have qualms about fighting cancer by promoting senescence. Given the many factors secreted by senescent cells, Campisi observes, “If I had a tumor and I was being treated by chemo, I would want those tumor cells to die.” And noting that cultured senescent cells can be genetically tweaked to reengage in mitosis, Bernards asserts, “The only good tumor cell is a dead tumor cell, as they say.”
Roninson responds that cytostatic drugs would avoid the cytotoxicity that “is the principal cause of the negative side effects” experienced by cancer patients. Stressing scientists’ longstanding quest for such drugs, he says that he is “not in the minority” in his goal of forcing tumor cells to undergo permanent growth arrest, rather than only apoptosis. Instead, he maintains, “it’s the aficionados of apoptosis who are in the minority by denying this as a goal.”
April 12, 2005
Original web page at The Scientist