The ability to identify markers associated with senescence is important for the use of this phenotype in clinical practice. Senescence has been routinely identified by staining for SA-β-gal activity (12
), and this has been used as a marker for senescence in aging tissues (12
) as well as in tumor tissues after chemotherapy (18
). It has been suggested that this staining may also be induced by transforming growth factor-β signaling independent of senescence, generating concern regarding its specificity (85
). SA-β-gal staining is dependent on increased lysosomal activity and requires fresh or frozen tissue for staining. Thus, this technique is incompatible with many immunohistological techniques routinely used in clinical pathology laboratories, especially with respect to archived tissues. The gene associated with SA-β-gal activity, the lysosomal galactosidase beta-1 (GLB1) is not required for senescence growth arrest and may be uncoupled from senescence in some cancer cell lines (86
). The development of immunohistological methods to detect GLB1 protein expression and localization in paraffin-embedded tissue, although not improving the reliability of this marker, would nonetheless facilitate its use in archival samples.
Although specific senescent biomarkers have yet to be fully developed, senescent cells may be identified on the basis of multiple characteristics (). When cells enter senescence, they develop a distinctive morphology, becoming enlarged, flattened, and multinucleated (). This morphology, however, is most easily identified in vitro and may not be apparent in tissues. Many senescent cells also develop extensive vacuoles in the cytoplasm associated with an increase in cellular complexity. This senescent morphology can be measured by flow cytometry as increased side scatter (4
). However, the most important characteristic of senescence is the irreversible loss of cell proliferative capacity. Flow cytometric cell cycle profiling typically shows that the number of cells in S phase decreases and the number in G1 or G2/M increases. In addition, cells become multinucleated, identified by the occurrence of additional 2N and 4N peaks. Taken together, these simple techniques can be used to identify characteristics of senescence in cultured cells.
Cellular characteristics and molecular markers of senescence in wild-type and cancer cells*
The analysis of senescence in tissue samples can be more challenging because many of the in vitro techniques are difficult to perform in patient samples, especially those that are paraffin embedded. Several classes of markers that can assist in the identification of senescent cells in tissues have been identified (). Senescence-associated heterochromatic foci (SAHF) are condensed regions of heterochromatin that accumulate during senescence (87
). These composite foci contain methylated and deacetylated histones and other associated proteins. SAHF have been used to identify senescence in vitro in fibroblasts and other non-immortalized cells. Widely tested markers in this category include methylation of histone 3 at lysines 9 and 27 and phosphorylation of H2A histone family, member X (γ-H2AX), all of which colocalize in SAHF (90
). In cancer, SAHF staining using homolog protein 1 gamma has been used to identify senescence in MCF7 cells (91
). In cancers in which chromatin maintenance is dysregulated, the occurrence and composition of these foci may vary. The utility of these markers to identify senescence in patient tissues is as yet unexplored.
A more promising class of markers include the CDKIs whose increased expression mediates senescence cell cycle arrest (). Amplified expression of the CDKIs p16Ink4a
and related Ink4 proteins, p57Kip2
, and notably p27Kip1
, has been observed in senescent cells and tissues (7
). However, many CDKIs are inactivated during senescence bypass, making them less reliable markers. In cancer, the downregulation of p27Kip1
and expression of its regulator ubiquitin ligase SKP2 has been identified in prostate and other cancers (92
), as well as in precancerous lesions (17
). In an AKT1 (v-akt murine thymoma viral oncogene homolog 1)-driven murine prostate cancer model, p27Kip1
is a key checkpoint for senescence (17
). The CDKN1B/p27Kip1
gene itself is infrequently mutated or deleted in many cancers, suggesting that its induction may represent a more promising marker of senescence.
Senescence is also characterized by a large protein secretory response. This phenotype in fibroblasts and some cancer cells includes proteins involved in IGF signaling (including IGF2 and IGFBPs 3, 5, 6, and 7) (41
), immuno-inflammatory cytokines (eg, IL-6, IL-8, and related proteins) (39
) and chemokine (C-X-C motif) ligand 14 (BRAK/CXCL14), whose function remains largely undefined (11
). IL-8, IGFBP7, and the IL-6 receptor chemokine (C-X-C motif) receptor 2 (CXCR2) have been shown by immunohistochemistry to be expressed in lesions undergoing tumor-suppressive oncogene-induced senescence (39
). The induction of these secreted factors in senescence may potentially serve as serum-based markers for the identification of patients undergoing senescence responses.
We used microarrays to screen a series of genes with increased expression during epithelial senescence for their role as markers of senescence in cancer (11
). In a series of cancer lines using a number of senescence-inducing drugs, transcripts of versican, filamin A–interacting protein 1–like (FILIP1L), and chromosome 5 open reading frame 13/P311 RNA were found to represent specific markers of senescence that are not induced during apoptosis. Changes in mitochondrial architecture may also be used. Mitochondria in proliferating fibroblasts are distinct and small, whereas in senescent cells, mitochondria fuse into elongated and integrated networks (38
). The expression and localization of the integral mitochondrial proteins human fission protein 1 (hFIS1) and optic atrophy 1 (OPA1) regulate these changes and the development of senescence (38
). Finally, the proteins Dec1 (BHLHB2) and DcR1 (TNFRSF10D) have been associated with senescence in noncancer tissues (15
). Although these proteins may be detected by immunohistochemistry, the utility of these potential markers in identifying senescence in fixed patient tumors has yet to be investigated.
Recently, quantitative modeling was used to assess validity of senescence markers in nontransformed cells as they become replicatively senescent (90
). Decreased proliferation of senescent cells was associated with measured increases in SA-β-gal activity and the combined detection of phosphorylated H2A histone family, member X (γ-H2AX, H2AFX) and decreased expression of the proliferation marker protein KI-67 (antigen identified by monoclonal antibody Ki-67, MKI67). This pattern of staining predicted the extent of senescence more closely than detection of other senescence-associated markers, individually or in combination. Dual-detection of KI-67 and extensive γ-H2AX phosphorylation was also associated with SA-β-gal staining in mouse intestinal crypts. Although these detection methods have not been used with other cell types or other mechanisms to induce senescence, they provide a computational framework for developing and validating senescence biomarkers and for predicting the frequency of markers in senescent tissue.
In summary, the identification of multiple markers in tissues currently provides more reliable evidence for senescence than that provided by a single marker. The most widely used marker of senescence is SA-β-gal, which provides strong evidence for identifying senescence when used in vitro with changes in morphology, increased side scatter, and accumulation in phases G0/G1 and G2/M. Other useful in vitro markers include the CDKIs p21Waf1, p16Ink4a and p27Kip1; versican and FILIP1L; and the increased expression of secreted cytokines. In vivo, SA-β-gal staining in conjunction with CDKI protein induction and other markers of decreased proliferation provide evidence for the presence of senescence.