Decreasing the pH will increase the area between CTP and UTP, whereas increasing the pH will increase the area between ATP and GTP at the expense of decreasing the CTP and UTP separation. In most cases, there are no detectable rNTPs when the boronate-purifi ed dNTP eluate is analyzed by HPLC. However, depending on the number of cells analyzed and the condition of the column, there may be slight contami- nation of the dNTP fraction with rNTPs. Thus, it is necessary to make sure that you can separate the rNTPs from the dNTPs by HPLC analysis.
Acknowledgment
This work has been supported by NIH grants CA120244 and CA190533 (MAN), CA083081 (DSS).
References
1. Courtois-Cox S, Jones SL, Cichowski K (2008) Many roads lead to oncogene-induced senes- cence. Oncogene 27(20):2801–2809
2. Bansal R, Nikiforov MA (2010) Pathways of oncogene-induced senescence in human mela- nocytic cells. Cell Cycle 9(14):2782–2788 3. Bianchi-Smiraglia A, Nikiforov MA (2012)
Controversial aspects of oncogene-induced senescence. Cell Cycle 11(22):4147–4151 4. Mooi WJ, Peeper DS (2006) Oncogene- induced
cell senescence—halting on the road to cancer. N Engl J Med 355(10):1037–1046
5. Bartkova J et al (2005) DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 434(7035):864–870 6. Bartkova J et al (2006) Oncogene-induced
senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 444(7119):633–637
7. Di Micco R et al (2006) Oncogene-induced senescence is a DNA damage response trig- gered by DNA hyper-replication. Nature 444(7119):638–642
8. Weyemi U, Dupuy C (2012) The emerging role of ROS-generating NADPH oxidase NOX4 in DNA-damage responses. Mutat Res 751(2):77–81
9. Liu YC et al (2008) Global regulation of nucle- otide biosynthetic genes by c-Myc. PLoS One 3(7):e2722
10. Mannava S et al (2008) Direct role of nucleo- tide metabolism in C-MYC-dependent prolif- eration of melanoma cells. Cell Cycle 7(15):2392–2400
11. Zhuang D et al (2008) C-MYC overexpression is required for continuous suppression of
oncogene- induced senescence in melanoma cells. Oncogene 27(52):6623–6634
12. Mannava S et al (2012) PP2A-B56alpha con- trols oncogene-induced senescence in normal and tumor human melanocytic cells. Oncogene 31(12):1484–1492
13. Bester AC et al (2011) Nucleotide defi ciency promotes genomic instability in early stages of cancer development. Cell 145(3):435–446 14. Mannava S et al (2012) Ribonucleotide reduc-
tase and thymidylate synthase or exogenous deoxyribonucleosides reduce DNA damage and senescence caused by C-MYC depletion.
Aging 4(12):917–922
15. Mannava S et al (2013) Depletion of deoxyri- bonucleotide pools is an endogenous source of DNA damage in cells undergoing oncogene- induced senescence. Am J Pathol 182(1):
142–151
16. Aird KM et al (2013) Suppression of nucleo- tide metabolism underlies the establishment and maintenance of oncogene-induced senes- cence. Cell Rep 3(4):1252–1265
17. Yarbro JW (1992) Mechanism of action of hydroxyurea. Semin Oncol 19(3 Suppl 9):1–10
18. Wu CH et al (2007) Cellular senescence is an important mechanism of tumor regression upon c-Myc inactivation. Proc Natl Acad Sci U S A 104(32):13028–13033
19. Pollock PM et al (2003) High frequency of BRAF mutations in nevi. Nat Genet 33(1):
19–20
20. Curtin JA et al (2005) Distinct sets of genetic alterations in melanoma. N Engl J Med 353(20):2135–2147
175
Mikhail A. Nikiforov (ed.), Oncogene-Induced Senescence: Methods and Protocols, Methods in Molecular Biology, vol. 1534, DOI 10.1007/978-1-4939-6670-7_17, © Springer Science+Business Media New York 2017
Chapter 17
Senescence-Like Phenotypes in Human Nevi
Andrew Joselow , Darren Lynn , Tamara Terzian , and Neil F. Box
Abstract
Cellular senescence is an irreversible arrest of cell proliferation at the G1 stage of the cell cycle in which cells become refractory to growth stimuli. Senescence is a critical and potent defense mechanism that mammalian cells use to suppress tumors. While there are many ways to induce a senescence response, oncogene-induced senescence (OIS) remains the key to inhibiting progression of cells that have acquired oncogenic mutations. In primary cells in culture, OIS induces a set of measurable phenotypic and behav- ioral changes, in addition to cell cycle exit. Senescence-associated β-Galactosidase (SA-β-Gal) activity is a main hallmark of senescent cells, along with morphological changes that may depend on the oncogene that is activated, or on the primary cell type. Characteristic cellular changes of senescence include increased size, fl attening, multinucleation, and extensive vacuolation. At the molecular level, tumor suppressor genes such as p53 and p16 INK4A may play a role in initiation or maintenance of OIS. Activation of a DNA damage response and a senescence-associated secretory phenotype could delineate the onset of senescence. Despite advances in our understanding of how OIS suppresses some tumor types, the in vivo role of OIS in mela- nocytic nevi and melanoma remains poorly understood and not validated. In an effort to stimulate research in this fi eld, we review in this chapter the known markers of senescence and provide experimental protocols for their identifi cation by immunofl uorescent staining in melanocytic nevi and malignant melanoma.
Key words Nevus , Oncogene , Senescence , DNA damage , Melanoma , Immunofl uorescence , Cancer , Protocol , Galactosidase , p16 INK4A , p53 , BRAF
1 Introduction
Cellular proliferation is highly regulated and key failsafe mecha- nisms prevent the unwanted expansion of cell number that can lead to cancer . Tumorigenesis may be constrained through two major mechanisms: apoptosis or cell cycle exit, including transient cell cycle arrest , quiescence and senescence [ 1 ]. Apoptosis (pro- grammed cell death) is a well-described pathway in humans involv- ing energy-dependent biochemical processes that ultimately lead to the cell’s own “suicide.” This process results in membrane bleb- bing, pyknosis, and eventual removal through the immune activity.
Quiescence is defi ned by reversible cell cycle exit and an extended period of metabolic inactivity [ 2 ]. Senescence, on the other hand,
is an irreversible arrest that restricts cellular proliferation in response to physiological stress signals, mitogen levels, or nutrients that would otherwise trigger cell duplication [ 3 – 5 ].
Senescence was fi rst identifi ed in cells with shortened telo- meres and telomeric dysfunction resulting in premature cell cycle arrest [ 6 ]. Subsequently, it was shown that overexpression of an oncogene, RAS , could induce senescence in cultured human cells through a telomerase-independent mechanism [ 7 ]. Numerous studies have since corroborated these fi ndings, now commonly referred to as oncogene-induced senescence, or OIS. OIS is thought to occur when the aberrant activation of oncogenes (e.g., RAS , MYC , etc.) leads to depletion of cellular energy and replica- tion reserves. The excess oncogenic load causes stalled replication forks and reactive oxygen species -induced damage, activating the cellular senescence program [ 3 – 5 ].
Melanocytic nevi were initially considered to be an excellent example of senescence in vivo. These pigmented lesions are viewed as precancerous, and result from a focal and limited proliferation of melanocytes that is driven by the mutational activation of an onco- gene such as BRAF . BRAF V600E is the most common oncogenic mutation found in human melanomas [ 8 , 9 ]. After forming a nevus, nevomelanocytes undergo a permanent exit from the cell cycle that prevents them from progressing to melanoma [ 3 , 10 ]. In support of this model, primary cultured melanocytes transduced with a BRAF V600E expressing lentivirus experience an initial burst of proliferation followed by morphologic changes and increased lev- els of SA-β-Gal [ 10 ]. These changes are one of the most obvious hallmarks of senescent melanocytes in culture and include loss of the elongated fusiform profi le characteristic of highly proliferative melanocytes, gain of a fl attened egg-shape, and an appearance of multinucleation and vacuolation. On the other hand, nevomelano- cytes present in common acquired melanocytic nevi have not been reported as morphologically distinct from adjacent normal skin melanocytes when examined histologically. Rather, it is the pres- ence of nesting, or clustered growth of melanocytes, that distin- guishes junctional nevi from solar lentigo. Analysis of the morphology of melanocytes in normal skin and in nevi using con- focal microscopy on thick sections (50 μm), and some of the anti- bodies and markers described here, will allow characterization of any true senescent features of nevomelanocytes.
Beyond morphology, numerous molecular markers of senes- cence have been utilized in various cell types that may be also use- ful for the study of senescence in nevus and melanoma formation.
Senescent cells, typically those with a fl attened egg-shape and a vacuole-rich cytoplasm, exhibit unusually high lysosomal β-Gal activity [ 2 ]. Some have speculated that oncogene activation in the presence of intact tumor suppressors may cause cell hypertrophy and subsequent compensatory activation of lysosomal enzymes,
including β-Gal [ 3 , 11 ]. β-Gal gene silencing experiments demon- strated that its expression functions as a marker or an indicator of senescence rather than as a contributor to the process [ 12 ].
Additionally, DNA damage (p53, ϒ-H2AX), proliferation ( Ki67 and C-MYC ), cell cycle regulation ( p16 INK4A , p14 ARF , p15 INK4B , FBX031 and p53 ), and chromatin structural change (Senescence- Associated Heterochromatin Foci or SAHF ) indicators were used in melanocytes and other cell types as putative senescence markers [ 2 , 13 , 14 ]. A tissue-specifi c hyper-secretory phenotype was also identifi ed in senescent cells [ 15 – 17 ]. A list of markers used in the literature is presented in Table 1 . To validate the utility of some of them in distinguishing nevi (senescence) from melanoma (senes- cence bypass or escape), we performed immunofl uorescent stain- ing on paraffi n sections from ten nevus and ten melanoma samples.
We selected Ki67 (proliferation) and ϒ-H2AX (DNA repair foci) as representative biomarkers for senescence ( see Fig. 1 ), and observed successful staining using the protocol provided below.
Table 1
Summary of literature fi ndings for cellular senescence associated markers
Marker Association Cell/tissue studied References
SA-β-Gal Senescence induction HUVEC a [ 22 ]
p16 (INK4A) Cell cycle regulation Melanocytes [ 23 , 24 ] p15 (INK4B) Cell cycle regulation Prostate tissue [ 19 ]
ARF (p14) Cell cycle regulation Melanocytes [ 23 ]
Ki67 Proliferation Melanocytes [ 14 ]
C-MYC Proliferation Cancer cells [ 25 ]
SAHF b Chromatin remodeling Melanoma cells [ 26 , 27 ]
γ-H2AX DNA damage Melanocytes [ 14 , 18 ]
p53 DNA damage Melanocytes [ 18 ]
FBXO31 Senescence induction Melanoma cells [ 24 ] IGFBP7 Senescence induction Nevi, melanoma cells [ 24 ] PML-IV Bodies Senescence induction Melanoma cells [ 26 ] miR-203 Senescence induction Melanoma cells [ 28 ]
Dec1 Senescence induction Cancer cells [ 29 ]
DcR2 Senescence induction Cancer cells [ 30 ]
RAS G12V Senescence induction Retroviral cells [ 31 ]
a Human umbilical vein endothelial cells (HUVEC)
b Senescence Associated Heterochromatic Foci (SAHF): H3K9Me, HP1, H2A
Fig. 1 Hematoxylin and Eosine (H&E) and immunofl uorescent staining of biomarkers in human nevus and melanoma sections, 20×. ( a ) H&E staining. In these examples, the nevus shows extensive nesting while the melanoma has a high density of melanocytes at the dermal-epidermal junction. ( b ) MART-1 and γ-H2AX immunostaining labeling melanocytes (cytoplasmic) and DNA damage (nuclear), respectively. ( c ) MART-1 and Ki-67 double immunostains label melanocytes and detect proliferation, respectively. Double positivity marks proliferative melanocytes
Positive staining for these proteins was detected in both nevus and melanoma samples, although it was clear that melanomas had con- siderably higher proliferation and DNA repair foci. We also observed successful staining for β-Galactosidase using the β-Gal antibody (data not shown), which may provide an alternative to the SA-β-Gal biochemical assay that requires the less commonly available frozen sample compared to paraffi n-embedded tissues generated in most pathology practices. MART-1 is used in clinical diagnosis and identifi es melanocytes and melanoma cells. Since known molecular indicators of senescence are not exclusive to this process, the use of multiple markers may be necessary for the iden- tifi cation of senescence in vivo.
There is a great need for further studies in this area, since con- fl icting reports have questioned the sensitivity and specifi city of these biomarkers for recognizing senescent nevus cells. Several pub- lications could not distinguish between nevi and melanoma using some of these reported markers [ 14 , 18 , 19 ]. Only Ki67 appeared to consistently distinguish human nevi from a panel of primary and metastatic melanomas. Likewise, p16 INK4A is reliably used in clinical diagnosis to distinguish between nevi and melanoma. It is possible that there are differences between in vivo with in vitro biomarkers of senescence. Alternatively, nevi and melanoma are not necessarily mutually exclusive, since approximately 25 % of melanomas appear in preexisting nevi [ 19 – 21 ]. Heterogeneity of cell properties and mutation content is a well- established idea for tumors that has not been considered in reference to nevus senescence and evasion or bypass of senescence. The concept that nevi are irreversibly senes- cent has not been well documented, particularly since low mitotic activity has been found in congenital, common acquired, and dys- plastic nevi [ 17 , 21 ]. It is anticipated that quantitative differences may be observed between nevi and melanoma, necessitating studies that consider staining patterns in greater detail. If senescence-like phenotypes can be reliably identifi ed in nevi, it will lead to improve- ments in histological detection of melanoma and to novel avenues for anti-melanoma therapy.
2 Materials
1. Primary Antibodies : Ki67 , ϒ-H2AX, p16 INK4a , MART-1, β-Gal.