Dr Jaskaren Kohli

The development of genetic tools allowed for the validation of the pro-aging and pro-disease functions of senescent cells in vivo. These discoveries prompted the development of senotherapies—pharmaceutical interventions aimed at interfering with the detrimental effect of senescent cells—that are now entering the clinical stage. However, unequivocal identification and examination of cellular senescence remains highly difficult because of the lack of universal and specific markers. Here, to overcome the limitation of measuring individual markers, we describe a detailed two-phase algorithmic assessment to quantify various senescence-associated parameters in the same specimen. In the first phase, we combine the measurement of lysosomal and proliferative features with the expression of general senescence-associated genes to validate the presence of senescent cells. In the second phase we measure the levels of pro-inflammatory markers for specification of the type of senescence. The protocol can help graduate-level basic scientists to improve the characterization of senescence-associated phenotypes and the identification of specific senescent subtypes. Moreover, it can serve as an important tool for the clinical validation of the role of senescent cells and the effectiveness of anti-senescence therapies.

p16Ink4a is a potent cell cycle inhibitor engaged to support cell cycle arrest during cellular senescence. However, in tumors carrying mutations in key downstream effectors, p16Ink4a is highly expressed but fails to block cell proliferation. p16Ink4a-overexpressing tumor cells are highly aggressive and no targeted interventions are available. To study the effect of specific therapies, we generated murine sarcomas by overexpressing RAS oncogene and disrupting p53 activity. We observed that p16Ink4a-overxpressing murine sarcoma cells were resistant to ABT-263 and ABT-737, anti-cancer small molecules previously shown to eliminate p16Ink4a+ senescent cells. We then generated sarcoma cells carrying a suicide and reporter gene, called 3MR, under the regulation of the full p16Ink4a promoter. Activation of the suicide efficiently killed p16Ink4a-overxpressing sarcoma cells in vitro and in vivo. These data suggest that suicide gene therapy could represent an important therapeutic approach for the treatment of highly aggressive p16Ink4a+ cancers.

ABSTRACT

Located in the basal epidermis and hair follicles, melanocytes of the integument are responsible for its coloration through production of melanin pigments. Melanin is produced in lysosomal‐like organelles called melanosomes. In humans, this skin pigmentation acts as an ultraviolet radiation filter. Abnormalities in the division of melanocytes are quite common, with potentially oncogenic growth usually followed by cell senescence producing benign naevi (moles), or occasionally melanoma. Therefore, melanocytes are a useful model for studying melanoma, as well as pigmentation and organelle transport and the diseases affecting these mechanisms. This chapter focuses on the isolation, culture, and transfection of human and murine melanocytes. The first basic protocol describes the primary culture of melanocytes from human skin and the maintenance of growing cultures. The second basic protocol details the subculture and preparation of mouse keratinocyte feeder cells. The primary culture of melanocytes from mouse skin is described in the third basic protocol, and, lastly, the fourth basic protocol outlines a technique for transfecting melanocytes and melanoma cells. Curr. Protoc. Cell Biol. 63:1.8.1‐1.8.20. © 2014 by John Wiley & Sons, Inc.

Cellular senescence is a permanent state of cell cycle arrest that occurs in proliferating cells subjected to different stresses. Senescence is, therefore, a cellular defense mechanism that prevents the cells to acquire an unnecessary damage. The senescent state is accompanied by a failure to re-enter the cell cycle in response to mitogenic stimuli, an enhanced secretory phenotype and resistance to cell death. Senescence takes place in several tissues during different physiological and pathological processes such as tissue remodeling, injury, cancer, and aging. Although senescence is one of the causative processes of aging and it is responsible of aging-related disorders, senescent cells can also play a positive role. In embryogenesis and tissue remodeling, senescent cells are required for the proper development of the embryo and tissue repair. In cancer, senescence works as a potent barrier to prevent tumorigenesis. Therefore, the identification and characterization of key features of senescence, the induction of senescence in cancer cells, or the elimination of senescent cells by pharmacological interventions in aging tissues is gaining consideration in several fields of research. Here, we describe the known key features of senescence, the cell-autonomous, and noncell-autonomous regulators of senescence, and we attempt to discuss the functional role of this fundamental process in different contexts in light of the development of novel therapeutic targets.

The transition from reversible to irreversible pulmonary arterial hypertension involves vascular senescence and can be countered by senolysis.

Cellular senescence is a state of stable cell cycle arrest associated with macromolecular alterations and secretion of pro‐inflammatory cytokines and molecules. Senescence‐associated phenotypes restrict damage propagation and activate immune responses, two essential processes involved in response to viral infections. However, excessive accumulation and persistence of senescent cells can become detrimental and promote pathology and dysfunctions. Various pharmacological interventions, including antiviral therapies, lead to aberrant and premature senescence. Here, we review the molecular mechanisms by which viral infections and antiviral therapy induce senescence. We highlight the importance of these processes in attenuating viral dissemination and damage propagation, but also how prematurely induced senescent cells can promote detrimental adverse effects in humans. We describe which sequelae due to viral infections and treatment can be partly due to excessive and aberrant senescence. Finally, we propose that pharmacological strategies which eliminate senescent cells or suppress their secretory phenotype could mitigate side effects and alleviate the onset of additional morbidities. These strategies can become extremely beneficial in patients recovering from viral infections or undergoing antiviral therapy.

TERT (telomerase reverse transcriptase) is the catalytic component of telomerase. TERT shows little expression in normal somatic cells but is commonly re-expressed in cancers, facilitating immortalization. Recently-discovered TERT promoter mutations create binding sites for ETS-family transcription factors to upregulate TERT. ETS1 is reported to be important for TERT upregulation in melanoma. However it is unclear when in melanoma progression TERT and ETS1 proteins are expressed. To elucidate this question, ETS1 and TERT immunohistochemistry were performed on a panel of benign (n=27) and dysplastic nevi (n=34), radial growth phase (n=29), vertical growth phase (n=25) and metastatic melanomas (n=27). Lesions were scored by percentage of positive cells. ETS1 was readily detectable in all lesions, but not in normal melanocytes. TERT was located in either the nucleolus, the nucleoplasm (non-nucleolar) or both. Non-nucleolar TERT increased in prevalence with progression, from 19% of benign nevi to 78% of metastases. It did not however correlate with cell proliferation (Ki-67 immunostaining), nor differ significantly in prevalence between primary melanomas with or without a TERT promoter mutation. These results demonstrate that ETS1 is expressed very early in melanoma progression, and interestingly only non-nucleolar TERT correlates clearly in prevalence with melanoma progression. It can be acquired at various stages and by mechanisms other than promoter mutations.

University Medical Center Groningen

Cutaneous melanoma is a deadly skin cancer that affects over 200 000 people worldwide each year. Progression from a normal melanocyte to a metastatic melanoma typically occurs in a step‐wise manner, where each stage results from the de‐regulation of certain cell signalling pathways. Cellular senescence is known to play a critical role in melanoma suppression, and genes encoding proteins responsible for cell senescence are commonly mutated in somatic cases of the disease. Germline mutations in these genes can also be found in individuals with familial melanoma. Nonetheless, cutaneous melanoma is not a homogeneous disease, and various sub‐types exist, each displaying variations in certain mutational signatures. Advances in sequencing technologies have allowed researchers to categorise melanoma sub‐types with more precision, as well as to identify novel recurrent gene mutations, which may lead to the development of more personalised therapies in the future. Mutational clonal evolution of melanoma can be correlated with histopathological lesion types. Oncogenic mutations in MAPK pathway components are the only identified mutations in benign naevi. Benign naevi are in an arrested state called senescence, which must be bypassed for melanoma progression. TERT promoter mutations are common in melanoma and can emerge in dysplastic naevi, although expression of TERT is not sufficient to confer immortality. Invasive melanomas evolve mechanisms to bypass apoptosis, often through excessive PI3K‐AKT signalling. Metastatic melanomas are usually immortal, typically through gain of TERT expression and defects of the p16 pathway. Mutations in familial melanoma genes commonly result in a lengthening of melanocyte proliferative lifespan.

Axon degeneration precedes cell body death in many age-related neurodegenerative disorders, often determining symptom onset and progression. A sensitive method for revealing axon pathology could indicate whether this is the case also in Huntington's disease (HD), a fatal, devastating neurodegenerative disorder causing progressive deterioration of both physical and mental abilities, and which brain region is affected first. We studied the spatio-temporal relationship between axon pathology, neuronal loss, and mutant Huntingtin aggregate formation in HD mouse models by crossing R6/2 transgenic and HdhQ140 knock-in mice with YFP-H mice expressing the yellow fluorescent protein in a subset of neurons. We found large axonal swellings developing age-dependently first in stria terminalis and then in corticostriatal axons of HdhQ140 mice, whereas alterations of other neuronal compartments could not be detected. Although mutant Huntingtin accumulated with age in several brain areas, inclusions in the soma did not correlate with swelling of the corresponding axons. Axon abnormalities were not a prominent feature of the rapid progressive pathology of R6/2 mice. Our findings in mice genetically similar to HD patients suggest that axon pathology is an early event in HD and indicate the importance of further studies of stria terminalis axons in man.

Several cancer interventions induce DNA damage and promote senescence in cancer and nonmalignant cells. Senescent cells secrete a collection of proinflammatory factors collectively termed the senescence-associated secretory phenotype (SASP). SASP factors are able to potentiate various aspects of tumorigenesis, including proliferation, metastasis, and immunosuppression. Moreover, the accumulation and persistence of therapy-induced senescent cells can promote tissue dysfunction and the early onset of various age-related symptoms in treated cancer patients. Here, we review in detail the mechanisms by which cellular senescence contributes to cancer development and the side effects of cancer therapies. We also review how pharmacological interventions to eliminate senescent cells or inhibit SASP production can mitigate these negative effects and propose therapeutic strategies based on the age of the patient.