Protocol for isolation of tumor-derived extracellular vesicles and functional studies on human T cell subsets.

Extracellular vesicles (EVs) enable communication between cells and tissues and are implicated in modulation of tumor immunosuppression. Here, we present a protocol for isolating tumor-derived EVs and assessing their functional influence in cultures with different subsets of human T cells. We describe steps for differential ultracentrifugation, size exclusion chromatography, EVs quantification, and fluorescence-activated cell sorting of human T cells. We then detail procedures for culturing T cells with EVs and using high-resolution spectral flow cytometry phenotyping for the analysis thereof. For complete details on the use and execution of this protocol, please refer to Swatler et al.1 and Swatler et al.2.

Extracellular Vesicles as Next-Generation Biomarkers in Lung Cancer Patients: A Case Report on Adenocarcinoma and Squamous Cell Carcinoma.

Extracellular vesicles (EVs) released from primary cell lines, originating from resected tissues during biopsies in patients with non-small cell lung cancer (NSCLC) revealing adenocarcinoma and squamous cell carcinoma subtypes, were examined for membrane proteomic fingerprints using a proximity barcoding assay. All the collected EVs expressed canonical tetraspanins (CD9, CD63, and CD81) highly coexpressed with molecules such as lysosome-associated membrane protein-1 (LAMP1-CD107a), sialomucin core protein 24 (CD164), Raph blood group (CD151), and integrins (ITGB1 and ITGA2). This representation of the protein molecules on the EV surface may provide valuable information on NSCLC subtypes and offer new diagnostic opportunities as next-generation biomarkers in personalized oncology.

Lack of activity of the mitochondrial large-conductance calcium-regulated potassium channels in senescent vascular smooth muscle cells.

A limited number of studies have shown functional changes in mitochondrial ion channels in aging and senescent cells. We have identified, for the first time, mitochondrial large-conductance calcium-regulated potassium channels in human smooth muscle mitochondria. This channel, with a conductance of 273 pS, was regulated by calcium ions and membrane potential. Additionally, it was activated by the potassium channel opener NS11021 and blocked by paxilline. Importantly, we have shown that senescence of these cells induced by hydrogen peroxide treatment leads to the disappearance of potassium channel protein levels and channel activity measured by the single channel patch-clamp technique. Our data suggest that disturbances in the expression of mitochondrial large conductance calcium-regulated potassium channels may be hallmarks of cellular senescence and contribute to the misregulation of mitochondrial function in senescent cells.

Exploring Extracellular Vesicles of Probiotic Yeast as Carriers of Biologically Active Molecules Transferred to Human Intestinal Cells.

Extracellular vesicles (EVs) are nanoparticles containing various bioactive cargos-e.g., proteins, RNAs, and lipids-that are released into the environment by all cell types. They are involved in, amongst other functions, intercellular communication. This article presents studies on EVs produced by the probiotic yeast Saccharomyces boulardii CNCM I-745. The size distribution and concentration of EVs in the liquid culture of yeast were estimated. Moreover, the vesicles of S. boulardii were tested for their cytotoxicity against three model human intestinal cell lines. This study did not show any significant negative effect of yeast EVs on these cells under tested conditions. In addition, EVs of S. boulardii were verified for their ability to internalize in vitro with human cells and transfer their cargo. The yeast vesicles were loaded with doxorubicin, an anticancer agent, and added to the cellular cultures. Subsequently, microscopic observations revealed that these EVs transferred the compound to human intestinal cell lines. A cytotoxicity test confirmed the activity of the transferred doxorubicin. Detailed information about the proteins present in EVs might be important in terms of exploring yeast EVs as carriers of active molecules. Thus, proteomic analysis of the EV content was also conducted within the present study, and it allowed the identification of 541 proteins after matching them to the Saccharomyces Genome Database (SGD). Altogether, this study provides strong evidence that the EVs of the probiotic CNCM I-745 strain could be considered a drug delivery system.

Parallel SPR and QCM-D Quantitative Analysis of CD9, CD63, and CD81 Tetraspanins: A Simple and Sensitive Way to Determine the Concentration of Extracellular Vesicles Isolated from Human Lung Cancer Cells.

Tetraspanins, including CD9, CD63, and CD81, are transmembrane biomarkers that play a crucial role in regulating cancer cell proliferation, invasion, and metastasis, as well as plasma membrane dynamics and protein trafficking. In this study, we developed simple, fast, and sensitive immunosensors to determine the concentration of extracellular vesicles (EVs) isolated from human lung cancer cells using tetraspanins as biomarkers. We employed surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation (QCM-D) as detectors. The monoclonal antibodies targeting CD9, CD63, and CD81 were oriented vertically in the receptor layer using either a protein A sensor chip (SPR) or a cysteamine layer that modified the gold crystal (QCM-D) without the use of amplifiers. The SPR studies demonstrated that the interaction of EVs with antibodies could be described by the two-state reaction model. Furthermore, the EVs' affinity to monoclonal antibodies against tetraspanins decreased in the following order: CD9, CD63, and CD81, as confirmed by the QCM-D studies. The results indicated that the developed immunosensors were characterized by high stability, a wide analytical range from 6.1 × 104 particles·mL-1 to 6.1 × 107 particles·mL-1, and a low detection limit (0.6-1.8) × 104 particles·mL-1. A very good agreement between the results obtained using the SPR and QCM-D detectors and nanoparticle tracking analysis demonstrated that the developed immunosensors could be successfully applied to clinical samples.

Fiji-Based Tool for Rapid and Unbiased Analysis of SA-ß-Gal Activity in Cultured Cells.

Normal cells under stressful conditions such as DNA damage or excessive mitogenic signaling may undergo senescence, which is associated with cell cycle arrest and induction of a proinflammatory phenotype. Accumulation of senescent cells may contribute to the shortening of the life span by accelerating aging and promoting chronic diseases. Cytochemical detection of the senescence-associated ß-galactosidase (SA-ß-gal) activity with 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside (X-gal) is a widely recognised marker of cell senescence. However, its simplicity and cost effectiveness lead to limitations in quantification, which is usually limited to manual counting of the positive cells. In order to address those limitations, we developed a Fiji-based macro extension that performs automatic and unbiased analysis of the integrated density of SA-ß-gal specific signal. Our tool is not only faster than manual counting but also provides extra resolution compared to the manual methods. Our macro extension could be a valuable tool in any senescence research laboratory.

Exosomes derived from lung cancer cells: Isolation, characterization, and stability studies.

Recent advances in nanomedicine have paved the way for developing targeted drug delivery systems. Nanoscale exosomes are present in almost every body fluid and represent a novel mechanism of intercellular communication. Because of their membrane origin, they easily fuse with cells, acting as a natural delivery system and maintaining the bioactivity and immunotolerance of cells. To develop a reconstitutable exosome-based drug candidate for clinical applications, quality assurance by preserving its physical and biological properties during storage is necessary. Therefore, this study aimed to determine the best storage conditions for exosomes derived from lung cancer cells (A549). This study established that the phosphate-buffered saline buffer enriched with 25 mM trehalose is an optimal cryoprotectant for A549-derived exosomes stored at -80°C. Under these conditions, the concentration, size distribution, zeta potential, and total cargo protein levels of the preserved exosomes remained constant.

Therapy-Induced Senescent/Polyploid Cancer Cells Undergo Atypical Divisions Associated with Altered Expression of Meiosis, Spermatogenesis and EMT Genes.

Upon anticancer treatment, cancer cells can undergo cellular senescence, i.e., the temporal arrest of cell division, accompanied by polyploidization and subsequent amitotic divisions, giving rise to mitotically dividing progeny. In this study, we sought to further characterize the cells undergoing senescence/polyploidization and their propensity for atypical divisions. We used p53-wild type MCF-7 cells treated with irinotecan (IRI), which we have previously shown undergo senescence/polyploidization. The propensity of cells to divide was measured by a BrdU incorporation assay, Ki67 protein level (cell cycle marker) and a time-lapse technique. Advanced electron microscopy-based cell visualization and bioinformatics for gene transcription analysis were also used. We found that after IRI-treatment of MCF-7 cells, the DNA replication and Ki67 level decreased temporally. Eventually, polyploid cells divided by budding. With the use of transmission electron microscopy, we showed the presence of mononuclear small cells inside senescent/polyploid ones. A comparison of the transcriptome of senescent cells at day three with day eight (when cells just start to escape senescence) revealed an altered expression of gene sets related to meiotic cell cycles, spermatogenesis and epithelial-mesenchymal transition. Although chemotherapy (DNA damage)-induced senescence is indispensable for temporary proliferation arrest of cancer cells, this response can be followed by their polyploidization and reprogramming, leading to more fit offspring.

Unbiased proteomic analysis of extracellular vesicles secreted by senescent human vascular smooth muscle cells reveals their ability to modulate immune cell functions.

Atherosclerosis, a common age-related disease, is characterized by intense immunological activity. Atherosclerotic plaque is composed of endothelial cells, vascular smooth muscle cells (VSMCs), lipids and immune cells infiltrating from the blood. During progression of the disease, VSMCs undergo senescence within the plaque and secrete SASP (senescence-associated secretory phenotype) factors that can actively modulate plaque microenvironment. We demonstrated that senescent VSMCs secrete increased number of extracellular vesicles (senEVs). Based on unbiased proteomic analysis of VMSC-derived EVs and of the soluble fraction of SASP (sSASP), more than 900 proteins were identified in each of SASP compartments. Comparison of the composition of VMSC-derived EVs with the SASP atlas revealed several proteins, including Serpin Family F Member 1 (SERPINF1) and Thrombospondin 1 (THBS1), as commonly upregulated components of EVs secreted by senescent VSMCs and fibroblasts. Among soluble SASP factors, only Growth Differentiation Factor 15 (GDF15) was universally increased in the secretome of senescent VSMCs, fibroblasts, and epithelial cells. Bioinformatics analysis of EV proteins distinguished functionally organized protein networks involved in immune cell function regulation. Accordingly, EVs released by senescent VSMCs induced secretion of IL-17, INFγ, and IL-10 by T cells and of TNFα produced by monocytes. Moreover senEVs influenced differentiation of monocytes favoring mix M1/M2 polarization with proinflammatory characteristics. Altogether, our studies provide a complex, unbiased analysis of VSMC SASP and prove that EVs derived from senescent VSMCs influence the cytokine milieu by modulating immune cell activity. Our results strengthen the role of senescent cells as an important inducer of inflammation in atherosclerosis.

Combination of dasatinib and quercetin improves cognitive abilities in aged male Wistar rats, alleviates inflammation and changes hippocampal synaptic plasticity and histone H3 methylation profile.

Aging is associated with cognitive decline and accumulation of senescent cells in various tissues and organs. Senolytic agents such as dasatinib and quercetin (D+Q) in combination have been shown to target senescent cells and ameliorate symptoms of aging-related disorders in mouse models. However, the mechanisms by which senolytics improve cognitive impairments have not been fully elucidated particularly in species other than mice. To study the effect of senolytics on aging-related multifactorial cognitive dysfunctions we tested the spatial memory of male Wistar rats in an active allothetic place avoidance task. Here we report that 8 weeks treatment with D+Q alleviated learning deficits and memory impairment observed in aged animals. Furthermore, treatment with D+Q resulted in a reduction of the peripheral inflammation measured by the levels of serum inflammatory mediators (including members of senescent cell secretome) in aged rats. Significant improvements in cognitive abilities observed in aged rats upon treatment with D+Q were associated with changes in the dendritic spine morphology of the apical dendritic tree from the hippocampal CA1 neurons and changes in the level of histone H3 trimethylation at lysine 9 and 27 in the hippocampus. The beneficial effects of D+Q on learning and memory in aged rats were long-lasting and persisted at least 5 weeks after the cessation of the drugs administration. Our results expand and provide new insights to the existing knowledge associated with effects of senolytics on alleviating age-related associated cognitive dysfunctions.

A common signature of cellular senescence; does it exist?

Cellular senescence is a stress response, which can be evoked in all type of somatic cells by different stimuli. Senescent cells accumulate in the body and participate in aging and aging-related diseases mainly by their secretory activity, commonly known as senescence-associated secretory phenotype-SASP. Senescence is typically described as cell cycle arrest. This definition stems from the original observation concerning limited cell division potential of human fibroblasts in vitro. At present, the process of cell senescence is attributed also to cancer cells and to non-proliferating post-mitotic cells. Many cellular signaling pathways and specific and unspecific markers contribute to the complex, dynamic and heterogeneous phenotype of senescent cells. Considering the diversity of cells that can undergo senescence upon different inducers and variety of mechanisms involved in the execution of this process, we ask if there is a common signature of cell senescence. It seems that cell cycle arrest in G0, G1 or G2 is indispensable for cell senescence; however, to ensure irreversibility of divisions, the exit from the cell cycle to the state, which we call a GS (Gero Stage), is necessary. The DNA damage, changes in nuclear architecture and chromatin rearrangement are involved in signaling pathways leading to altered gene transcription and secretion of SASP components. Thus, nuclear changes and SASP are vital features of cell senescence that, together with temporal arrest in the cell cycle (G1 or/and G2), which may be followed by polyploidisation/depolyploidisation or exit from the cell cycle leading to permanent proliferation arrest (GS), define the signature of cellular senescence.

Chromatin-Directed Proteomics Identifies ZNF84 as a p53-Independent Regulator of p21 in Genotoxic Stress Response.

The p21WAF1/Cip1 protein, encoded by CDKN1A, plays a vital role in senescence, and its transcriptional control by the tumour suppressor p53 is well-established. However, p21 can also be regulated in a p53-independent manner, by mechanisms that still remain less understood. We aimed to expand the knowledge about p53-independent senescence by looking for novel players involved in CDKN1A regulation. We used a chromatin-directed proteomic approach and identified ZNF84 as a novel regulator of p21 in various p53-deficient cell lines treated with cytostatic dose of doxorubicin. Knock-down of ZNF84, an as-yet un-characterized protein, inhibited p21 gene and protein expression in response to doxorubicin, it attenuated senescence and was associated with enhanced proliferation, indicating that ZNF84-deficiency can favor senescence bypass. ZNF84 deficiency was also associated with transcriptomic changes in genes governing various cancer-relevant processes e.g., mitosis. In cells with ZNF84 knock-down we discovered significantly lower level of H2AX Ser139 phosphorylation (γH2AX), which is triggered by DNA double strand breaks. Intriguingly, we observed a reverse correlation between the level of ZNF84 expression and survival rate of colon cancer patients. In conclusion, ZNF84, whose function was previously not recognized, was identified here as a critical p53-independent regulator of senescence, opening possibilities for its targeting in novel therapies of p53-null cancers.

Cellular Senescence in Brain Aging.

Aging of the brain can manifest itself as a memory and cognitive decline, which has been shown to frequently coincide with changes in the structural plasticity of dendritic spines. Decreased number and maturity of spines in aged animals and humans, together with changes in synaptic transmission, may reflect aberrant neuronal plasticity directly associated with impaired brain functions. In extreme, a neurodegenerative disease, which completely devastates the basic functions of the brain, may develop. While cellular senescence in peripheral tissues has recently been linked to aging and a number of aging-related disorders, its involvement in brain aging is just beginning to be explored. However, accumulated evidence suggests that cell senescence may play a role in the aging of the brain, as it has been documented in other organs. Senescent cells stop dividing and shift their activity to strengthen the secretory function, which leads to the acquisition of the so called senescence-associated secretory phenotype (SASP). Senescent cells have also other characteristics, such as altered morphology and proteostasis, decreased propensity to undergo apoptosis, autophagy impairment, accumulation of lipid droplets, increased activity of senescence-associated-ß-galactosidase (SA-ß-gal), and epigenetic alterations, including DNA methylation, chromatin remodeling, and histone post-translational modifications that, in consequence, result in altered gene expression. Proliferation-competent glial cells can undergo senescence both in vitro and in vivo, and they likely participate in neuroinflammation, which is characteristic for the aging brain. However, apart from proliferation-competent glial cells, the brain consists of post-mitotic neurons. Interestingly, it has emerged recently, that non-proliferating neuronal cells present in the brain or cultivated in vitro can also have some hallmarks, including SASP, typical for senescent cells that ceased to divide. It has been documented that so called senolytics, which by definition, eliminate senescent cells, can improve cognitive ability in mice models. In this review, we ask questions about the role of senescent brain cells in brain plasticity and cognitive functions impairments and how senolytics can improve them. We will discuss whether neuronal plasticity, defined as morphological and functional changes at the level of neurons and dendritic spines, can be the hallmark of neuronal senescence susceptible to the effects of senolytics.

Inhibition of NADPH Oxidases Activity by Diphenyleneiodonium Chloride as a Mechanism of Senescence Induction in Human Cancer Cells.

NADPH oxidases (NOX) are commonly expressed ROS-producing enzymes that participate in the regulation of many signaling pathways, which influence cell metabolism, survival, and proliferation. Due to their high expression in several different types of cancer it was postulated that NOX promote tumor progression, growth, and survival. Thus, the inhibition of NOX activity was considered to have therapeutic potential. One of the possible outcomes of anticancer therapy, which has recently gained much interest, is cancer cell senescence. The induction of senescence leads to prolonged inhibition of proliferation and contributes to tumor growth restriction. The aim of our studies was to investigate the influence of low, non-toxic doses of diphenyleneiodonium chloride (DPI), a potent inhibitor of flavoenzymes including NADPH oxidases, on p53-proficient and p53-deficient HCT116 human colon cancer cells and MCF-7 breast cancer cells. We demonstrated that the temporal treatment of HCT116 and MCF-7 cancer cells (both p53 wild-type) with DPI caused induction of senescence, that was correlated with decreased level of ROS and upregulation of p53/p21 proteins. On the contrary, in the case of p53-/- HCT116 cells, apoptosis was shown to be the prevailing effect of DPI treatment. Thus, our studies provided a proof that inhibiting ROS production, and by this means influencing ROS sensitive pathways, remains an alternative strategy to facilitate so called therapy-induced senescence in cancers.

Trimethylamine But Not Trimethylamine Oxide Increases With Age in Rat Plasma and Affects Smooth Muscle Cells Viability.

It has been suggested that trimethylamine oxide (TMAO), a liver oxygenation product of gut bacteria-produced trimethylamine (TMA), is a marker of cardiovascular risk. However, mechanisms of the increase and biological effects of TMAO are obscure. Furthermore, the potential role of TMAO precursor, that is TMA, has not been investigated. We evaluated the effect of age, a cardiovascular risk factor, on plasma levels of TMA and TMAO, gut bacteria composition, gut-to-blood penetration of TMA, histological and hemodynamic parameters in 3-month-old and 18-month-old, male, Sprague-Dawley and Wistar-Kyoto rats. Cytotoxicity of TMA and TMAO was studied in human vascular smooth muscle cells. Older rats showed significantly different gut bacteria composition, a significantly higher gut-to-blood TMA penetration, and morphological and hemodynamic alterations in intestines. In vitro, TMA at concentration of 500 µmol/L (2-fold higher than in portal blood) decreased human vascular smooth muscle cells viability. In contrast, TMAO at 1,000-fold higher concentration than physiological one had no effect on human vascular smooth muscle cells viability. In conclusion, older rats show higher plasma level of TMA due to a "leaky gut". TMA but not TMAO affects human vascular smooth muscle cells viability. We propose that TMA but not TMAO may be a marker and mediator of cardiovascular risk.

Curcumin induces multiple signaling pathways leading to vascular smooth muscle cell senescence.

Curcumin, a phytochemical present in the spice named turmeric, and one of the promising anti-aging factors, is itself able to induce cellular senescence. We have recently shown that cells building the vasculature senesced as a result of curcumin treatment. Curcumin-induced senescence was DNA damage-independent; however, activation of ATM was observed. Moreover, neither increased ROS production, nor even ATM were indispensable for senescence progression. In this paper we tried to elucidate the mechanism of curcumin-induced senescence. We analyzed the time-dependence of the level and activity of numerous proteins involved in senescence progression in vascular smooth muscle cells and how inhibition p38 or p38 together with ATM, two proteins involved in canonical signaling pathways, influenced cell senescence. We showed that curcumin was able to influence many signaling pathways of which probably none was dominant and sufficient to induce senescence by itself. However, we cannot exclude that the switch between initiation and progression of senescence is the result of the impact of curcumin on signaling pathways engaging AMPK, ATM, sirtuin 1 and p300 and on their reciprocal interplay. Cytostatic concentration of curcumin induced cellular stress, which exceeded the adaptive response and, in consequence, led to cellular senescence, which is triggered by time dependent activation of several signaling pathways playing diverse roles in different phases of senescence progression. We also showed that activity of ß-glucuronidase, the enzyme involved in deconjugation of the main metabolites of curcumin, glucuronides, increased in senescent cells. It suggests a possible local elevation of curcumin concentration in the organism.

Targeting normal and cancer senescent cells as a strategy of senotherapy.

Senotherapy is an antiageing strategy. It refers to selective killing of senescent cells by senolytic agents, strengthening the activity of immune cells that eliminate senescent cells or alleviating the secretory phenotype (SASP) of senescent cells. As senescent cells accumulate with age and are considered to be at the root of age-related disorders, senotherapy seems to be very promising in improving healthspan. Genetic approaches, which allowed to selectively induce death of senescent cells in transgenic mice, provided proof-of-concept evidence that elimination of senescent cells can be a therapeutic approach for treating many age-related diseases. Translating these results into humans is based on searching for synthetic and natural compounds, which are able to exert such beneficial effects. The major challenge in the field is to show efficacy, safety and tolerability of senotherapy in humans. The question is how these therapeutics can influence senescence of non-dividing post-mitotic cells. Another issue concerns senescence of cancer cells induced during therapy as there is a risk of resumption of senescent cell division that could terminate in cancer renewal. Thus, development of an effective senotherapeutic strategy is also an urgent issue in cancer treatment. Different aspects, both beneficial and potentially detrimental, will be discussed in this review.

TMA, A Forgotten Uremic Toxin, but Not TMAO, Is Involved in Cardiovascular Pathology.

Trimethylamine-N-oxide (TMAO) has been suggested as a marker and mediator of cardiovascular diseases. However, data are contradictory, and the mechanisms are obscure. Strikingly, the role of the TMAO precursor trimethylamine (TMA) has not drawn attention in cardiovascular studies even though toxic effects of TMA were proposed several decades ago. We assessed plasma TMA and TMAO levels in healthy humans (HH) and cardiovascular patients qualified for aortic valve replacement (CP). The cytotoxicity of TMA and TMAO in rat cardiomyocytes was evaluated using an MTT test. The effects of TMA and TMAO on albumin and lactate dehydrogenase (LDH) were assessed using fluorescence correlation spectroscopy. In comparison to HH, CP had a two-fold higher plasma TMA (p < 0.001) and a trend towards higher plasma TMAO (p = 0.07). In CP plasma, TMA was inversely correlated with an estimated glomerular filtration rate (eGFR, p = 0.002). TMA but not TMAO reduced cardiomyocytes viability. Incubation with TMA but not TMAO resulted in the degradation of the protein structure of LDH and albumin. In conclusion, CP show increased plasma TMA, which is inversely correlated with eGFR. TMA but not TMAO exerts negative effects on cardiomyocytes, likely due to its disturbing effect on proteins. Therefore, TMA but not TMAO may be a toxin and a marker of cardiovascular risk.

The Role of Curcumin in the Modulation of Ageing.

It is believed that postponing ageing is more effective and less expensive than the treatment of particular age-related diseases. Compounds which could delay symptoms of ageing, especially natural products present in a daily diet, are intensively studied. One of them is curcumin. It causes the elongation of the lifespan of model organisms, alleviates ageing symptoms and postpones the progression of age-related diseases in which cellular senescence is directly involved. It has been demonstrated that the elimination of senescent cells significantly improves the quality of life of mice. There is a continuous search for compounds, named senolytic drugs, that selectively eliminate senescent cells from organisms. In this paper, we endeavor to review the current knowledge about the anti-ageing role of curcumin and discuss its senolytic potential.

Insight into the role of PIKK family members and NF-кB in DNAdamage-induced senescence and senescence-associated secretory phenotype of colon cancer cells.

Senescence of cancer cells is an important outcome of treatment of many cancer types. Cell senescence is a permanent cell cycle arrest induced by stress conditions, including DNA damage. DNA damage activates DNA damage response (DDR), which involves members of the phosphatidylinositol 3-kinase-related kinase (PIKK) superfamily: protein kinases ATM, ATR, and DNA-PKcs. The so-far collected data indicate that ATM, with its downstream targets CHK2, p53, and p21, is the key protein involved in DDR-dependent senescence. It was also documented that the so-called senescence-associated secretory phenotype-SASP relies on ATM/CHK2, and not on p53 signaling. Moreover, genotoxic agents used in cancer treatment can activate NF-κB, which also induces transcription of SASP genes. In this paper, we have studied the involvement of three PIKK family members in colon cancer cell senescence and connection between DNA-damage-induced senescence and NF-κB-regulated SASP in p53-proficient and p53-deficient colon cancer cells treated with doxorubicin. We showed that doxorubicin induced cell senescence in both p53+/+ and p53-/- HCT116 cells, proving that this process is p53-independent. Senescence was successfully abrogated by a PIKK inhibitor, caffeine, or by simultaneous silencing of three PIKKs by specific siRNAs. By silencing individual members of PIKK family and analyzing common markers of senescence, the level of p21 and SA-ß-Gal activity, we came to the conclusion that ATR kinase is crucial for the onset of senescence as, in contrast to ATM and DNA-PKsc, it could not be fully substituted by other PIKKs. Moreover, we showed that in case of silencing the three PIKKs, there was no SASP reduction accompanying the decrease in the level of p21 and SA-ß-Gal (Senescence-Associated-ß-Galactosidase) activity; whereas knocking down the NF-κB component, p65, abrogated SASP, but did not affect other markers of senescence, proving that DNA damage regulated senescence independently and NF-κB evoked SASP.