Liferation And Survival

In this review, we analyze selective (poly)phenol mechanisms in senescent and cancer cells to discriminate between in vitro and in vivo evidence and human applications considering (poly)phenol bioavailability, the influence of the gut microbiota, and their dose-response effects.

Mechanisms that regulate cell survival and proliferation are important for both the development and homeostasis of normal tissue, and as well as for the emergence and expansion of malignant cell populations. Caspase-3 (CASP3) has long been recognized for its proteolytic role in orchestrating cell death-initiated pathways and related processes; however, whether CASP3 has other functions in mammalian cells that do not depend on its known catalytic activity have remained unknown. To investigate this possibility, we examined the biological and molecular consequences of reducing CASP3 levels in normal and transformed human cells using lentiviral-mediated short hairpin-based knockdown experiments in combination with approaches designed to test the potential rescue capability of different components of the CASP3 protein. The results showed that a 50% reduction in CASP3 levels rapidly and consistently arrested cell cycle progression and survival in all cell types tested. Mass spectrometry-based proteomic analyses and more specific flow cytometric measurements strongly implicated CASP3 as playing an essential role in regulating intracellular protein aggregate clearance. Intriguingly, the rescue experiments utilizing different forms of the CASP3 protein showed its prosurvival function and effective removal of protein aggregates did not require its well-known catalytic capability, and pinpointed the N-terminal prodomain of CASP3 as the exclusive component needed in a diversity of human cell types. These findings identify a new mechanism that regulates human cell survival and proliferation and thus expands the complexity of how these processes can be controlled. The graphical abstract illustrates the critical role of CASP3 for sustained proliferation and survival of human cells through the clearance of protein aggregates. See full list on nature.com Caspase-3, also known as CASP3 in humans, belongs to a family of Cysteine-ASPartic proteASES (cysteine proteases). Initially, like related caspases, it is produced as an inactive zymogen. These are generically referred to as procaspases, whose latent proteolytic activity is then activated upon exposure to specific internal and/or external signals [1,2,3]. CASP3 is best known for its role in mediating the cleavage of specific proteins following the activation of apoptosis. The possibility that caspase-3 has other functions was first suggested from studies of model organisms, e.g., yeast [4], Drosophila [5, 6], and mice [7, 8] with selective deletion of caspase-3 gene sequences that include its conserved catalytic site [7,8,9,10]. These showed that, in mice, caspase-3-deficiency leads to abnormalities in osteogenesis [11] and cardiac [12] and skeletal muscle features [13], suggesting non-apoptotic roles, nevertheless shown to be mediated by caspase-3-dependent proteolytic degradation of intracellular proteins. [7,8,9,10]. However, if and how caspase 3 may have functional properties that are not dependent on its known catalytic domain has remained undefined. These historic findings prompted us to investigate the potential range of activities of human CASP3 initially in normal and malignant human mammary cells as models, anticipating potential exploitable differences associated with their transformation. The findings led to the identification of multiple non-proteolytic biological activities (cell survival and proliferation) and biochemical activities (protein aggregate removal) of CASP3. This then prompted a further investigation of the molecular control of these activities of CASP3 and a demonstration of their generality to other human cell types. See full list on nature.com CASP3 is required for the expansion of normal and malignant human mammary cell populations The mammary gland in normal adult human women consists of a two-cell layered tree-like structure with branching ducts that terminate in expanded lobules. The inner layer is composed of phenotypically distinct luminal progenitors (LPs) and luminal cells (LCs), and the outer basally positioned layer contains basal cells (BCs) [14]. LPs and BCs proliferate in vitro in the presence of EGF [14] and are thought to be the origin of most triple-negative and poorly treatable human breast cancers [15, 16]. To investigate the potential ability of CASP3 expression to modulate the growth properties of human mammary cells, we employed a knockdown (KD) strategy using lentiviral short hairpin (sh) vectors targeting CASP3, along with scrambled (Scr) controls (Fig. 1A). Western blot (WB) and flow cytometric analyses were initially performed on a triple negative breast cancer (TNBC) cell line MDA-MB-231 [17], and an immortalized but non-tumorigenic MCF10A [18] mammary epithelial cell line over 2 days following their transduction. The KD consistently produced a significant 57-fold reduction in PROCASPASE-3 levels in the shCASP3-treated cells compared to controls (Fig. 1B and S1A, B). Examination of the impact of CASP3 KD on the in vitro expansion of the MCF10A and MDA-MB-231 cell lines, and BT-20 [19], another TNBC cell line, consistently demonstrated a significant albeit variable decrease in the output of the shCASP3 cells compared to their shScr-transduced controls (Fig. 1C). These results provided the first indication that maintenance of physiological levels of CASP3 expression are required to support the proliferation of both normal and malignant human mammary cells in vitro. Examination of the impact of CASP3 KD on FACS-purified normal EGF-responsive human mammary BCs and LPs also showed a significant reduction in their clonogenic output of these primary cells (Fig. 1DF).A Experimental plan to assess the effect of CASP3 KD on human mammary cell population expansion in vitro. Viable cell numbers were measured 4 and 8 days after sorting transduced cells. B WB and flow cytometric analysis of changes in PROCASPASE3 levels in shCASP3- or shScr-transduced MCF10A cells measured 2 days after sorting transduced cells (104/cm2). C Reduced expansion of shCASP3- vs shSCR-transduced human mammary cells in 2D cultures. Data shown are the mean SEM of the Annexin V-PI- cell yields measured 4 and 8 days later in 3 experiments, each using a different CASP3 shRNA and the same shScr shRNA (***P 40% of the shScr-transduced controls (Fig. 3E and Fig. S1C).A Experimental plan for tracking the loss of viability in shCASP3- and shScr-transduced cells in bulk cultures. B Flow cytometrically determined proportions of Annexin V+ and/or PI + MCF10A and MDA-MB-231 cells assessed 3 days after FACS isolation of CASP3 shRNAs or shScr transduced cells. Values shown are the mean SEM of data from 3 separate experiments, each using CASP3 shRNA#1 and the same shScr shRNA (***P < 0.001, Students t-test). C Experimental plan for assessing the proliferation and death of single cells in culture. D KaplanMeier (KM) survival plots showing rapidly induced death in individually tracked shCASP3-transduced cells. Data are from 72 wells each initially containing a single shCASP3-transduced cell and 72 wells similarly initiated with a single shScr-transduced cell. Results have been compared using a log-rank test (***P < 0.001). E Cumulative plots of the timing of the first division (2 cells/well) of shCASP3- and shScr-transduced cells plated in (D) and compared using the KomoglerovSmirnov test (***P < 0.001).Full size image See full list on nature.com The key finding from this study is that both normal and malignant human cells of multiple types, and likely all cell types, given their validation in human iPSCs, rely on expression of the CASP3 prodomain for their survival and proliferation. Initially, this finding was developed from a detailed examination of the responses of a variety of normal and malignant human mammary cells. However, the failure to identify any human mammary cells whose viability was not sensitive to CASP3 KD when assayed either in vitro or in vivo in transplanted immunodeficient mice suggested a potentially more preserved generic function of CASP3, which experiments with a variety of human cell types confirmed. The fact that our CASP3 KD and prodomain rescue experiments in every cell type tested also showed an exclusive and specific pro-survival function of the N-terminal prodomain reinforces the concept that this pro-survival function of CASP3 is likely an evolutionarily conserved component and activity of CASP3 (Fig S5G). Until recently, most non-apoptotic functions of caspase-3 were believed to be linked to its known proteolytic activity, allowing CASP3 to cleave various intracellular proteins to impact signaling pathways and thereby altering intrinsic or neighboring cell behavior [1, 33]. A comparable catalytic-independent role of Procaspase-3 has been documented in mouse embryonic fibroblasts (MEFs), where caspase-3 was found to influence fibronectin secretion, cell morphology, adhesion, and migration independent of cell cycle changes. This study suggested an involvement of ERGolgi transport or vesicle trafficking, but the precise mechanisms by which procaspase-3 regulates these properties remained unexplored [34]. The second significant finding from this work is the evidence that the pro-survival role of the CASP3 prodomain appears to act by regulating protein aggregate accumulation possibly via a CASP3-dependent activation of autophagy. Our first results suggesting this mechanism came from comparative global proteome analyses of CASP3 KD and intact human mammary cells. Additional evidence from intracellular FACS analyses of protein aggregate accumulation provided more extensive documentation of the correlation of selective presence of an intact CASP3 prodomain and decreased aggregate levels. While considerable research has focused on understanding the functions o the large (p20) and small (p10) subunits of procaspase-3 that contain its well-recognized proteolytically active site, the functional properties of the CASP3 N-terminal prodomain have remained largely unexplored. However, a suggested regulatory role of the caspase-3 prodomain in the context of mouse embryonic fibroblast apoptosis control was reported [35]. In that study, it was shown that a region within caspase-3 prodomain negatively regulates the activation of caspase-3 and its cleavage by caspase-9 is required for complete activation of caspase-3. This provides additional support for the concept of an evolutionary conservation of caspase-3 prodomain to regulate caspase-3 activation and induction of apoptosis [35]. Our findings now add another rationale for the evolutionary conservation of CASP3 prodomain in relation to its pro-survival function during evolution. Protein aggregates are known for their toxic impact on cells, disrupting crucial cellular processes by interacting with cellular membranes or sequestering essential protein complexes [36, 37]. The accumulation of protein aggregates observed here in shCASP3-transduced cells is consistent with this result causing the observed initial delay in cell cycle progression, and decreasing cell viability and cell proliferation. Remarkably, the forced overexpression of catalytically active, inactive, or just the N-terminal prodomain of CASP3 were all found to reduce the otherwise increased levels of protein aggregates. This suggests a previously unknown mechanism for clearing protein aggregates from cells requiring the CASP3 N-terminal prodomain. It is interesting to note how these findings build on previous evidence from studies of the role of ScMCA1 in yeast. ScMCA1 is the forerunner of CASP3 in yeast and ScMCA1-deficient yeast cells also accumulate misfolded protein aggregates [38, 39], leading to delays in cell cycle progression [40, 41]. This role in removing protein aggregates in yeast involves both the catalytic and non-catalytic functions of ScMCA1 [38, 39]. However, the specific part of ScMCA1 responsible for this action has remained uncertain, as has the mechanisms through which ScMCA1 reduces protein aggregates independently of its catalytic activity. ScMCA1 may have a chaperone-like function through its Q/N rich prodomain, thereby aiding in the reduction of misfolded aggregates within cells and a similar mechanism may have been preserved through evolution in cells of many organisms including humans. Interestingly, a recent study has suggested that CASP3 may exert both catalytically dependent and independent effects on proteostasis [42]. It revealed that CASP3 can induce disaggregation of TDP43 independent of cell cycle kinetics, ultimately promoting normal mitochondrial function in skeletal muscle myocytes. Apoptosis and proteostasis also exhibit considerable crosstalk, as a number of recent studies have identified multifunctional proteins that mediate the two cellular functions in evolutionarily distant species [43, 44]. For example, Mca1 and Mca2 have been reported to regulate stress responses in Magnaporthe oryzae by promoting the clearance of insoluble aggregates [44]. Similar reports suggest that Ustilago maydis Mca1 N-terminal subunit plays a role in the proteostasis of this organism by inducing the clearance of stress-induced intracellular insoluble protein aggregates by directing Mca1 localization to protein aggregates [45]. These results add further support to the concept that the CASP3 N-terminal prodomain functions as a regulator of protein aggregate accumulation. In addition to the enrichment of pathways related to protein aggregation, our proteome data showed a possible effect of CASP3 KD on autophagy and ER stress as shown by changes in the levels of proteins involved in these pathways (i.e., ATF6, HSPs proteins). Future studies will clearly be of interest to elucidate how CASP3 may be involved in regulating cell behavior via effects on autophagy and the precise mechanism(s) of cell death involved. See full list on nature.com The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org/) via the PRIDE partner repository with the dataset identifier PXD045234. See full list on nature.com The authors thank M. Hale, G. Edin, and A. Toner for consenting donors of normal human mammary samples, collecting these and technical assistance, including some of their initial processing and cryopreservation and other procedures. This work was supported by grants from CIHR (453079, 505665, 388450), CCSRI (706040), and CEEHRC (348593). EE held a ... See full list on nature.com Authors and Affiliations Terry Fox Laboratory, British Columbia Cancer Research Institute, Vancouver, BC, Canada Ebrahim Eskandari, Susanna Tan, Margarita E. MacAldaz, Shengsen Ding, Justin Long & Connie J. EavesDepartment of Medical Genetics, University of British Columbia, Vancouver, BC, Canada Ebrahim Eskandari, Gregg B. Morin & Connie J. EavesCanadas Michael Smith Genome Sciences Centre, BC Cancer Research Institute, University of British Columbia, Vancouver, BC, Canada Gian Luca Negri, Karina Nielsen, Sandra E. Spencer & Gregg B. MorinSchool of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada Connie J. EavesAuthors Ebrahim Eskandari View author publicationsYou can also search for this author in PubMed Google ScholarGian Luca Negri View author publicationsYou can also search for this author in PubMed Google ScholarSusanna Tan View author publicationsYou can also search for this author in PubMed Google ScholarMargarita E. MacAldaz View author publicationsYou can also search for this author in PubMed Google ScholarShengsen Ding View author publicationsYou can also search for this author in PubMed Google ScholarJustin Long View author publicationsYou can also search for this author in PubMed Google ScholarKarina Nielsen View author publicationsYou can also search for this author in PubMed Google ScholarSandra E. Spencer View author publicationsYou can also search for this author in PubMed Google ScholarGregg B. Morin View author publicationsYou can also search for this author in PubMed Google ScholarConnie J. Eaves View author publicationsYou can also search for this author in PubMed Google Scholar Contributions EE and CJE conceptualized the project and critiqued all of the data. EE designed and executed most of the biological experiments and analyzed the derived data. ST, SD, and JL assisted with cell culture and Fucci-tracking experiments. MM assisted with culturing iPSCs. GBM, GLN, SES, and KN assisted with proteomics design and execution. SES and GLN carried out the computational and bioinformatics analysis of the proteomics data. EE prepared all of the figures and Tables and, with CE, wrote the manuscript, which all authors read and approved. Corresponding author Correspondence to Connie J. Eaves. See full list on nature.com Competing interests The authors declare no competing interests. See full list on nature.com Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. See full list on nature.com There are fundamental cellular processes, such as proliferation, survival, necrosis and apoptosis that play a role in physiological and pathological processes. These pathways are described and discussed in the light of cell mechanics. Cell proliferation refers to an increase in cell number due to cell division (cytokinesis), which occurs as the final step of the cell cycle. Healthy cells actively proliferate whereas growth-arrested, senescent, and dead or dying cells do not. In this review, we describe these unconventional ways in which cells have evolved to die or survive, as well as the contributions that these processes make to homeostasis and cancer. Cell proliferation and survival are fundamental biological processes. Cell proliferation refers to cell division, while cell survival indicates the maintenance of cell viability. Together, these processes are essential for tissue growth, development, and overall functionality within organisms. However, the molecular mechanisms are still largely elusive for other cellular processes that are regulated by TGF- and determine a cells proliferation and survival, apoptosis, dormancy, autophagy, and senescence.

Such details provide a deeper understanding and appreciation for Liferation And Survival.

Cell proliferation refers to an increase in cell number due to cell division (cytokinesis), which occurs as the final step of the cell cycle. Healthy cells actively proliferate whereas growth-arrested, senescent, and dead or dying cells do not.

Cell proliferation and survival are fundamental biological processes. Cell proliferation refers to cell division, while cell survival indicates the maintenance of cell viability. Together, these processes are essential for tissue growth, development, and overall functionality within organisms.

However, the molecular mechanisms are still largely elusive for other cellular processes that are regulated by TGF- and determine a cells proliferation and survival, apoptosis, dormancy, autophagy, and senescence.

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