Why does serum starvation induced differentiation

Serum deprivation in vitro induces proliferation arrest to protect cells from toxicities; consequently, a reversible cell cycle arrest quiescence is associated with this phenotype and cellular consequences are multi-drug chemo-resistance and a propensity to evade apoptosis 18 , Serum-deprived PC3 Fig. Moreover, cell cycle arrest was also observed when cells were grown with serum and stimulated with H 2 O 2.

These observations simply report that a stress event via loss of serum or OS halts proliferation. Doxorubicin, a potent inducer of apoptosis, served as a control. Proliferation profile of serum-deprived cells.

One million 1. It is reported, cells which display a shrunken, round, and flat morphology is indicative of a quiescent phenotype 18 , Indeed, we observed PC3 and DU cells cultured without serum, as well serum-deprived cells with H 2 O 2 , displayed a rounder and flatter phenotype compared to cells in serum suggesting that cells submit to a quiescent phenotype for survival and adaption to stress that may be a consequence of nutrient deprivation or downstream stress.

Quiescence was determined via Western blot analysis for phosphorylated-retinoblastoma pRB or p27 Kip1 protein expression. To confirm the quiescent phenotype on a molecular level, serum-deprived PC3 and DU prostate cancer cells were examined for expression of quiescent markers: 1 tumor suppressor gene, retinoblastoma RB 23 , 47 ; and 2 CDK inhibitor, cyclin-dependent kinase inhibitor 1B p27 Kip1 It is well known that RB is an enforcer of quiescence 47 , and as such, the diminishing expression of phosphorylated-RB pRB in serum-deprived cells and serum-deprived cells exposed to H 2 O 2 indicates the onset of quiescence under stress Fig.

Furthermore, we detected more accumulation of p27 Kip1 in the nucleus of PC3 Fig. Compared to prior results Fig. We examined p27 Kip1 nuclear accumulation in the presence of each inhibitor in DU prostate cancer cells Fig.

Cells were harvested for western blotting and apoptosis. DMSO served as a control. DMSO was utilized as a control. Oxidative stress is considered to be one of the mechanisms that trigger early stages of prostate disease lesions, particularly prostatitic hyperplasia, benign prostatitic hyperplasia 58 , 59 , 60 , proliferative inflammatory atrophy PIA 61 , 62 , and others.

Overall, if not subsided, the consequence s of oxidative stress result in a significant decrease in the antioxidant systems leading to lipid, protein, and DNA damage. However, at levels that are still under investigation, the resulting ROS during a stressful event can prime biochemical molecules to allow prostate cancer to develop and progress, such as deactivating tumor suppressors 35 , 63 or increasing expression of pro-migratory signaling axes These dichotomic roles for ROS make it difficult to assess its clinical efficacy.

For instance, questions that may arise during a clinical observation are: 1 is the observance of oxidative stress in a clinical BPH simply the result of aging tissue and vascular deterioration; 2 or is it the onset of tumor development; or 3 is the signaling potential of ROS collateral damage in cancer chemotherapy with the eventual outcome of a migratory tumor cell 34 , 64 , 65 , 66 , The preventative role of oxidative stress regulators is thought to protect the prostate from tumor development; however, chronic stress over time induces somatic mutations in DNA, lipids and proteins resulting in neoplastic transformation due to alterations in metabolic checkpoints.

Additionally, the byproducts of ROS-based therapy are now being acknowledged to help propagate, amplify, and create a mutagenic and oncogenic microenvironment that is beneficial to a transforming metastatic tumor cell Thus, the metabolic relationships that are regulated by oxidative stress and the onset of prostate tumorigenesis remain an enigma. In general, an advanced tumor is conditioned to survive in the poorest conditions i.

Angiogenesis is one of the hallmarks of survivability because neovascularization provides the nutrients and oxygen necessary for tumor sustainability. However, considering that tumors survive very well during the metastasis process and in a heterogeneous tumor mass with a limited vascular supply, there must be additional markers to access tumor survivability. Izuishi et al. In brief, one way is by increasing the supply through angiogenesis, and the other way is by developing tolerance and alternate coping mechanisms to survive.

Eventually, tumors grow beyond its ability to coordinate a vascular supply, and possibly, only the tumors cells that have learned to cope or adapt in a deprived environment might be the phenotype that advances to malignancy. Much therapeutic attention is paid to angiogenesis since this is a crucial event for survival and migration; however, therapeutic focus should expand to tumor populations that outgrow angiogenesis-dependent survival and can tolerate nutrient deprivation and oxidative stress.

We observed that starvation primed prostate cancer cells for further insult to oxidative stress. Herein, our data support the notion that nutrient deprivation primes tumors for adaptation to stress and may serve as a determinant for tumor survival during stress and tumor progression Particularly, prostate cancer cells treated with both serum and H 2 O 2 were apoptotic, a phenotype that was not observed in cells that were initially starved.

For instance, Longo et al. Additionally, Hill et al. Furthermore, quiescent and self-renewing stem cells easily reside in quiescence during hypoxia and oxidative stress 76 , Perhaps, this is what we are observing in cancer. Autophagy is another consideration for phenotypic adaptation to stress Pancreatic cancer cell lines, which are notoriously hypo-vascular as malignancy increases, survived for considerably longer periods under extremely low nutrient conditions than cell lines of liver cancer suggesting that tumor cells that have acquired the ability to survive an unfavorable microenvironment might be the most aggressive malignancy, and correlates with poor differentiation of tumors.

In comparison, we observed that nutrient deprivation primed prostate cancer cells for tolerance of oxidative stress and adaptation for longer periods compared to cells with serum.

For instance, Babaei et al. Early in our study, LNCaP demonstrated poor survivability during oxidative stress even when initially starved.

The LNCaP cell line has a wild-type, functional p53 while DU bears a mutant p53, and PC3 bears a frameshift producing a stop codon and an allele deletion One of the most important unknowns in investigating p53 is how it determines a cellular outcome cell cycle arrest vs. ROS act as both an up-stream signal that triggers p53 activation and as a downstream factor that mediates apoptosis Death is not the only outcome of p53 signaling during oxidative stress; however, Zhao et al.

In addition, basal levels of p53 also has an antioxidant role, and the outcome depends on the context of the cell How do cells recognize nutrient starvation?

Therefore, it is also probable that as cells increase in malignancy, they have already acquired constitutive tolerance for nutrient and oxygen starvation through multiple carcinogenesis steps Early works attribute much of cellular survivability during oxidative stress to AKT 90 where Song et al.

In this case, a disulfide bridge formed within the catalytic cleft of PTEN, inhibiting its suppressive functions, which allowed ROS to freely orchestrate signaling.

Despite these events, cells still underwent apoptosis. Further investigation into apoptosis revealed that expression of the tumor suppressor pVHL increased and contains a target site for p-AKT phosphorylation. With all of this literature describing the relationship between ROS, AKT and cell survival, we believe our data is novel because there are no studies that describe the physical state of surviving epithelial tumor cells during an oxidative stress event.

In literature, the phenom is reserved in cancer stem cells. With regards to therapy, trans-arterial chemoembolization, damaging the blood supply of a tumor to prevent delivery of oxygen, growth factors, nutrients and others, has shown great success in providing significant improvement in overall survival, disease-free survival, and recurrence rates Although not considered curative, this intervention is dominant in liver cancer. The procedure is being considered for prostate cancer with hopes of causing irreversible necrosis of prostate tissue and causing the gland to shrink and soften 96 , Nevertheless, our results bring to the forefront a tumor cell phenotype that is often underappreciated, yet critical to metastasis, relapse and likely death.

Treatment times varied per experiment see figure legends. Cells were analyzed by fluorescent microscopy with a Zeiss Axio Vert. A1 Microscope.

Live: emission at nm; excitation at nm. Dead: emission at nm; excitation at nm. Annexin-V Apoptosis Detection Kit Cell Signaling Technology was used to quantify the levels of apoptosis according to the manufacturer's instructions.

Briefly, one million 1. Data was analyzed using FlowJo v Cells were fixed with 3. Two million 2. Briefly, cells were lysed in a series of buffers, centrifuged to obtain a non-nuclear fraction and an intact nuclear pellet, and further lysed to isolate the nuclear fraction.

Cells were stimulated as previously described above. Statistical analyses and graphs were generated using GraphPad Prism 6. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Zhang, C. Neuro-oncology 15 , — DeSantis, C. Cancer statistics for African Americans, progress and opportunities in reducing racial disparities. CA Cancer J. Article PubMed Google Scholar. Bouayed, J. Exogenous antioxidants—double-edged swords in cellular redox state: health beneficial effects at physiologic doses versus deleterious effects at high doses.

Tu, W. Aging Dis. Kumari, S. Reactive oxygen species: a key constituent in cancer survival. Insights 13 , Toyokuni, S. Persistent oxidative stress in cancer. FEBS Lett. Panieri, E. ROS homeostasis and metabolism: a dangerous liason in cancer cells.

Cell Death Dis. Mercurio, F. NF-kappaB as a primary regulator of the stress response. Oncogene 18 , — Takeuchi, T.

Carcinogenesis 14 , — Pervaiz, S. Tumor intracellular redox status and drug resistance—serendipity or a causal relationship?. Zhou, D.

Reactive oxygen species in normal and tumor stem cells. Cancer Res. Szatrowski, T. Production of large amounts of hydrogen peroxide by human tumor cells.

Zelenka, J. Targeting of stress response pathways in the prevention and treatment of cancer. Freitas, M. Oxidative stress adaptation in aggressive prostate cancer may be counteracted by the reduction of glutathione reductase.

Tiligada, E. Chemotherapy: induction of stress responses. Cancer 13 Suppl 1 , S Wu, C. Acta — , Boutin, A. Oncogenic Kras drives invasion and maintains metastases in colorectal cancer. Genes Dev. Li, B. Autophagy mediates serum starvation-induced quiescence in nucleus pulposus stem cells by the regulation of P Stem Cell Res. Legesse-Miller, A. Quiescent fibroblasts are protected from proteasome inhibition-mediated toxicity. Cell 23 , — E Solanas, G. Aged stem cells reprogram their daily rhythmic functions to adapt to stress.

Cell , Jin, K. CAN Yao, G. Modelling mammalian cellular quiescence. Interface Focus 4 , Guo, J. Retinoblastoma protein pRb , but not p or p, is required for maintenance of enterocyte quiescence and differentiation in small intestine. M Oesterle, E. Cell Cycle 10 , — Hills, S. DNA replication and oncogene-induced replicative stress. CB 24 , R Nakagawa, M. Constitutive activation of NF-kappaB pathway in hematopoietic stem cells causes loss of quiescence and deregulated transcription factor networks.

We demonstrated that cell cycle synchronization could stimulate rDNA transcription reactivation during somatic cell reprogramming into iPSCs. Our findings offer new insights into the regulation of rDNA transcriptional activity during somatic cell reprogramming and will benefit partially reprogrammed cells to overcome the epigenetic barrier to pluripotency.

Complete sequence of the kb human ribosomal DNA repeat: analysis of the intergenic spacer. Complete sequence of the kb mouse ribosomal DNA repeat: analysis of the intergenic spacer. Grummt I. Life on a planet of its own: regulation of RNA polymerase I transcription in the nucleolus. Genes Dev. Human rRNA transcription is modulated by the coordinate binding of two factors to an upstream control element.

A housekeeper with power of attorney: the rRNA genes in ribosome biogenesis. Cell Mol Life Sci. EMBO J. J Cell Biol. EMBO Rep. The nucleolar remodeling complex NoRC mediates heterochromatin formation and silencing of ribosomal gene transcription. Nat Genet. The chromatin remodeling complex NuRD establishes the poised state of rRNA genes characterized by bivalent histone modifications and altered nucleosome positions.

McStay B, Grummt I. The epigenetics of rRNA genes: from molecular to chromosome biology. Annu Rev Cell Dev Biol. Moss T. At the crossroads of growth control; making ribosomal RNA.

Curr Opin Genet Dev. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.

Berdasco M, Esteller M. DNA methylation in stem cell renewal and multipotency. Stem Cell Res Ther. Genomic characterization of the mouse ribosomal DNA locus. G3 Genes, Genomes, Genetics. J Biol Chem. Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc. Eichler DC, Craig N. Processing of eukaryotic ribosomal RNA. Biosynthesis of ribosomal RNA in nucleoli regulates pluripotency and differentiation ability of pluripotent stem cells.

Stem Cells. Serum starvation induced cell cycle synchronization facilitates human somatic cells reprogramming. PloS One. Cai K, Dynlacht BD. Activity and nature of p21 WAF1 complexes during the cell cycle. Genetic background affects induced pluripotent stem cell generation. Integration of murine leukemia virus DNA depends on mitosis. Dissecting direct reprogramming through integrative genomic analysis. Promotion of reprogramming to ground state pluripotency by signal inhibition.

PLoS Biol. A molecular roadmap of reprogramming somatic cells into iPS cells. Nanog overcomes reprogramming barriers and induces pluripotency in minimal conditions.

Curr Biol. NuRD blocks reprogramming of mouse somatic cells into pluripotent stem cells. Klein J, Grummt I. Feng Q, Zhang Y. The NuRD complex: linking histone modification to nucleosome remodeling. Curr Top Microbiol Immunol. NuRD suppresses pluripotency gene expression to promote transcriptional heterogeneity and lineage commitment. Cell Stem Cell. The NuRD component Mbd3 is required for pluripotency of embryonic stem cells. Nat Cell Biol. Mbd3, a component of the NuRD co-repressor complex, is required for development of pluripotent cells.

Nucleic Acids Res. Cis-existence of H3K27me3 and H3K36me2 in mouse embryonic stem cells revealed by specific ions of isobaric modification chromatogram. H3K9 methylation is a barrier during somatic cell reprogramming into iPSCs.

The RNA polymerase I transcription machinery: an emerging target for the treatment of cancer. Annu Rev Pharmacol Toxicol. Gurdon JB. The transplantation of nuclei between two species of Xenopus.

Dev Biol. Viable offspring derived from fetal and adult mammalian cells. Cytoplasmic activation of human nuclear genes in stable heterocaryons. Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm. Exp Cell Res. Nuclear reprogramming of human somatic cells by xenopus egg extract requires BRG1.

We wondered whether and how autophagy was involved in the regulation of quiescence in NPSCs. Twenty-four hours after infection, cells were switched to either normal or serum starvation conditions.

Previously, Lemons et al. Representative images of GFP and mRFP puncta under normal and serum starvation conditions are shown a , together with the quantification of autophagosomes and autolysosomes b. GAPDH served as an internal control. NC normal culture condition, SS serum starvation condition. Representative images of GFP and mRFP puncta under serum starvation conditions with or without rapamycin are shown e , together with the quantification of autophagosomes and autolysosomes f.

SS serum starvation condition, Rapa rapamycin. Representative images of GFP and mRFP puncta under serum starvation conditions with or without chloroquine are shown k , together with the quantification of autophagosomes and autolysosomes l. As expected, serum starvation with rapamycin treatment induced more red puncta autolysosomes than no rapamycin treatment, indicating that autophagy was enhanced Fig.

These results illustrated that rapamycin further enhanced autophagic activity in serum-starved NPSCs, and rapamycin also enhanced the entrance of NPSCs into quiescence by increasing the level of the protein P27 Fig.

In addition, we also determined that enhanced autophagy drives cells into quiescence with Ki67 staining. The rapamycin-treated group showed a significantly lower proportion of cells positive for Ki67 than the control group, indicating the inhibition of proliferation Fig. To further confirm the effect of autophagy on NPSC quiescence, we inhibited autophagy activity when the induced cells entered G0 by the serum starvation treatment. As expected, chloroquine treatment induced more yellow autophagosome puncta and less red autolysosome puncta, indicating reduced autophagy Fig.

As such, these results illustrated that chloroquine inhibited autophagic activity in serum-starved NPSCs; importantly, chloroquine also prevented NPSCs from entering quiescence by decreasing the level of the protein P27 Fig. Consistent with the Western blot analyses, the addition of chloroquine increased cell proliferation by 1.

According to the results described above, the protein expression of P27 not only increased in quiescent NPSCs but could also be regulated by autophagy. We wondered whether p27 is required for NPSC quiescence as in other cell types.

Both the cell cycle and EdU incorporation analyses showed that proliferation was inhibited under serum-starvation; however, P27 siRNA reduced the percentage of the population in the G0 state and increased EdU incorporation in both the normal and serum-starved conditions Fig.

Cellular quiescence, a reversible mode of exiting the cell cycle, is a novel area of research within the field of stem cell biology. As a critical characteristic of adult stem cells in vivo, quiescence maintains the capacity for cell cycle re-entry and subsequent differentiation in response to environmental stimuli [ 5 , 9 , 16 ]. Several studies have proposed the existence of stem cells in human, mouse, rat, rabbit, porcine, canine, bovine and rhesus monkey nucleus pulposus tissues [ 7 , 17 , 18 , 19 , 20 , 21 ].

However, recent studies demonstrated that the number and functions of NPSCs declined with age due to cellular senescence [ 7 , 8 ]. Given that previous studies suggested cellular quiescence maintains stemness and prevents premature exhaustion and senescence of stem cells, we hypothesise that quiescence may play a role in age-related changes in NPSCs. Mammalian cells can be induced to enter a quiescent state by a variety of in vitro models, including anchorage deprivation, serum starvation, contact inhibition, suspension culture and culture on soft polyacrylamide substrate.

However, as noted by Rumman [ 16 ], a key consideration concerns the type of growth arrest attained by the abrogation of mitogenic signalling in different cell types. For example, contact inhibition is a well-established model for cellular quiescence in primary fibroblasts retinal pigment epithelium cells [ 4 , 22 ]. Considering the avascular structure of nucleus pulposus tissue, we chose serum starvation to induce NPSC quiescence in vitro.

Reversibility is a defining characteristic of cellular quiescence: in contrast to other non-dividing cells, such as terminally differentiated cells and senescent cells, only quiescent cells retain the ability to re-enter the cell cycle in response to normal physiological stimuli [ 9 ].

Coller [ 24 ] proposed a new description of cellular quiescence that one mechanism in which the reversibility of quiescence is insured by the suppression of terminal differentiation. A recent report also suggested that the preservation of quiescence protects muscle stem cell function [ 3 ]. Therefore, we further examined the self-renewal abilities and differentiation potential of reactivated NPSCs. All results support that the induction of quiescence maintains the properties of NPSCs.

It is not difficult to understand why previous reports have associated the loss of quiescence with accelerated ageing and pathological cellular changes. Therefore, we have a reason to infer the role of quiescence in NPSC senescence. It is especially important to explore the mechanisms that maintain NPSC quiescence. In the absence of autophagy, long-lived, non-dividing quiescent stem cells may accumulate damage from environmental stress [ 9 ]. In the skeletal muscle, autophagy is a key factor in maintaining the regenerative capacity of muscle stem cells by promoting quiescence and preventing senescence [ 13 ].

Human clinical trials and animal model studies have also documented that autophagy significantly contributes to IVD degeneration [ 25 , 26 , 27 ]. Similarly, we also detected the activation of autophagy in NPSCs induced into quiescence by serum starvation. Under environmental stresses, the induction of autophagy seems to be important in the regulation of stem cell activation [ 9 ].

For example, autophagy regulates the entry of glioblastoma cells and ovarian cancer spheroid cells into the quiescent state [ 10 , 28 ]. However, the detailed mechanism of autophagy in the regulation of quiescence remains to be fully elucidated. In our experiments, we also examined the entry into the quiescent state by activating and inhibiting autophagy. Intriguingly, we observed that the expression of the cyclin-dependent kinase inhibitor P27 increased in quiescent NPSCs. During the process of inducing quiescence, rapamycin treatment significantly increased the level of P27, while chloroquine treatment decreased the level of P The molecular mechanisms of adult stem cell quiescence involve both cell-intrinsic factors, such as the P53 and RB proteins and CDK inhibitors P21, P27 and P57 , and cell-extrinsic factors, such as Notch signalling and Wnt signalling [ 16 ].

Cellular quiescence markers differ in different cell types. P27 has been proven to maintain quiescence in some cell types, such as fibroblasts, radial stem cells and haematopoietic stem cells [ 29 , 30 , 31 ]. To investigate the specific contribution of p27 in the control of serum starvation-induced G0 arrest, we specifically reduced its synthesis by the transfection of short interfering RNAs.

These results suggested that p27 is required for NPSCs to enter a quiescent state. Therefore, we confirm the above inference that autophagy regulates NPSC quiescence by regulating the expression of P This study also has some limitations.

We confirmed the existence of quiescence in NPSCs by serum starvation in vitro. Furthermore, the conclusion was also based on in vitro evidence. There is no precise animal model to examine quiescence and its regulatory mechanism in NPSCs in vivo. Although in vitro models provide a simple, readily scalable and reproducible system for the analysis of quiescence, they do lack the complexity of the in vivo environment.

Further studies are required to assess how closely the molecular phenotype can be extrapolated to NPSCs in vivo. In addition, although the positive effect of autophagy on quiescence was shown, the exact mechanism is still unclear and requires further study. In conclusion, serum starvation efficiently induces quiescence in NPSCs.

The induction of quiescence in NPSCs maintains stem cell properties, including clonogenic self-renewal, osteogenic differentiation and chondrogenic differentiation. These findings are relevant for future studies addressing aspects of the in vivo biology of NPSCs and provide novel insights into the decline of function in endogenous NPSCs during ageing and degeneration of IVD. Genes Dev. The aged niche disrupts muscle stem cell quiescence. Quiescent fibroblasts exhibit high metabolic activity.

PLoS Biol. Article Google Scholar. Induction of quiescence G0 in bone marrow stromal stem cells enhances their stem cell characteristics. Stem Cell Res. Sakai D, Andersson GB. Stem cell therapy for intervertebral disc regeneration: obstacles and solutions. Nat Rev Rheumatol. Age-related changes in nucleus pulposus mesenchymal stem cells: an in vitro study in rats. Stem Cells Int. Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc.

Nat Commun. Molecular regulation of stem cell quiescence. Nat Rev Mol Cell Biol. Autophagy mediates glucose starvation-induced glioblastoma cell quiescence and chemoresistance through coordinating cell metabolism, cell cycle, and survival. Cell Death Dis. Ischemia induces quiescence and autophagy dependence in hepatocellular carcinoma.