Low cell number proteomic analysis using in-cell protease digests reveals a robust signature for cell cycle state classification

AbstractProteomic analysis of rare cell states is a major challenge. We report an advance to our PRoteomics of Intracellular iMMUnostained cell Subsets (PRIMMUS) workflow whereby fixed cells are directly digested by proteases in cellulo for mass spectrometry-based proteomics. This decreased the cell number requirement by two orders of magnitude to <2,000 human lymphoblasts. We quantitatively measured the proteomes of 8 interphase and 8 mitotic states, avoiding synchronization. From 8 replicate pseudo-timecourses, we identify a core set of 119 cell cycle-regulated proteins that segregated into five clusters. These clusters varied in mitotic abundance patterns and regulatory short linear sequence motifs controlling their localisation and interaction with E3 ubiquitin ligases. We identified protein signatures that allowed accurate cell cycle state classification. We use this classification to stage an unexpected cell population as similar in proteome to early G0/G1 and telophase cells. Our data indicate DNA damage responses and premature APC/C activation in these cells, consistent with a DNA damage-induced senescent state. The advanced PRIMMUS approach is readily and broadly applicable to characterise rare and abundant cell states.

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Identification of potential pathways involved in the induction of cell cycle arrest and apoptosis by a new 4-arylidene curcumin analogue T63 in lung cancer cells: a comparative proteomic analysis

Molecular BioSystems ◽

10.1039/c3mb70553f ◽

2014 ◽

Vol 10 (6) ◽

pp. 1320 ◽

Cited By ~ 18

Author(s):

Hao Liu ◽

Yan-Zhen Liu ◽

Fan Zhang ◽

Hong-Sheng Wang ◽

Ge Zhang ◽

...

Keyword(s):

Lung Cancer ◽

Cell Cycle ◽

Cell Cycle Arrest ◽

Cancer Cells ◽

Proteomic Analysis ◽

Lung Cancer Cells ◽

Comparative Proteomic Analysis ◽

Curcumin Analogue ◽

Cycle Arrest

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Cell number in relation to primary pattern formation in the embryo of Xenopus laevis

Development ◽

10.1242/dev.53.1.269 ◽

1979 ◽

Vol 53 (1) ◽

pp. 269-289

Author(s):

Jonathan Cooke

Keyword(s):

Cell Cycle ◽

Pattern Formation ◽

Cell Cycle Control ◽

Cell Number ◽

The Body ◽

Morphological Evidence ◽

Tail Bud ◽

Cell Cycle Time ◽

Mesodermal Cell ◽

Body Pattern

Morphological evidence is presented that definitive mesoderm formation in Xenopus is best understood as extending to the end of the neurula phase of development. A process of recruitment of cells from the deep neurectoderm layers into mesodermal position and behaviour, strictly comparable with that already agreed to occur around the internal blastoporal ‘lip’ during gastrula stages, can be shown to continue at the posterior end of the presumptive body pattern up to stage 20 (earliest tail bud). Spatial patterns of incidence of mitosis are described for the fifteen hours of development between the late gastrula and stage 20–22. These are related to the onset of new cell behaviours and overt cyto-differentiations characterizing the dorsal axial pattern,which occur in cranio-caudal and then medio-lateral spatial sequence as development proceeds. A relatively abrupt cessation of mitosis, among hitherto asynchronously cycling cells,precedes the other changes at each level in the presumptive axial pattern. The widespread incidence of cells still in DNA synthesis, anterior to the last mitoses in the posterior-to-anteriordevelopmental sequence of axial tissue, strongly suggests that cells of notochord and somites in their prolonged, non-cycling phase are G2-arrested, and thus tetraploid. This is discussed in relation to what is known of cell-cycle control in other situations. Best estimates for cell-cycle time in the still-dividing, posterior mesoderm of the neurula lie between 10 and 15 h. The supposition of continuing recruitment from neurectoderm can resolve an apparent discrepancy whereby total mesodermal cell number nevertheless contrives to double over a period of approximately 12 h during neurulation when most of the cells are leaving the cycle. Because of pre-existing evidence that cells maintain their relative positions (despite distortion)during the movements that form the mesodermal mantle, the patterns presented in this paper can be understood in two ways: as a temporal sequence of developmental events undergone by individual, posteriorly recruited cells as they achieve their final positions in the body pattern, or alternatively as a succession of wavefronts with respect to changes of cellstate, passing obliquely across the presumptive body pattern in antero-posterior direction. These concepts are discussed briefly in relation to recent ideas about pattern formation in growing systems.

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Classification of Five Uremic Solutes according to Their Effects on Renal Tubular Cells

International Journal of Nephrology ◽

10.1155/2014/512178 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 9

Author(s):

Takeo Edamatsu ◽

Ayako Fujieda ◽

Atsuko Ezawa ◽

Yoshiharu Itoh

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Viable Cell ◽

Cell Number ◽

Viable Cell Number ◽

Cycle Progression ◽

Tubular Cells ◽

Renal Tubular Cells ◽

Uremic Solutes ◽

Renal Tubular

Background/Aims. Uremic solutes, which are known to be retained in patients with chronic kidney disease, are considered to have deleterious effects on disease progression. Among these uremic solutes, indoxyl sulfate (IS) has been extensively studied, while other solutes have been studied less to state. We conducted a comparative study to examine the similarities and differences between IS,p-cresyl sulfate (PCS), phenyl sulfate (PhS), hippuric acid (HA), and indoleacetic acid (IAA).Methods. We used LLC-PK1 cells to evaluate the effects of these solutes on viable cell number, cell cycle progression, and cell death.Results. All the solutes reduced viable cell number after 48-hour incubation. N-Acetyl-L-cysteine inhibited this effect induced by all solutes except HA. At the concentration that reduced the cell number to almost 50% of vehicle control, IAA induced apoptosis but not cell cycle delay, whereas other solutes induced delay in cell cycle progression with marginal impact on apoptosis. Phosphorylation of p53 and Chk1 and expression of ATF4 and CHOP genes were detected in IS-, PCS-, and PhS-treated cells, but not in IAA-treated cells.Conclusions. Taken together, the adverse effects of PCS and PhS on renal tubular cells are similar to those of IS, while those of HA and IAA differ.

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Does Heme Oxygenase-1 Have a Role in Caco-2 Cell Cycle Progression?

Experimental Biology and Medicine ◽

10.1177/15353702-0322805-52 ◽

2003 ◽

Vol 228 (5) ◽

pp. 590-595 ◽

Cited By ~ 13

Author(s):

Aliye Uc ◽

Bradley E. Britigan

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Renewal Process ◽

Cell Cycle Progression ◽

Intestinal Epithelial Cell ◽

Cell Number ◽

Heme Oxygenase 1 ◽

Intestinal Epithelial ◽

Cycle Progression ◽

Proliferation And Differentiation

Intestinal epithelium undergoes a rapid self-renewal process characterized by the proliferation of the crypt cells, their differentiation into mature enterocytes as they migrate up to the villi, followed by their shedding as they become senescent villus enterocytes. The exact mechanism that regulates the intestinal epithelium renewal process is not well understood, but the differential expression of regulatory genes along the crypt-villus axis may have a role. Heme oxygenase-1 (HO-1) is involved in endothelial cell cycle progression, but its role in the intestinal epithelial cell turnover has not been explored. With its effects on cell proliferation and its differential expression along the crypt-villus axis, HO-1 may play a role in the intestinal epithelial cell renewal process. In this study, we examined the role of HO-1 in the proliferation and differentiation of Caco-2 cells, a well-established in vitro model for human enterocytes. After confluence, Caco-2 cells undergo spontaneous differentiation and mimic the crypt to villus maturation observed in vivo. In preconfluent and confluent Caco-2 cells, HO-1 protein expression was determined with the immunoblot. HO-1 activity was determined by the ability of the enzyme to generate bilirubin from hemin. The effect of a HO-1 enzyme activity inhibitor, tin protoporphyrin (SnPP), on Caco-2 cell proliferation and differentiation was examined. In preconfluent cells, cell number was determined periodically as a marker of proliferation. Cell viability was measured with MTT assay. Cell differentiation was assessed by the expression of a brush border enzyme, alkaline phophatase (ALP). HO-1 was expressed in subconfluent Caco-2 cells and remained detectable until 2 days postconfluency. This timing was consistent with cells starting their differentiation and taking the features of normal intestinal epithelial cells. HO-1 was inducible in confluent Caco-2 cells by the enzyme substrate, hemin in a dose- and time-dependent manner. SnPP decreased the cell number and viability of preconfluent cells and delayed the ALP enzyme activity of confluent cells. HO-1 may be involved in intestinal cell cycle progression.

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Proteomic Analysis of Breast Cancer Resistance to the Anticancer Drug RH1 Reveals the Importance of Cancer Stem Cells

Cancers ◽

10.3390/cancers11070972 ◽

2019 ◽

Vol 11 (7) ◽

pp. 972

Author(s):

Dalius Kuciauskas ◽

Nadezda Dreize ◽

Marija Ger ◽

Algirdas Kaupinis ◽

Kristijonas Zemaitis ◽

...

Keyword(s):

Breast Cancer ◽

Cell Cycle ◽

Stem Cells ◽

Stem Cell ◽

Cancer Stem Cells ◽

Cancer Cells ◽

Proteomic Analysis ◽

Acquired Resistance ◽

Cancer Resistance ◽

Resistant Cells

Antitumor drug resistance remains a major challenge in cancer chemotherapy. Here we investigated the mechanism of acquired resistance to a novel anticancer agent RH1 designed to be activated in cancer cells by the NQO1 enzyme. Data show that in some cancer cells RH1 may act in an NQO1-independent way. Differential proteomic analysis of breast cancer cells with acquired resistance to RH1 revealed changes in cell energy, amino acid metabolism and G2/M cell cycle transition regulation. Analysis of phosphoproteomics and protein kinase activity by multiplexed kinase inhibitor beads showed an increase in the activity of protein kinases involved in the cell cycle and stemness regulation and downregulation of proapoptotic kinases such as JNK in RH1-resistant cells. Suppression of JNK leads to the increase of cancer cell resistance to RH1. Moreover, resistant cells have enhanced expression of stem cell factor (SCF) and stem cell markers. Inhibition of SCF receptor c-KIT resulted in the attenuation of cancer stem cell enrichment and decreased amounts of tumor-initiating cells. RH1-resistant cells also acquire resistance to conventional therapeutics while remaining susceptible to c-KIT-targeted therapy. Data show that RH1 can be useful to treat cancers in the NQO1-independent way, and targeting of the cancer stem cells might be an effective approach for combating resistance to RH1 therapy.

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Label-Free Proteomics of the Fetal Pancreas Identifies Deficits in the Peroxisome in Rats with Intrauterine Growth Restriction

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/1520753 ◽

2019 ◽

Vol 2019 ◽

pp. 1-15

Author(s):

Xiaomei Liu ◽

Yanyan Guo ◽

Jun Wang ◽

Linlin Gao ◽

Caixia Liu

Keyword(s):

Oxidative Stress ◽

Cell Cycle ◽

Western Blotting ◽

Proteomic Analysis ◽

Intrauterine Growth Restriction ◽

Bioinformatics Analysis ◽

Growth Restriction ◽

Label Free ◽

Fetal Pancreas ◽

Intrauterine Growth

Aim. The objective of the present study was to identify differentially expressed proteins (DEPs) in the pancreas of a fetus with intrauterine growth restriction (IUGR) and to investigate the molecular mechanisms leading to adulthood diabetes in IUGR. Methods. The IUGR rat model was induced by maternal protein malnutrition. The fetal pancreas was collected at embryonic day 20 (E20). Protein was extracted, pooled, and subjected to label-free quantitative proteomic analysis. Bioinformatics analysis (GO and IPA) was performed to define the pathways and networks associated with DEPs. LC-MS results were confirmed by western blotting and/or quantitative PCR (q-PCR). The principal parameters of oxidative stress-superoxide dismutase (Sod) were determined in blood samples of fetal rats. Results. A total of 57 DEPs (27 upregulated, 30 downregulated) were identified with a 1.5-fold change threshold and a p value ≤ 0.05 between the IUGR and the control pancreas. Bioinformatics analysis revealed that these proteins play important roles in peroxisome biogenesis and fission, fatty acid beta-oxidation (FAO), mitotic cell cycle, and histone modification. The peroxin Pex14 was downregulated in the IUGR pancreas as confirmed by western blotting and q-PCR. Pmp70, a peroxisomal membrane protein involved in the transport of fatty acids, was upregulated. Hsd17b4 and Acox1/2, which catalyze different steps of peroxisomal FAO, were dysregulated. Sod plasma concentrations in the IUGR fetus were higher than those in the control, suggesting partial compensation for oxidative stress. Multiple DEPs were related to the regulation of the cell cycle, including reduced Cdk1, Mcm2, and Brd4. The histone acetylation regulators Hdac1/2 were downregulated, whereas Sirt1/3 and acetylated H3K56 were increased in the IUGR fetal pancreas. Conclusion. The present study identified DEPs in the fetal pancreas of IUGR rats by proteomic analysis. Downregulation of pancreas peroxins and dysregulation of enzymes involved in peroxisomal FAO may impair the biogenesis and function of the peroxisome and may underlie the development of T2 diabetes mellitus in adult IUGR rats. Disorders of cell cycle regulators may induce cell division arrest and lead to smaller islets. The present data provide new insight into the role of the peroxisome in the development of the pancreas and may be valuable in furthering our understanding of the pathogenesis of IUGR-induced diabetes.

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An automaton model for the cell cycle

Interface Focus ◽

10.1098/rsfs.2010.0009 ◽

2010 ◽

Vol 1 (1) ◽

pp. 36-47 ◽

Cited By ~ 28

Author(s):

Atilla Altinok ◽

Didier Gonze ◽

Francis Lévi ◽

Albert Goldbeter

Keyword(s):

Cell Cycle ◽

Steady State ◽

Deterministic Model ◽

Initial Conditions ◽

Cell Number ◽

Time Step ◽

M Phase ◽

Random Manner ◽

The Mean ◽

Cell Cycle Phases

We consider an automaton model that progresses spontaneously through the four successive phases of the cell cycle: G1, S (DNA replication), G2 and M (mitosis). Each phase is characterized by a mean duration τ and a variability V . As soon as the prescribed duration of a given phase has passed, the transition to the next phase of the cell cycle occurs. The time at which the transition takes place varies in a random manner according to a distribution of durations of the cell cycle phases. Upon completion of the M phase, the cell divides into two cells, which immediately enter a new cycle in G1. The duration of each phase is reinitialized for the two newborn cells. At each time step in any phase of the cycle, the cell has a certain probability to be marked for exiting the cycle and dying at the nearest G1/S or G2/M transition. To allow for homeostasis, which corresponds to maintenance of the total cell number, we assume that cell death counterbalances cell replication at mitosis. In studying the dynamics of this automaton model, we examine the effect of factors such as the mean durations of the cell cycle phases and their variability, the type of distribution of the durations, the number of cells, the regulation of the cell population size and the independence of steady-state proportions of cells in each phase with respect to initial conditions. We apply the stochastic automaton model for the cell cycle to the progressive desynchronization of cell populations and to their entrainment by the circadian clock. A simple deterministic model leads to the same steady-state proportions of cells in the four phases of the cell cycle.

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