Shedding Light on Intracellular Proteins using Flow Cytometry.

Intracellular protein abundance is routinely measured in mammalian cells using population-based techniques such as western blotting which fail to capture single cell protein levels or using fluorescence microscopy which is although suitable for single cell protein detection but not for rapid analysis of large no. of cells. Flow cytometry offers rapid, high-throughput, multiparameter-based analysis of intracellular protein expression in statistically significant no. of cells at single cell resolution. In past few decades, customized assays have been developed for flow cytometric detection of specific intracellular proteins. This review discusses the scope of flow cytometry for intracellular protein detection in mammalian cells along with specific applications. Technological advancements to overcome the limitations of traditional flow cytometry for the same are also discussed.

Dynamic Roles of Signaling Pathways in Maintaining Pluripotency of Mouse and Human Embryonic Stem Cells.

Culturing of mouse and human embryonic stem cells (ESCs) in vitro was a major breakthrough in the field of stem cell biology. These models gained popularity very soon mainly due to their pluripotency. Evidently, the ESCs of mouse and human origin share typical phenotypic responses due to their pluripotent nature, such as self-renewal capacity and potency. The conserved network of core transcription factors regulates these responses. However, significantly different signaling pathways and upstream transcriptional networks regulate expression and activity of these core pluripotency factors in ESCs of both the species. In fact, ample evidence shows that a pathway, which maintains pluripotency in mouse ESCs, promotes differentiation in human ESCs. In this review, we discuss the role of canonical signaling pathways implicated in regulation of pluripotency and differentiation particularly in mouse and human ESCs. We believe that understanding these distinct and at times-opposite mechanisms-is critical for the progress in the field of stem cell biology and regenerative medicine.

Molecular Milieu of Autophagy in Cervical Cancer and its Therapeutic Implications.

Cervical cancer is a common death-causing cancer among women in developing countries. Majority of the cases are triggered by persistent infections with high-risk Human Papillomavirus (HPV16 and 18). Metastasis, disease relapse, and drug resistance are common among patients in advanced stages of cancer despite the available therapies. Consequently, new prospective targets are needed for this disease. Autophagy is professed to have implications in cervical cancer progression as well as cancer dormancy. This article reviews the role of autophagy in cervical cancer progression and the modulation of the autophagy pathway by HPV. Further, various therapeutic agents that target autophagy in cervical cancer are discussed.

Molecular pharmacology of multitarget cyclin-dependent kinase inhibitors in human colorectal carcinoma cells.

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer death. Certain signaling pathways are implicated in colorectal carcinogenesis. Cyclin-dependent kinases (CDKs) are commonly hyperactivated in CRC and hence multitarget CDK inhibitors serve as promising therapeutic drugs against CRC. OBJECTIVE: Off-target effects of multitarget CDK inhibitors with differential CDK inhibitory spectrum viz. P276-00 (also known as riviciclib), roscovitine and UCN-01 on CRC cell lines of varied genetic background were delineated. METHOD: Protein expression was analyzed for key signaling proteins by western blotting. ß-catenin localization was assessed using immunofluorescence. HIF-1 transcriptional activity and target gene expression were studied by reporter gene assay and RT-PCR respectively. Anti-migratory and anti-angiogenic potential was evaluated by wound healing assay and endothelial tube formation assay. RESULTS: CDK inhibitors modulated various signaling pathways in CRC and for certain proteins showed a highly cell line-dependent response. Riviciclib and roscovitine inhibited HIF-1 transcriptional activity and HIF-1α accumulation in hypoxic HCT116 cells. Both of these drugs also abrogated migration of HCT116 and in vitro angiogenesis in HUVECs. CONCLUSION: Anticancer activity of multitarget CDK inhibitors can be certainly attributed to their off-target effects and should be analyzed while assessing their therapeutic utility against CRC.

The story of EGFR: from signaling pathways to a potent anticancer target.

EGFR is a member of the ERBB family. It plays a significant role in cellular processes such as growth, survival and differentiation via the activation of various signaling pathways. EGFR deregulation is implicated in various human malignancies, and therefore EGFR has emerged as an attractive anticancer target. EGFR inhibition using strategies such as tyrosine kinase inhibitors and monoclonal antibodies hinders cellular proliferation and promotes apoptosis in cancer cells in vitro and in vivo. EGFR inhibition by tyrosine kinase inhibitors has been shown to be a better treatment option than chemotherapy for advanced-stage EGFR-driven non-small-cell lung cancer, yet de novo and acquired resistance limits the clinical benefit of these therapeutic molecules. This review discusses the cellular signaling pathways activated by EGFR. Further, current therapeutic strategies to target aberrant EGFR signaling in cancer and mechanisms of resistance to them are highlighted.

Cyclin-dependent kinases as potential targets for colorectal cancer: past, present and future.

Colorectal cancer (CRC) is a common cancer in the world and its prevalence is increasing in developing countries. Deregulated cell cycle traverse is a hallmark of malignant transformation and is often observed in CRC as a result of imprecise activity of cell cycle regulatory components, viz. cyclins and cyclin-dependent kinases (CDKs). Apart from cell cycle regulation, some CDKs also regulate processes such as transcription and have also been shown to be involved in colorectal carcinogenesis. This article aims to review cyclin-dependent kinases as potential targets for CRC. Furthermore, therapeutic candidates to target CDKs are also discussed.

Promising Anticancer Activity of Multitarget Cyclin Dependent Kinase Inhibitors against Human Colorectal Carcinoma Cells.

BACKGROUND: Colorectal cancer (CRC) is the third leading cause of cancer death worldwide, and its incidence is steadily rising in developing nations. Cell cycle aberrations due to deregulation of cyclin dependent kinases (CDKs) and cyclins are common events during colorectal carcinogenesis. Yet, efficacy of multitarget CDK inhibitors as therapeutic agents has not been much explored against CRC. OBJECTIVE: The anticancer potential of multitarget CDK inhibitor riviciclib (also known as P276-00), was investigated against CRC cell lines of varied genetic background. METHODS: Cytotoxicity of riviciclib - potent CDK1, CDK4 and CDK9-specific inhibitor was evaluated in vitro. Further, its effect on clonogenic potential, cell cycle, apoptosis and transcription was tested using colony forming assay, flow cytometry and western blot analysis, respectively. Also, efficacy of riviciclib in combination with standard chemotherapeutic agents was assessed. Dependency of CRC cells on specific CDKs for their survival was confirmed using siRNA studies. RESULTS: Riviciclib exerted significant cytotoxicity against CRC cells and inhibited their colony forming potential. It induced apoptosis along with inhibition of cell cycle CDKs and cyclins as well as transcriptional CDKs and cyclins. Moreover, dual combination of riviciclib with standard chemotherapeutic drugs exhibited synergism in CRC cells. siRNA studies indicated that CRC cells are dependent on specific CDKs for their survival which are targets of riviciclib. CONCLUSION: This study provides evidence that multitarget CDK inhibitors can serve as promising therapeutic agents against CRC alone or in combination.

A flow cytometric journey into cell cycle analysis.

Cell cycle involves a series of changes that lead to cell growth and division. Cell cycle analysis is crucial to understand cellular responses to changing environmental conditions. Since its inception, flow cytometry has been particularly useful for cell cycle analysis at single cell level due to its speed and precision. Previously, flow cytometric cell cycle analysis relied solely on the measurement of cellular DNA content. Later, methods were developed for multiparametric analysis. This review explains the journey of flow cytometry to understand different molecular and cellular events underlying cell cycle using various protocols. Recent advances in the field that overcome the shortcomings of traditional flow cytometry and expand its scope for cell cycle studies are also discussed.

Flow cytometry: principles, applications and recent advances.

Flow cytometry (FCM) is a sophisticated technique that works on the principle of light scattering and fluorescence emission by the specific fluorescent probe-labeled cells as they pass through a laser beam. It offers several unique advantages as it allows fast, relatively quantitative, multiparametric analysis of cell populations at the single cell level. In addition, it also enables physical sorting of the cells to separate the subpopulations based on different parameters. In this constantly evolving field, innovative technologies such as imaging FCM, mass cytometry and Raman FCM are being developed in order to address limitations of traditional FCM. This review explains the general principles, main applications and recent advances in the field of FCM.

Combining fluorescent cell barcoding and flow cytometry-based phospho-ERK1/2 detection at short time scales in adherent cells.

Detection of levels of intracellular phospho-proteins is key to analyzing the dynamics of signal transduction in cellular systems. Cell-to-cell variability in the form of differences in protein level in each cell affects signaling and is implicated in prognosis of many diseases. Quantitative analysis of such variability necessitate measuring the protein levels at single-cell resolution. Single-cell intracellular protein abundance detection in statistically significant number of adherent cells for short time sampling points post stimulation using classical flow cytometry (FCM) technique has thus far been a challenge due to the detrimental effects of cell detachment methods on the cellular machinery. We systematically show that cell suspension obtained by noninvasive temperature-sensitive detachment of adherent cells is amenable to high-throughput phospho-ERK1/2 protein detection at single-cell level using FCM in these short time sampling points. We demonstrate this on three adherent cell lines, viz., HeLa, A549, and MCF7, from distinct lineages having characteristically different elasticity at 37 °C. In particular, we use a right combination of multiplexing via fluorescent cell barcoding (FCB) and intracellular antibody staining for simultaneous detection of phospho-ERK1/2 (pERK) stimulated by epidermal growth factor (EGF) in multiple samples. Based on systematic characterization using Alexa 350 dye, we arrive at two conditions that must be satisfied for correct implementation of FCB. Our study reveals that the temperature-sensitive detachment of HeLa cells correctly captures the expected pronounced bimodal pERK distribution as an early response to EGF, which the enzymatic treatment methods fail to detect. © 2018 International Society for Advancement of Cytometry.