Live-cell Studies Research Articles

Dual photon excitation microscopy and image threshold segmentation in live cell imaging during compression testing

Morphological studies of live connective tissue cells are imperative to helping understand cellular responses to mechanical stimuli. However, photobleaching is a constant problem to accurate and reliable live cell fluorescent imaging, and various image thresholding methods have been adopted to account for photobleaching effects. Previous studies showed that dual photon excitation (DPE) techniques are superior over conventional one photon excitation (OPE) confocal techniques in minimizing photobleaching. In this study, we investigated the effects of photobleaching resulting from OPE and DPE on morphology of in situ articular cartilage chondrocytes across repeat laser exposures. Additionally, we compared the effectiveness of three commonly-used image thresholding methods in accounting for photobleaching effects, with and without tissue loading through compression. In general, photobleaching leads to an apparent volume reduction for subsequent image scans. Performing seven consecutive scans of chondrocytes in unloaded cartilage, we found that the apparent cell volume loss caused by DPE microscopy is much smaller than that observed using OPE microscopy. Applying scan-specific image thresholds did not prevent the photobleaching-induced volume loss, and volume reductions were non-uniform over the seven repeat scans. During cartilage loading through compression, cell fluorescence increased and, depending on the thresholding method used, led to different volume changes. Therefore, different conclusions on cell volume changes may be drawn during tissue compression, depending on the image thresholding methods used. In conclusion, our findings confirm that photobleaching directly affects cell morphology measurements, and that DPE causes less photobleaching artifacts than OPE for uncompressed cells. When cells are compressed during tissue loading, a complicated interplay between photobleaching effects and compression-induced fluorescence increase may lead to interpretations in cell responses to mechanical stimuli that depend on the microscopic approach and the thresholding methods used and may result in contradictory interpretations.

A Method for Improving the Optical Properties of a Fluoregenic Di-metal Chelator as a Zn2+Ion Fluorescent Probe by Using a Bridging Substrate

Fluorescent probe based detection methods for metal ions are indispensable tools in many fields, including medical diagnostics, environmental monitoring, living cell studies, and electronics. These methods have multiple advantages over other methods, such as high sensitivity, low cost, ease of application, and versatility. Numerous fluorescent metal ion probes have been designed using many strategies, including fluorogenic metal chelators, fluorescent dye tagged oligonucleotides, catalytic signal amplification, and chemodosimetors. Fluorogenic metal chelators, which consist of a fluorogenic unit (signaling site) covalently linked to chelating moieties (receptor units) with an appropriate spacer, are a general type metal ion probe. The recognition of chelating moieties with metal ions induces a change in the photophysical properties of the fluorescent probe. This is converted into an optical signal expressed as an enhancement or quenching of the fluorophore emission. The recognition can be enhanced to utilize additive reagents that provide additional binding sites for metal ions. For example, 8-aminoquinolino-β-cyclodextrin, developed by Liu et al., exhibited cooperative binding to Zn ion with 1-adamatanoic acid. It also detected Zn ions more efficiently than without 1adamatanoic acid because 1-adamatanoic acid bound to βcyclodextrin to provide additional binding site for Zn ions. This strategy can easily expand to improve the optical properties of metal ion sensors with two metal binding sites. Di-metal complexes may include bridging substrates to complete the metal coordination sphere, and bridging substrates can modulate the properties of the resulting cascade complexes. In particular, bridging substrates provided additional metal ion binding sites and enhanced the binding properties of metal ions to metal ion ligands. Various fluorescent molecules with two metal binding sites have been synthesized as efficient probes for detecting metal ions, such as Zn and Ag. Scaffolds of di-metalic fluoregenic probes have also been devised to detect anions. Most of these molecules can be used as metal ion probes. The optical properties of these molecules can be improved by bridging substrates, which provide additional metal ion binding sites. To validate this concept, (9,10-bis[(2,2-dipicolylamino)methyl]anthracene) (1) was adapted as a Zn ion probe because [Zn2(1)] 4+ efficiently detects phosphate derivatives as potential bridging substrates to provide additional metal binding sites. In various phosphate derivatives, ATP was adapted as a bridging substrate to provide additional metal binding sites because [Zn2(1)] 4+ fluorescence is most enhanced by binding with ATP. Therefore, a combination of 1 and ATP (1/ATP) was prepared as a Zn fluorescent probe. The optical properties of 1/ATP, compared to those of 1, can be improved by providing additional binding sites for metal ions using ATP, as depicted in Scheme 1. To determine the optimal ratio of 1 and ATP, fluorescence titration of ATP was conducted using a 5 μM solution of 1 with 2 equivalents of Zn ions. The fluorescence emission spectra of [Zn2(1)] 4+ in the presence of varied ATP concentrations were evaluated. [Zn2(1)] 4+ fluorescence increased nearly proportionally to ATP concentration and was maximized at one equivalent of ATP (see Supporting information). A sensing system (1/ATP) consiting of 1 (5 μM) and ATP (5 μM) was prepared to evaluate the cooperative binding effect of the bridging substrate by providing additional binding sites for Zn ions. Fluorescence changes of 1/ATP in the presence of various concentrations of Zn were examined. The addition of Zn ions induced greatly enhanced fluorescence. The binding zinc ions within the bis-(2-picolyl)amine in 1 prevents the photo induced electron transfer process

Calcein-effluxing human colon cancer cells are enriched for self-renewal capacity and depend on β-catenin

Putative cancer stem cells are a subpopulation of cancer cells that give rise to chemotherapy resistance and are therefore of prognostic and therapeutic interest, though their identification remains elusive in colon cancer due to lack of reliable and accurate markers. We previously identified a p53-dependent putative cancer stem cell population, the calcein low population (C(lo)P), based on their exclusive efflux of the fluorescent dye Calcein. This functional identification method enables comparative live cell studies of subpopulations without differential toxicity that occurs with traditional Hoechst methods, which has confounded conclusions and limited the utility of this cancer stem cell marker. In this study, we examined the cancer stem cell-like properties of the C(lo)P population in vivo in comparison with the parental and calcein-high population (C(hi)P) in human colon cancer xenografts. Serial dilution xenograft experiments in NOD/SCID mice revealed that the C(lo)P is only marginally more tumorigenic compared to the C(hi)P or parental cells. However, serial passage of these tumors revealed that the C(lo)P is uniquely enriched for self-renewal capacity in vivo compared to the other populations. Immunohistochemical analysis of these tumors revealed that the C(lo)P possesses increased levels of nuclear β-catenin and furthermore, siRNA-mediated knockdown of β-catenin significantly reduced the C(lo)P population. These findings highlight the C(lo)P as an important subpopulation of tumor cells that are exclusively endowed with the ability to self-renew and propagate tumors. The dependency of the C(lo)P on β-catenin provides a molecular explanation for this ability and suggests that this population can and should be therapeutically targeted by inhibition of Wnt signaling.

Single-Molecule Fluorescence Imaging in Living Cells

The transition of single-molecule fluorescence detection and imaging from in vitro to living cells has greatly enriched our knowledge on the behavior of single biomolecules in their native environments and their roles in cellular processes. Here we review recent advances of single-molecule biophysical approaches to live-cell studies based on fluorescence imaging. We start by discussing the practical considerations in designing single-molecule fluorescence imaging in cells, including the choice of fluorescent probes, labeling methods, instrumentation, and imaging techniques. We then describe representative examples in detail to illustrate the physicochemical parameters that can be obtained by imaging individually labeled biomolecules in cells and what can be learned from such characterizations.

Rab11-family interacting proteins define spatially and temporally distinct regions within the dynamic Rab11a-dependent recycling system.

The Rab11-family interacting proteins (Rab11-FIPs) facilitate Rab11-dependent vesicle recycling. We hypothesized that Rab11-FIPs define discrete subdomains and carry out temporally distinct roles within the recycling system. We used live-cell deconvolution microscopy of HeLa cells expressing chimeric fluorescent Rab11-FIPs to examine Rab11-FIP localization, transferrin passage through Rab11-FIP-containing compartments, and overlap among Rab11-FIPs within the recycling system. FIP1A, FIP2, and FIP5 occupy widely distributed mobile tubules and vesicles, whereas FIP1B, FIP1C, and FIP3 localize to perinuclear tubules. Internalized transferrin entered Rab11-FIP-containing compartments within 5 min, reaching maximum colocalization with FIP1B and FIP2 early in the time course, whereas localization with FIP1A, FIP1C, FIP3, and FIP5 was delayed until 10 min or later. Whereas direct interactions with FIP1A were only observed for FIP1B and FIP1C, FIP1A also associated with membranes containing FIP3. Live-cell dual-expression studies of Rab11-FIPs revealed the tubular dynamics of Rab11-FIP-containing compartments and demonstrated a series of selective associations among Rab11-FIPs in real time. These findings suggest that Rab11-FIP1 proteins participate in spatially and temporally distinct steps of the recycling process along a complex and dynamic tubular network in which Rab11-FIPs occupy discrete domains.

Recent advances in fluorescent nucleic acid probes for living cell studies

Living cell studies can offer tremendous opportunities for biological and disease studies. Due to their high sensitivity and selectivity, minimum interference with living biological systems, ease of design and synthesis, fluorescent nucleic acid probes (FNAPs) have been widely used in living cell studies, such as for intracellular detection, cell detection, and cell-to-cell communication. Here, we review the general requirements and the recent developments in FNAPs for living cell studies. We broadly classify these designs as hybridization probes and aptamer probes. For hybridization probes, we describe recently developed designs, such as nanomaterial-based and amplification-based hybridization probes. For aptamer probes, we discuss four general paradigms that have appeared most frequently in the literature: nanomaterial-based, nanomachine-based, cell surface-anchored and activatable aptamer probe designs in vivo. FNAPs promise to open up new and exciting opportunities in biological marks detection for a wide range of biological and medical applications.

Aberration-free FTIR spectroscopic imaging of live cells in microfluidic devices

The label-free, non-destructive chemical analysis offered by FTIR spectroscopic imaging is a very attractive and potentially powerful tool for studies of live biological cells. FTIR imaging of live cells is a challenging task, due to the fact that cells are cultured in an aqueous environment. While the synchrotron facility has proven to be a valuable tool for FTIR microspectroscopic studies of single live cells, we have demonstrated that high quality infrared spectra of single live cells using an ordinary Globar source can also be obtained by adding a pair of lenses to a common transmission liquid cell. The lenses, when placed on the transmission cell window, form pseudo hemispheres which removes the refraction of light and hence improve the imaging and spectral quality of the obtained data. This study demonstrates that infrared spectra of single live cells can be obtained without the focus shifting effect at different wavenumbers, caused by the chromatic aberration. Spectra of the single cells have confirmed that the measured spectral region remains in focus across the whole range, while spectra of the single cells measured without the lenses have shown some erroneous features as a result of the shift of focus. It has also been demonstrated that the addition of lenses can be applied to the imaging of cells in microfabricated devices. We have shown that it was not possible to obtain a focused image of an isolated cell in a droplet of DPBS in oil unless the lenses are applied. The use of the approach described herein allows for well focused images of single cells in DPBS droplets to be obtained.

Dynamics of the HP1β-PCNA-containing complexes in DNA replication and repair

Heterochromatin protein 1 (HP1), a small non-histone chromosomal protein, was recently shown to form a complex in vivo with Proliferating Cell Nuclear Antigen (PCNA), a key factor in DNA replication. The complex, which requires HP1β in a form of a dimer, is engaged in DNA repair and replication. We now provide further evidence based on FRET-FLIM live cell studies confirming the association and close proximity between HP1β and PCNA in the complex. We also demonstrate using FRAP, that although HP1β-PCNA complexes are highly mobile in nonreplicating nuclei, when engaged in DNA replication, they become bound and do not exchange with the mobile pool. These observations are in agreement with a notion that a subpopulation of HP1 molecules interact with PCNA in vivo during DNA replication. Similarly, HP1β which is associated with PCNA in regions of DNA repair, is bound and does not exchange with the mobile pool, suggesting that HP1β in association with PCNA may be a component of a DNA repair complex.

Correcting the Effect of Refraction and Dispersion of Light in FT-IR Spectroscopic Imaging in Transmission through Thick Infrared Windows

Transmission mode is one of the most common sampling methods for FT-IR spectroscopic imaging because the spectra obtained generally have a reasonable signal-to-noise ratio. However, dispersion and refraction of infrared light occurs when samples are sandwiched between infrared windows or placed underneath a layer of liquid. Dispersion and refraction cause infrared light to focus with different focal lengths depending on the wavelength (wavenumber) of the light. As a result, images obtained are in focus only at a particular wavenumber while they are defocused at other wavenumber values. In this work, a solution to correct this spread of focus by means of adding a lens on top of the infrared transparent window, such that a pseudo hemisphere is formed, has been investigated. Through this lens (or pseudo hemisphere), refraction of light is removed and the light across the spectral range has the same focal depth. Furthermore, the lens acts as a solid immersion objective and an increase of both magnification and spatial resolution (by 1.4 times) is demonstrated. The spatial resolution was investigated using an USAF resolution target, showing that the Rayleigh criterion can be achieved, as well as a sample with a sharp polymer interface to indicate the spatial resolution that can be expected in real samples. The reported approach was used to obtain chemical images of cross sections of cancer tissue and hair samples sandwiched between infrared windows showing the versatility and applicability of the method. In addition to the improved spatial resolution, the results reported herein also demonstrate that the lens can reduce the effect of scattering near the edges of tissue samples. The advantages of the presented approach, obtaining FT-IR spectroscopic images in transmission mode with the same focus across all wavenumber values and simultaneous improvement in spatial resolution, will have wide implications ranging from studies of live cells to sorption of drugs into tissues.

Cysteineless non-glycosylated monomeric blue fluorescent protein, secBFP2, for studies in the eukaryotic secretory pathway

Fluorescent protein (FP) technologies suitable for use within the eukaryotic secretory pathway are essential for live cell and protein dynamic studies. Localization of FPs within the endoplasmic reticulum (ER) lumen has potentially significant consequences for FP function. All FPs are resident cytoplasmic proteins and have rarely been evolved for the chemically distinct environment of the ER lumen. In contrast to the cytoplasm, the ER lumen is oxidizing and the site where secretory proteins are post-translationally modified by disulfide bond formation and N-glycosylation on select asparagine residues. Cysteine residues and N-linked glycosylation consensus sequences were identified within many commonly utilized FPs. Here, we report mTagBFP is post-translationally modified when localized to the ER lumen. Our findings suggest these modifications can grossly affect the sensitivity and reliability of FP tools within the secretory pathway. To optimize tools for studying events in this important intracellular environment, we modified mTagBFP by mutating its cysteines and consensus N-glycosylation sites. We report successful creation of a secretory pathway-optimized blue FP, secBFP2.

Transferrin receptor specific nanocarriers conjugated with functional 7peptide for oral drug delivery

In an attempt to increase the interaction of a nanocarrier system with gastrointestinal epithelial cells, a transferrin receptor (TfR) specific 7peptide was conjugated to PEG-b-PCL copolymer and the functional nanocarriers were constructed and characterized. The endocytosis, intracellular trafficking and transcytosis of such nanocarriers loaded with coumarin 6 (7pep-M-C6) in a human colon carcinoma cell line (Caco-2) were investigated, followed by the in vivo intestine distribution study. The real-time imaging of live cell, three dimensional reconstruction of confocal image, quantitative colocalization analysis and other techniques were applied. First, the TfR expression was confirmed in Caco-2. Then, 7pep-M-C6 exhibited higher intracellular uptake compared with unmodified nanocarriers. In a live cell study, 7pep-M-C6 demonstrated faster uptake kinetics especially in the surface of cells. Together with a competition study using TfR antibody, it was proved that the increased cellular uptake was due to a receptor-mediated mechanism. Besides the unspecific endocytosis pathway, 7pep-M-C6 was found to enter the cells through a specific clathrin-mediated mechanism, related to the expression of TfR on Caco-2 cells. Possibly for this reason, 7pep-M-C6 tended to colocalize more with late endosomes and lysosomes than the control micelles. Also for the same mechanism, the increased transport of 7pep-M-C6 across Caco-2 monolayer was found, through a transcellular but not a paracellular pathway, while an increased in vivo intestinal distribution of 7pep-M-C6 was observed. In conclusion, the functional nanocarriers could specifically interact with gastrointestinal endothelial cells, increase their transport and alter their pathway as a result.

Live‐Cell Studies of p300/CBP Histone Acetyltransferase Activity and Inhibition

Histone acetyltransferase enzymes (HATs) are important therapeutic targets, but there are few cell-based assays available for evaluating the pharmacodynamics of HAT inhibitors. Here we present the application of a FRET-based reporter, Histac, in live-cell studies of p300/CBP HAT inhibition, by both genetic and pharmacologic disruption. shRNA knockdown of p300/CBP led to increased Histac FRET, thus suggesting a role for p300/CBP in the acetylation of the histone H4 tail. Additionally, we describe a new p300/CBP HAT inhibitor, C107, and show that it can also increase cellular Histac FRET. Taken together, these studies provide a live-cell strategy for identifying and evaluating p300/CBP inhibitors.

Melanosomal Dynamics Assessed with a Live-Cell Fluorescent Melanosomal Marker

Melanocytes present in skin and other organs synthesize and store melanin pigment within membrane-delimited organelles called melanosomes. Exposure of human skin to ultraviolet radiation (UV) stimulates melanin production in melanosomes, followed by transfer of melanosomes from melanocytes to neighboring keratinocytes. Melanosomal function is critical for protecting skin against UV radiation, but the mechanisms underlying melanosomal movement and transfer are not well understood. Here we report a novel fluorescent melanosomal marker, which we used to measure real-time melanosomal dynamics in live human epidermal melanocytes (HEMs) and transfer in melanocyte-keratinocyte co-cultures. A fluorescent fusion protein of Ocular Albinism 1 (OA1) localized to melanosomes in both B16-F1 cells and HEMs, and its expression did not significantly alter melanosomal distribution. Live-cell tracking of OA1-GFP-tagged melanosomes revealed a bimodal kinetic profile, with melanosomes exhibiting combinations of slow and fast movement. We also found that exposure to UV radiation increased the fraction of melanosomes exhibiting fast versus slow movement. In addition, using OA1-GFP in live co-cultures, we monitored melanosomal transfer using time-lapse microscopy. These results highlight OA1-GFP as a specific and effective melanosomal marker for live-cell studies, reveal new aspects of melanosomal dynamics and transfer, and are relevant to understanding the skin’s physiological response to UV radiation.

Overview of Live‐Cell Imaging: Requirements and Methods Used

Overview of Live‐Cell Imaging: Requirements and Methods Used

Photothermal Microscopy of the Core of Dextran-Coated Iron Oxide Nanoparticles During Cell Uptake

A detailed understanding of cellular interactions with superparamagnetic iron oxide nanoparticles (SPIONs) is critical when their biomedical applications are considered. We demonstrate how photothermal microscopy can be used to follow the cellular uptake of SPIONs by direct imaging of the iron oxide core. This offers two important advantages when compared with current strategies employed to image magnetic cores: first, it is nondestructive and is therefore suitable for studies of live cells and, second, it offers a higher sensitivity and resolution, thus allowing for the identification of low levels of SPIONs within a precise subcellular location. We have shown that this technique may be applied to the imaging of both cell monolayers and cryosections. In the former we have demonstrated the role of temperature on the rate of endocytosis, while in the latter we have been able to identify cells labeled with SPIONs from a mixed population containing predominantly unlabeled cells. Direct imaging of the SPION core is of particular relevance for research involving clinically approved SPIONs, which do not contain fluorescent tags and therefore cannot be detected via fluorescence microscopy.

Analysis of Protein Turnover by Quantitative SNAP‐Based Pulse‐Chase Imaging

Assessment of protein dynamics in living cells is crucial for understanding their biological properties and functions. The SNAP-tag, a self labeling suicide enzyme, presents a tool with unique features that can be adopted for determining protein dynamics in living cells. Here we present detailed protocols for the use of SNAP in fluorescent pulse-chase and quench-chase-pulse experiments. These time-slicing methods provide powerful tools to assay and quantify the fate and turnover rate of proteins of different ages. We cover advantages and pitfalls of SNAP-tagging in fixed- and live-cell studies and evaluate the recently developed fast-acting SNAPf variant. In addition, to facilitate the analysis of protein turnover datasets, we present an automated algorithm for spot recognition and quantification.

Modulation of Growth Factor Receptors in Membrane Microdomains

Membrane lateral heterogeneity is accepted as a requirement for the function of biological membranes, and the notion of the "raft/microdomain" gives specificity to this concept. Recently, fluorescence-based techniques such as fluorescence recovery after photobleaching (FRAP), single particle tracking (SPT), and fluorescence correlation spectroscopy (FCS) have shown promise for application to the dynamics of membrane molecules in microdomains. We previously revealed, by performing live-cell FRAP and SPT studies, a mechanism of insulin resistance in which dissociation of the insulin receptor (IR)-caveolin-1 (Cav1) complex was caused by an interaction between the IRβ subunit and the ganglioside GM3 cluster, a glycolipid-enriched membrane microdomain. We hoped to demonstrate that an alteration in the lipid component of microdomains affects lateral diffusion of membrane receptors. We therefore established an experimental system for monitoring the membrane organization of receptors by analyzing their lateral diffusion parameters in the plasma membranes of living cells using FRAP and SPT. In this study, measurement of the lateral diffusion of the IR was performed by fitting analysis to fluorescence recovery curves and trace analysis to individual fluorescent spots, which provided the diffusion constant. The results show how fast IR molecules diffuse before and after a change in membrane environment, such as stimulation by cholesterol depression or treatment with a glycosphingolipid (GSL) inhibitor. Using these techniques, we have established a method for determining the diffusion constant for the lateral movement of IR-EGFP, expressed in CHO-K1 cells. We will use these techniques for the lateral diffusion analysis of membrane receptors under other assay conditions, such as use of GSL-deficient cells or pathologic samples.

Cell-Based Proteome Profiling of Potential Dasatinib Targets by Use of Affinity-Based Probes

Protein kinases (PKs) play an important role in the development and progression of cancer by regulating cell growth, survival, invasion, metastasis, and angiogenesis. Dasatinib (BMS-354825), a dual Src/Abl inhibitor, is a promising therapeutic agent with oral bioavailability. It has been used for the treatment of imatinib-resistant chronic myelogenous leukemia (CML). Most kinase inhibitors, including Dasatinib, inhibit multiple cellular targets and do not possess exquisite cellular specificity. Recent efforts in kinase research thus focus on the development of large-scale, proteome-wide chemical profiling methods capable of rapid identification of potential cellular (on- and off-) targets of kinase inhibitors. Most existing approaches, however, are still problematic and in many cases not compatible with live-cell studies. In this work, we have successfully developed a cell-permeable kinase probe (DA-2) capable of proteome-wide profiling of potential cellular targets of Dasatinib. In this way, highly regulated, compartmentalized kinase-drug interactions were maintained. By comparing results obtained from different proteomic setups (live cells, cell lysates, and immobilized affinity matrix), we found DA-2 was able to identify significantly more putative kinase targets. In addition to Abl and Src family tyrosine kinases, a number of previously unknown Dasatinib targets have been identified, including several serine/threonine kinases (PCTK3, STK25, eIF-2A, PIM-3, PKA C-α, and PKN2). They were further validated by pull-down/immunoblotting experiments as well as kinase inhibition assays. Further studies are needed to better understand the exact relevance of Dasatinib and its pharmacological effects in relation to these newly identified cellular targets. The approach developed herein should be amenable to the study of many of the existing reversible drugs/drug candidates.

H2-Calponin Dynamics and Function in Cytokinesis

H2-calponin (h2-CaP) is an actin filament-associated protein and plays a role in regulating cytoskeletal function such as phagocytosis, cell migration and cell division (Hossain et al., AJP Cell Physiol 284:C156-67,2003; Huang et al., JBC 283:25887-99, 2008). Our previous studies showed that the levels of h2-CaP affected the rate of cell proliferation. To understand the mechanisms, we investigated a hypothesis that regulated expression and degradation of h2-CaP is critical to the normal process of cytokinesis. Western blotting and immunofluorescence microscopic studies showed that the levels of endogenous h2-CaP in NIH3T3 cells and HEK 293 cells decreased prior to cell division characterized by an absence in the F-actin-based contractile ring. GFP-fusion h2-calponin was constructed for dynamic studies in live cells. Control experiments using NIH3T3 cells and HEK 293 cells showed minimal difference between non-fusion and GFP-fusion h2-CaP in cytoskeleton association and effects on cell proliferation. A smooth muscle-originated cell line SM3 lacking endogenous calponin was transiently transfected with a GFP-h2-CaP expression plasmid and imaged using fluorescent microscopy at a series of time points. The results showed that 1) the level of h2-CaP decreased prior to cell division, especially at the site contractile ring; 2) the overall level of h2-CaP expression had an inverted correlation to the duration of cytokinesis; and 3) cells had high level transfective expression of h2-CaP could not complete cytokinesis, which was followed by cell death. The data suggest that h2-CaP is a factor regulating cytokinesis. This novel function of h2-CaP may be targeted to control cell proliferation during development, wound healing, inflammation, pathological remodeling, and tumor growth and metastasis.

Hepatocellular carcinoma stem cells origins and roles in hepatocarcinogenesis and disease progression

Hepatocellular carcinoma (HCC) is a treatment-resistant malignancy with an increasing incidence worldwide. More than 500,000 individuals suffer from this disease annually. Risk factors for human HCC include hepatitis B and C infections, dietary aflatoxin, alcohol abuse, smoking, and oral contraceptive use. Accumulating evidence suggests that liver stem cells play a critical role in HCC development and progression. Dedifferentiated hepatocytes, hepatic oval cells and bone marrow cells are the three major types of liver stem cells, and CD133, CD90, and EpCAM are identified as specific antigenic markers for HCC stem cells. Wnt, Hedgehog, and the angiogenic signalings are main pathways that regulate the HCC stem cell self-renewal and pluripotential, and may be potential targets for novel therapeutic strategies of this malignancy. This review article provides an update in the studies of live and HCC stem cells.