Exposure to hyperbaric oxygen induces cell cycle perturbation in prostate cancer cells

Summary Cell cycle synchronization of tumor cells by exposure to hyperbaric oxygenation (HBO) may increase the efficacy of chemotherapy or radiation by placing cells into a chemosensitive portion of the cycle. The purpose of the current study was to examine oxygen pressure-dependent relationships with respect to the cell cycle in prostate tumor cells in vitro. LNCaP cells were grown in an incubator at 21% O₂ and then exposed to 100% oxygen at pressures up to 6 atmospheres (atm) for 1.5 h. Cells were then returned to the incubator and evaluated for DNA content by propidium iodide and new DNA synthesis with a pulse-chase experiment. Cell cycle effects were evaluated by flow cytometry. Exposure to HBO increased the percentage of cells synthesizing new DNA in a dose-dependent fashion: 0 atm, 44%; 6 atm, 65%. Cells that synthesize new DNA accumulate in G₂/M as a function of partial pressure of oxygen. These results suggest that HBO induces cells to enter the cell cycle and accumulate in G₂/M. Cell cycle synchronization and entry of senescent cells into the cell cycle suggest that HBO may be a useful adjuvant to chemotherapy or radiation in the treatment of prostate cancer. There are two potential mechanisms of action that may make HBO efficacious in the treatment of prostate cancer. HBO may potentiate cancer chemotherapeutic agents that cause damage to DNA during DNA synthesis or HBO may inhibit cell division causing accumulation in G₂/M.     

Tyrosine phosphorylations specific to mitosis in human and hamster cells

Changes in protein tyrosine phosphorylation are known to be important for regulating cell cycle progression. With the aim of identifying new proteins involved in the regulation of mitosis, we used an antibody against phosphotyrosine to analyze proteins from synchronized human and hamster cells. At least seven proteins were found that displayed mitosis-specific tyrosine phosphorylation in HeLa cells (pp165, 205, 240, 250, 270, 290, and ～ 400) and one such protein in hamster BHK cells (pp155). In synchronized HeLa and BHK cells, all proteins except HeLa pp165, pp205, and pp250 were readily detectable only in mitosis. Tyrosine phosphorylation of pp165, pp205, and pp250 was apparent during arrest in S phase, suggesting that cell cycle perturbations can affect the phosphorylation state of some of these proteins. In a related finding in BHK cells, pp155 underwent tyrosine phosphorylation when cells were forced into premature mitosis by caffeine treatment. Only one protein (pp135 in HeLa cells) was found to be dephosphorylated on tyrosine during mitosis. The above findings may prove helpful for isolating new cell cycle proteins that are important for both the normal regulation of mitosis and the mitotic aberrations associated with cell cycle perturbations and chemical treatments.     

Ras-dependent cell cycle commitment during G2 phase

Synchronization used to study cell cycle progression may change the characteristics of rapidly proliferating cells. By combining time-lapse, quantitative fluorescent microscopy and microinjection, we have established a method to analyze the cell cycle progression of individual cells without synchronization. This new approach revealed that rapidly growing NIH3T3 cells make a Ras-dependent commitment for completion of the next cell cycle while they are in G2 phase of the preceding cell cycle. Thus, Ras activity during G2 phase induces cyclin D1 expression. This expression continues through the next G1 phase even in the absence of Ras activity, and drives cells into S phase.     

Loss of p53 expression in cancer cells alters cell cycle response after inhibition of exportin-1 but does not prevent cell death

The tumor suppressor protein p53 is central to the cellular stress response and may be a predictive biomarker for cancer treatments. Upon stress, wildtype p53 accumulates in the nucleus where it enforces cellular responses, including cell cycle arrest and cell death. p53 is so dominant in its effects, that p53 enforcement – or – restoration therapy is being studied for anti-cancer therapy. Two mechanistically distinct small molecules that act via p53 are the selective inhibitor of nuclear export, selinexor, and MDM2 inhibitor, nutlin-3a. Here, individual cells are studied to define cell cycle response signatures, which captures the variability of responses and includes the impact of loss of p53 expression on cell fates. The individual responses are then used to build the population level response. Matched cell lines with and without p53 expression indicate that while loss-of-function results in altered cell cycle signatures to selinexor treatment, it does not diminish overall cell loss. On the contrary, response to single-agent nutlin-3a shows a strong p53-dependence. Upon treatment with both selinexor and nutlin-3a there are combination effects in at least some cell lines – even when p53 is absent. Collectively, the findings indicate that p53 does act downstream of selinexor and nutlin-3a, and that p53 expression is dispensable for selinexor to cause cell death, but nutlin-3a response is more p53-dependent. Thus, TP53 disruption and lack of expression may not predict poor cell response to selinexor, and selinexor’s mechanism of action potentially provides for strong efficacy regardless of p53 function.     

角膜内皮细胞再生研究新进展

人角膜内皮细胞的主要功能是维持角膜透明性,角膜内皮单层发育成熟形成细胞接触后,内皮细胞会停止分裂增殖,但并没有退出细胞周期。角膜内皮细胞的增殖有多种因素的参与和影响,接触抑制和G1期抑制使细胞增殖暂时停止;细胞因子TGF-β2抑制人角膜内皮细胞进入细胞周期S期,而EGF、FGF、NGF则能够促进细胞的增殖;ROCK抑制剂Y-27632能够促进角膜内皮细胞的粘连,有助于内皮细胞的损伤修复。体外培养角膜内皮前体细胞、诱导多潜能干细胞向角膜内皮细胞分化,为今后治疗角膜内皮失代偿提供了新方向。     

microRNA-16-5p enhances radiosensitivity through modulating Cyclin D1/E1–pRb–E2F1 pathway in prostate cancer cells

Prostate cancer (CaP) is the second most common cancer in men worldwide in 2012, and radiation therapy is one of the most common definitive treatment options for localized CaP. However, radioresistance is a major challenge for the current radiotherapy, accumulating evidences suggest microRNAs (miRNAs), as an important regulator in cellular ionizing radiation (IR) responses, are closely correlated with radiosensitivity in many cancers. Here, we identified microRNA-16-5p(miR-16-5p) is significantly upregulated in CaP LNCaP cells following IR and can enhance radiosensitivity through modulating Cyclin D1/E1–pRb–E2F1 pathway. To identify the expression profile of miRNAs in CaP cells exposed to IR, we performed human miRNA probe hybridization chip analysis and miR-16-5p was found to be significantly overexpressed in all treatment groups that irradiated with different doses of X-rays and heavy ions (¹²C⁶⁺). Furthermore, overexpression of miR-16-5p suppressed cell proliferation, reduced cell viability, and induced cell cycle arrest at G0/G1 phase, resulting in enhanced radiosensitivity in LNCaP cells. Additionally, miR-16-5p specifically targeted the Cyclin D1/E1–3′-UTR in LNCaP cells and affected the expression of Cyclin D1/E1 in both mRNA and protein levels. Taken together, miR-16-5p enhanced radiosensitivity of CaP cells, the mechanism may be through modulating Cyclin D1/Cyclin E1/pRb/E2F1 pathway to cause cell cycle arrest at G0/G1 phase. These findings provided new insight into the correlation between miR-16-5p, cell cycle arrest, and radiosensitivity in CaP, revealed a previously unrecognized function of miR-16-5p–Cyclin D1/E1–pRb–E2F1 regulation in response to IR and may offer an alternative therapy to improve the efficiency of conventional radiotherapy.     

Fluorescence kinetics in HeLa cells after treatment with cell cycle arrest inducers visualized with Fucci (fluorescent ubiquitination-based cell cycle indicator)

Fucci (fluorescent ubiquitination-based cell cycle indicator) is able to visualize dynamics of cell cycle progression in live cells; G1- and S-/G2-/M-phase cells expressing Fucci emit red and green fluorescence, respectively. This system could be applied to cell kinetic analysis of tumour cells in the field of cancer therapy; however, it is still unclear how fluorescence kinetics change after various treatments, including exposure to anticancer agents. To explore this, we arrested live HeLa cells expressing the Fucci probes at various cell cycle stages and observed the fluorescence, in conjunction with flow cytometric analysis. X-irradiation, HU (hydroxyurea) and nocodazole arrest cells at G2/M boundary, early S-phase and early M-phase, respectively. Although X-irradiation and HU treatment induced similar accumulation kinetics of green fluorescent cells, nocodazole treatment induced an abnormal red fluorescence at M phase, followed by accumulation of both red and green fluorescent cells with 4N DNA content. We conclude that certain agents that disrupt normal cell cycle regulation could cause unexpected fluorescence kinetics in the Fucci system.     

Relationship between cell proliferation, cell-cycle phase, and retroviral vector production in FLYRD18 human packaging cells

The relatively low concentrations of retroviral vectors produced by most packaging cells requires the optimization and intensification of their production to make a commercially viable product for gene therapy. While a number of reports exist concerning target cell-cycle effects on retroviral vector infection efficiency, no studies have been reported on the effects of packaging cell cycle on vector production. We have studied the effect of proliferation of the human packaging cell line, FLYRD18, on vector production. In addition, the titer levels of vector produced by cells in each phase of the cell cycle were compared. Numerous studies suggested progression of the cells through the cell cycle to be essential for vector production. However, vector release was found not to be predominant in any particular phase of the cell cycle. These findings indicate that packaging cell proliferation is important for optimal virus production and that arrest of the cells in any particular phase of the cell cycle affords no benefits in retroviral vector production. In contrast to previous reports (using other cell lines), we observed no temporary inhibition of cell cycle progression after detachment of cells from their substratum and that virus production occurred immediately after re-plating of the cells. The findings in this report are important for determining the optimal culture conditions for vector production by packaging cells in vitro.     

Apoptosis and cell cycle arrest of hepatocellular carcinoma spheroids treated by an alternating electric field

Most of the current cancer therapies may induce serious side effects and affect patient quality of life. Recently, a novel treatment using an alternating low-intensity and intermediate-frequency electric field was proposed and found to be a noninvasive and minimally toxic approach. However, additional fundamental studies and scientific evidence are required to further support the development of this treatment into a standard cancer therapy. In the current work, an in-house fabricated culture plate was developed to study the responses of hepatocellular carcinoma spheroids to treatment with an alternating electric field. From the results of the viability study, the electric field was confirmed to influence the dividing cells in the spheroids. Fluorescent staining of live and dead cells revealed that a fraction of the cells were damaged in the field-treated spheroids. Moreover, flow cytometry analyses were conducted and showed that a fraction of the cells in the spheroids underwent apoptosis and cell cycle arrest. Additionally, the apoptosis pathway (Bax/caspase) and cell cycle arrest pathway (p53/p21) were found to be activated after exposure to the electric field. In summary, the results further elucidated the cellular and molecular mechanism inducing apoptosis and cell cycle arrest in the field-treated hepatocellular carcinoma spheroids. This study provides more evidence to support the efficacy of electric-field-based cancer therapy.     

Antiproliferative activity of CCN3: involvement of the C-terminal module and post-translational regulation

Previous work had suggested that recombinant CCN3 was partially inhibiting cell proliferation. Here we show that native CCN3 protein secreted into the conditioned medium of glioma transfected cells indeed induces a reduction in cell proliferation. Large amounts of CCN3 are shown to accumulate both cytoplasmically and extracellularly as cells reach high density, therefore highlighting new aspects on how cell growth may be regulated by CCN proteins. Evidence is presented establishing that the amount of CCN3 secreted into cell culture medium is regulated by post-translational proteolysis. As a consequence, the production of CCN3 varies throughout the cell cycle and CCN3 accumulates at the G2/M transition of the cycle. We also show that CCN3-induced inhibition of cell growth can be partially reversed by specific antibodies raised against a C-terminal peptide of CCN3. The use of several clones expressing various portions of CCN3 established that the CT module of CCN3 is sufficient to induce cell growth inhibition.     

番茄红素对胰腺alpha和beta细胞的差异作用

番茄红素是一种强抗氧化剂,在糖尿病的治疗实验中显示出对机体的保护作用,但是其细胞机制尚不明确。本研究采用不同浓度梯度的番茄红素处理胰腺alpha和beta细胞系,检测细胞的生长、凋亡、周期、活性氧、ATP水平和相关细胞因子的表达变化。结果显示,番茄红素不影响alpha细胞的生长、凋亡、周期、活性氧和ATP水平,但番茄红素可以促进beta细胞的生长、上调其S期比例、降低活性氧水平并提升ATP水平。与此同时,番茄红素可以提升beta细胞tnfα、tgfβ和hif1αmRNA的表达。这些结果表明番茄红素的作用具有细胞特异性,可以活化胰腺beta细胞,为其在糖尿病防治的临床应用提供数据支撑。     

The modulation of radiation-induced cell death by genistein in K562 cells：Activation of thymidine kinase 1

Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML) cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization (SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression ofTK1 mRNA and TK 1 enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition, the expression of cell cycle-related proteins, cyclin A and cyclin B 1, provided the evidences of G I/S progression and G2-arrest, and their relationship with TKI in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.     

Analysis of the Cell Cycle and a Method Employing Synchronized Cells for Study of Protein Expression at Various Stages of the Cell Cycle

Study of protein expression during the cell cycle requires preparation of pure fractions of cells at various phases of the cell cycle. This was achieved by the development of methods for cell synchronization. Successful cell synchronization requires knowledge of the duration of all phases of the cell cycle. So, in the present review these interrelated problems are considered together. The first part of this review deals with basic methods employed for analysis of duration of cell cycle phases. The second summarizes data on treatments used for cell synchronization. Methods for calculation of percent of cells at various stages of the cell cycle in fractions of synchronized cells are considered in the third part. The fourth part of this review deals with a method of study of protein expression during the cell cycle by means of immunoblotting of synchronized cell fractions. In the Appendix, basic principles are illustrated with practical examples of analysis of the cell cycle, synchronization, and study of expression of some proteins at various stages of the cell cycle using synchronized XL2 (Xenopus laevis) cells.     

Blocking the NOTCH pathway can inhibit the growth of CD133-positive A549 cells and sensitize to chemotherapy

Cancer stem cells (CSCs) are believed to play an important role in tumor growth and recurrence. These cells exhibit self-renewal and proliferation properties. CSCs also exhibit significant drug resistance compared with normal tumor cells. Finding new treatments that target CSCs could significantly enhance the effect of chemotherapy and improve patient survival. Notch signaling is known to regulate the development of the lungs by controlling the cell-fate determination of normal stem cells. In this study, we isolated CSCs from the human lung adenocarcinoma cell line A549. CD133 was used as a stem cell marker for fluorescence-activated cell sorting (FACS). We compared the expression of Notch signaling in both CD133+ and CD133− cells and blocked Notch signaling using the γ-secretase inhibitor DAPT (GSI-IX). The effect of combining GSI and cisplatin (CDDP) was also examined in these two types of cells. We observed that both CD133+ and CD133− cells proliferated at similar rates, but the cells exhibited distinctive differences in cell cycle progression. Few CD133+ cells were observed in the G₂/M phase, and there were half as many cells in S phase compared with the CD133− cells. Furthermore, CD133+ cells exhibited significant resistance to chemotherapy when treated with CDDP. The expression of Notch signaling pathway members, such as Notch1, Notch2 and Hes1, was lower in CD133+ cells. GSI slightly inhibited the proliferation of both cell types and exhibited little effect on the cell cycle. The inhibitory effects of DPP on these two types of cells were enhanced when combined with GSI. Interestingly, this effect was especially significant in CD133+ cells, suggesting that Notch pathway blockade may be a useful CSC-targeted therapy in lung cancer.     

Pocket proteins and cell cycle regulation in inner ear development

Loss of neurosensory cells of the ear, caused by genetic and non-genetic factors, is becoming an increasing problem as people age, resulting in deafness and vestibular disorders. Unveiling useful mechanisms of cell cycle regulation may offer the possibility to generate new cells out of remaining ones, thus providing the cellular basis to induce new hair cell differentiation in the mammalian ear. Here, we provide an overview of cell cycle regulating genes in general and of those studied in the ear in particular. We categorize those genes into regulators that act upstream of the pocket proteins and into those that act downstream of the pocket proteins. The three members of the pocket protein family essentially determine, through interaction with the eight members of the E2F family, whether or not the cell cycle will progress to the S-phase and thus cell division. The abundant presence of one or more members of these families in adult hair cells supports the notion that inhibition of cell cycle progression through these proteins is a lifelong process. Indeed, manipulating some of those proteins, unfortunately, leads to abortive entry into the cell cycle. Combined with recent success to induce hair cell differentiation through molecular therapy, these approaches may provide a viable strategy to restore lost hair cells in the inner ear.     

Safeguarding clinical translation of pluripotent stem cells with suicide genes

The generation of human induced pluripotent stem cells (hiPSCs) opens a new avenue in regenerative medicine. However, transplantation of hiPSC-derived cells carries a risk of tumor formation by residual pluripotent stem cells. Numerous adaptive strategies have been developed to prevent or minimize adverse events and control the in vivo behavior of transplanted stem cells and their progeny. Among them, the application of suicide gene modifications, which is conceptually similar to cancer gene therapy, is considered an ideal means to control wayward stem cell progeny in vivo. In this review, the choices of vectors, promoters, and genes for use in suicide gene approaches for improving the safety of hiPSCs-based cell therapy are introduced and possible new strategies for improvements are discussed. Safety-enhancing strategies that can selectively ablate undifferentiated cells without inducing virus infection or insertional mutations may greatly aid in translating human pluripotent stem cells into cell therapies in the future.     

Cell cycle and apoptosis: Common pathways to life and death

Programmed cell death, or apoptosis, is a highly regulated process used to eliminate unwanted or damaged cells from multicellular organisms. The morphology of cells undergoing apoptosis is similar to cells undergoing both normal mitosis and an aberrant form of mitosis called mitotic catastrophe. During each of these processes, cells release substrate attachments, lose cell volume, condense their chromatin, and disassemble the nuclear lamina. The morphological similarities among cells undergoing these processes suggest that the underlying biochemical changes also may be related. The susceptibility of cells to apoptosis frequently depends on the differentiation state of the cell. Additionally, cell cycle checkpoints appear to link the cell cycle to apoptosis. Deregulation of the cell cycle components has been shown to induce mitotic catastrophe and also may be involved in triggering apoptosis. Some apoptotic cells express abnormal levels of cell cycle proteins and often contain active Cdc2, the primary kinase active during mitosis. Although cell cycle components may not be involved in all forms of apoptosis, in many instances cell proliferation and cell death may share common pathways.     

Enhancing therapeutic efficacy by targeting non-oncogene addicted cells with combinations of signal transduction inhibitors and chemotherapy

The effects of inhibition of the Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways and chemotherapeutic drugs on cell cycle progression and drug sensitivity were examined in cytokine-dependent FL5.12 hematopoietic cells. We examined their effects, as these cells resemble normal hematopoietic precursor cells as they do not exhibit “oncogene-addicted” growth, while they do display “cytokine-addicted” proliferation as cytokine removal resulted in apoptosis in greater than 80% of the cells within 48 h. When cytokine-dependent FL5.12 cells were cultured in the presence of IL-3, which stimulated multiple proliferation and anti-apoptotic cascades, MEK, PI3K and mTOR inhibitors transiently suppressed but did not totally inhibit cell cycle progression or induce apoptosis while chemotherapeutic drugs such as doxorubicin and paclitaxel were more effective in inducing cell cycle arrest and apoptosis. Doxorubicin induced a G₁ block, while paclitaxel triggered a G₂/M block. Doxorubicin was more effective in inducing cell death than paclitaxel. Furthermore the effects of doxorubicin could be enhanced by addition of MEK, PI3K or mTOR inhibitors. Cytokine-dependent cells which proliferate in vitro and are not “oncogene-addicted” may represent a pre-malignant stage, more refractory to treatment with targeted therapy. However, these cells are sensitive to chemotherapeutic drugs. It is important to develop methods to inhibit the growth of such cytokine-dependent cells as they may resemble the leukemia stem cell and other cancer initiating cells. These results demonstrate the enhanced effectiveness of targeting early hematopoietic progenitor cells with combinations of chemotherapeutic drugs and signal transduction inhibitors.     

Possible role of cell cycle-dependent morphology,geometry, and mechanical properties in tumor cell metastasis

Studies that examine the shear- and abrasion-sensitivity of proliferating cells are important in order to understand the behavior of hybridoma cells in bioreactor culture and metastasizing cancer cells in the bloodstream. Little is known about the link between morphology, structure, and mechanical properties of a given cell line, especially with respect to variations throughout the cell cycle. In our experiments with GAP A3 hybridoma cells, distinct cell morphologies were identified and correlated with phases of the cell cycle by video microscopic observation of synchronized cells, and of individual cells that were followed throughout their cell cycle. Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (apparent cell viscosity) properties of single cells. As the cell cycle progressed at 37°C, an increase in cell volume from 1400 μm³ to 5700 μm³ was accompanied by an increase in apparent cell viscosity from 430 poise to 12,000 poise, consistent with an accumulation of more cytoplasmic material in the “older” cells. Hybridomas are representative of the various leukemias derived from hemopoietic cells, and even though as a whole, they appeared to be rather shear-insensitive, the wide range of property values demonstrates that a given cell line cannot be characterized by a single value for any one property, and that properties must be related to the cell cycle when considering proliferating cells. It is interesting to see if distinct stages in the metastatic sequence of events might correlate with any of these physical features of the cell cycle, irrespective of cell type or cell line. For example, the cytokinetic doublet could represent a fragile structure that may fail and produce cell death under fluid-shear conditions that would not affect the cells at any other stage in the cell cycle. Identifying such cell cycle-dependent features in metastasizing cancer cells could lead to a better understanding of the metastatic process and to possible clinical treatments directed at making cells more shear- and abrasion-sensitive, and therefore, more likely to be killed by the natural hydrodynamic forces of the circulatory system.