Ecdysone and the cell cycle: Investigations in a mosquito cell line

Cell lines provide a tool for investigating basic biological processes that underlie the complex interactions among the tissues and organs of an intact organism. We compare the evolution of insect and mammalian populations as they progress from diploid cell strains to continuous cell lines, and review the history of the well-characterized Aedes albopictus mosquito cell line, C7-10. Like Kc and S3 cells from Drosophila melanogaster, C7-10 cells are sensitive to the insect steroid hormone, 20-hydroxyecdysone (20E), and express 20E-inducible proteins as well as the EcR and USP components of the ecdysteroid receptor. The decrease in growth associated with 20E treatment results in an accumulation of cells in the G1 phase of the cycle, and a concomitant decrease in levels of cyclin A. In contrast, 20E induces a G2 arrest in a well-studied imaginal disc cell line from the moth, Plodia interpunctella. We hypothesize that 20E-mediated events associated with molting and metamorphosis include effects on regulatory proteins that modulate the mitotic cell cycle and that differences between the 20E response in diverse insect cell lines reflect an interplay between classical receptor-mediated effects on gene expression and non-classical effects on signaling pathways similar to those recently described for the vertebrate steroid hormone, estrogen.     

Dual effects of estrogen on vascular smooth muscle cells: receptor-mediated proliferative vs. metabolite-induced pro-senescent actions

Objective

To investigate the mechanism for the dual effects of estrogen on vascular smooth muscle cells (VSMCs).

Methods

Cultured rat VSMCs were exposed to gradient concentrations (10⁻⁹-10⁻⁵ M) of 17β-estradiol (E₂) with or without pre-administration of a broad-spectrum CYP450 inhibitor 1-aminobenzotriazole (ABT) (10 × 10⁻⁶ M) and an estrogen receptor (ER) antagonist ICI 182,780 (10⁻⁶ M), respectively. The growth, cell cycle progression, premature senescence, estrogen metabolites, reactive oxygen species (ROS) and DNA damage of the cells were analyzed with cell counting assay, flow cytometry, Western blot, liquid chromatography-mass spectrometry and comet assay, respectively.

Results

E₂ in its physiological levels from 10⁻⁹ M to 10⁻⁸ M had a concentration-dependent promoting effect on growth of VSMCs. However, when the concentration increased over 10⁻⁸ M, the growth-promoting effect gradually reversed to a growth-inhibiting action. When the activity of CYP450s was blocked by ABT, the growth-promoting effect of E₂ increased and did not reverse at high concentrations. Whereas when the ERs were blocked by ICI 182,780, E₂ showed a pure growth-inhibiting effect. The E₂ metabolites 2- and 4-hydroxyestradiols accumulated with the increase of E₂ over 10⁻⁸ M, which accompanied by increased ROS, DNA damage and cellular senescence. All of these changes were eliminated by block of CYP450s, indicating that the VSMC growth inhibition by E₂ is due to an increased production of ROS from accumulated E₂ metabolites which induces DNA damage, leading to VSMC premature senescence.

Conclusion

The complex effect of E₂ is due to two opposite actions: one ER-mediated and proliferative, and the other estrogen metabolite-induced and pro-senescent.     

The in vitro growth of a cord blood–derived cell population enriched for CD34+ cells is influenced by its cell cycle status and treatment with hydroxyurea

Objective

Cell cycle plays a fundamental role in the physiology of hematopoietic stem and progenitor cells. In the present study we used a negative selection system to obtain an immature cell population—enriched for cord blood–derived CD34⁺ cells—and we determined its proliferation, expansion and differentiation patterns as a function of the cell cycle status. The effects of hydroxyurea (HU) were also assessed.

Accumulation of lead by free and immobilized cyanobacteria with special reference to accumulation factor and recovery

Lead accumulation by free and immobilized cyanobacteria, Lyngbya majuscula and Spirulina subsalsa was studied. Exponentially growing biomass was exposed to 1-20 mg L⁻¹ of Pb(II) solution at pH 6, 7 and 8 for time periods ranging from 10 min to 48 h. L. majuscula accumulated 10 times more Pb (13.5 mg g⁻¹) than S. subsalsa (1.32 mg g⁻¹) at pH 6 within 3 h of exposure to 20 mg L⁻¹ Pb(II) solution and 76% of the Pb could be recovered using 0.1 M EDTA. This chelator (2 μM) did not influence Pb accumulation whereas 100 μM citrate increased that of S. subsalsa 6- to 8-fold. L. majuscula filaments enmeshed in a glass wool packed in a column removed 95.8% of the Pb from a 5 mg L⁻¹ Pb solution compared to free and dead biomass which removed 64 and 33.6% Pb respectively. A 92.5% recovery of accumulated Pb from the immobilized biomass suggests that repeated absorption-desorption is possible.     

Cell cycle parameters inAedes albopictus mosquito cells

Summary We have analyzed cell cycle parameters for theAedes albopictus C7-10 mosquito cell line, which has been systematically developed for somatic cell genetics, expression of transfected genes, and synthesis of hormone-inducible proteins. In rapidly cycling cells, we measured a generation time of 10–12 h. The duration of mitosis (M) was ≤1 h, and the DNA synthesis phase (S) required 6 h. UnlikeDrosophila melanogaster Kc cells, in which the G2 gap is substantially longer than G1, in C7-10 cells G1 and G2 each lasted approximately 2h. In these cells, the duration of both S and G2 was independent of the population doubling time, and the increase in population doubling time as cells approached confluency was due to prolongation of G1. When treated with the insect steroid hormone, 20-hydroxyecdysone, C7-10 mosquito cells complete the cycle in progress before undergoing a reversible arrest.     

Parathyroid hormone and the regulation of cell cycle in colon adenocarcinoma cells

Parathyroid hormone (PTH) functions as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. In this study, we investigated the role of PTH in the regulation of the cell cycle in human colon adenocarcinoma Caco-2 cells. Flow cytometry analysis revealed that PTH (10^− 8 M, 12-24 h) treatment increases the number of cells in the G0/G1 phase and diminishes the number in both phases S and G2/M. In addition, analysis by Western blot showed that the hormone increases the expression of the inhibitory protein p27Kip1 and diminishes the expression of cyclin D1, cyclin D3 and CDK6. However, the amounts of CDK4, p21Cip1, p15INK4B and p16INK4A were not different in the absence or presence of PTH. Inhibitors of PKC (Ro-318220, bisindolylmaleimide and chelerythine), but not JNK (SP600125) and PP2A (okadaic acid and calyculin A), reversed PTH response in Caco-2 cells. Taken together, our results suggest that PTH induces G0/G1 phase arrest of Caco-2 intestinal cells and changes the expression of proteins involved in cell cycle regulation via the PKC signaling pathway.     

Modulation of fibroblast response to maitotoxin along the cell division cycle

Maitotoxin (MTX) induces an increase of [Ca²⁺]_i and of phosphoinositide breakdown in various cell types. The [Ca²⁺]_i increase followed with fluorescent probes on cell suspensions has been described as slow and lasting, in contrast to the signal induced by calcium ionophores such as ionomycin. MTX effects have been studied on two fibroblastic cell lines, BHK21 C13 and FR 3T3, synchronized by serum deprivation treatment performed in an isoleucine-free medium for BHK21 C13 cells. In BHK21 C13 cells, flow cytometry analysis showed that two stages, G1/S and G2/M, were particularly susceptible to MTX treatment. Scanning laser cytometry demonstrated that calcium response of FR 3T3 fibroblasts followed with Indo-1 varied during the cell division cycle. The [Ca²⁺]_i increase was almost always vertical, but its delay after MTX addition lasted from zero (S and G2/M transition) to 10–20 min (G1) or more (G2). No [Ca²⁺]_i change could be detected during mitosis. The [Ca²⁺]_i response at the S phase was biphasic. These observations suggest that (1) the lasting response described in the literature represents a global cell population effect, and (2) cells are more sensitive to MTX at specific stages of the cell division cycle, which could correspond to periods when calcium signals have been detected in different cell types.Abbreviations MTX maitotoxin - [Ca²⁺]_i intracellular calcium concentration - IP₃ inositol triphosphate     

D-Lys-GHRP-6 antagonizes the effect of unacylated but not of acylated ghrelin on the growth of HECa10 murine endothelial cells

Recent studies demonstrate that ghrelin can be an endogenous regulator of angiogenesis. We studied direct effects of human acylated (hAG) and unacylated (hUAG) ghrelin, as well as of rat acylated ghrelin (rAG) on the growth of HECa10 murine endothelial cells. Ghrelin was applied separately or together with D-Lys³-GHRP-6, which is commonly used as an antagonist of ghrelin receptor type 1a – GHS-R1a. The growth of HECa10 cells was assessed with Mosmann and in selected study conditions also with BrdU and TUNEL methods. Both hAG and hUAG (10⁻⁵ M to 10⁻¹² M) inhibited the growth of HECa10 cells in 24 h and 72 h cultures. Similarly, rAG decreased the growth of the cells after 24 h (10⁻⁷ M and 10⁻¹¹ M), and after 72 h (10⁻⁷ M, 10⁻⁸ M and 10⁻¹¹ M). Unexpectedly, D-Lys³-GHRP-6 itself also inhibited the growth of these cells at 10⁻⁴ to 10⁻⁶ M in 24 h, 48 h (dose–response effect) and 72 h cultures. D-Lys³-GHRP-6 did not modify the inhibitory effect of rAG. However, D-Lys³-GHRP-6 at the concentration of 10⁻⁴ M diminished, abolished or even reversed the inhibitory effect of hUAG in 72 h culture and this was dependent on ghrelin concentrations. These data indicate that both AG and UAG have antiangiogenic properties at least at the level of endothelial growth, through decreased metabolic activity of the cells or stimulation of apoptosis. D-Lys³-GHRP-6 (inhibitor of GHS-R1a) seems not to be an appropriate antagonist in this experimental condition. Similar effects of these substances on HECa10 cells suggest that they are not mediated by GHS-R1a.     

Signaling related with biphasic effects of bisphenol A (BPA) on Sertoli cell proliferation: A comparative proteomic analysis

Background

Biphasic effects on cell proliferation of bisphenol A (BPA) can occur at lesser or greater exposures. Sertoli cells play a pivotal role in supporting proliferation and differentiation of germ cells. The mechanisms responsible for inverse effects of great and low concentrations of BPA on Sertoli cell proliferation need further study.

Methods

We utilized proteomic study to indentify the protein expression changes of Sertoli TM4 cells treated with 10^− 8 M and 10^− 5 M BPA. The further mechanisms related to mitochondria, energy metabolism and oxidative stress were investigated by qRT-PCR and Western-blotting analysis.

Results

Proteomic studies identified 36 proteins and two major clusters of proteins including energy metabolism and oxidative stress expressed with opposite changes in Sertoli cells treated with 10^− 8 M and 10^− 5 M BPA, respectively, for 24 h. Exposure to 10^− 5 M BPA resulted in greater oxidative stress and then inhibited cell proliferation, while ROS scavenger NAC effectively blocked these effects. Exposure to 10^− 8 M BPA caused higher intercellular ATP, greater activities of mitochondria, and resulted in significant proliferation of TM4 cells, while oligomycin A, an inhibitor of ATP synthase, abolished these growth advantages.

Conclusions

Our study demonstrated that micromolar BPA inhibits proliferation of Sertoli cells by elevating oxidative stress while nanomolar BPA stimulates proliferation by promoting energy metabolism.

General significance

Micromolar BPA inhibits cell proliferation by elevating oxidative stress while nanomolar BPA stimulates cell proliferation by promoting energy metabolism.     

Adenine-induced inhibition of Na(+)-ATPase activity: Evidence for involvement of the Gi protein-coupled receptor in the cAMP signaling pathway

In the present work, we demonstrate that adenine reduced Na⁺-ATPase activity in isolated basolateral membrane (BLM) of proximal tubule in a dose-dependent manner. Adenine metabolism was ruled out by TLC analysis of the potential [³H]adenine derived-metabolites. Specific binding of [³H]adenine to isolated BLM was observed in a dose-dependent manner with K_d and B_max of 242.6 ± 27.6 nM and 2749.9 ± 104.9 fmol mg⁻¹, respectively. Adenine increased the [³⁵S]GTPγS specific binding and it was completely abolished by 10⁻⁶ M GDPβS (G protein inhibitor) but it was not modified by DPCPX, DMPX and MRS1523, selective antagonists for A₁, A₂ and A₃ receptors, respectively. Furthermore, the inhibitory effect of adenine on the Na⁺-ATPase activity was blocked by 10⁻⁶ M GDPβS, 1 μg/ml pertussis toxin (Gi protein inhibitor), 10⁻⁶ M foskolin (adenylyl cyclase activator) and 10⁻⁸ M cAMP. These data demonstrate that adenine inhibits the proximal tubule Na⁺-ATPase activity through the Gi protein-coupled receptor.     

Increased stringency of the 1,25-dihydroxyvitamin D3-induced G1 to S phase block in polyploid HL60 cells

Treatment of mammalian cells with 1,25-dihydroxyvitamin D₃ (1,25D₃) produces a G1 to S (G1/S) phase cell cycle block. In addition, it has been noted that a smaller proportion of cells accumulates in the G2/M compartment in 1,25D₃-treated cultures. Since cyclins have a major influence on the regulation of cell cycle progression, we determined the expression of cyclins A and B as markers of the G2 phase and of cyclin E as the marker of G1/S transition. No increase in the steady-state levels of cyclin A or cyclin B mRNA was detected in the total cell population or in the cyclin B1 protein in the G2/M cell cycle compartment. In contrast, immunodetectable cyclin E protein was increased in cell cultures as a whole and specifically in the G2/M compartment cells. Determination of BrdU incorporation into DNA by flow cytometry showed marked inhibition of DNA replication in cells with DNA content higher than 4C, and autoradiography of ³H-TdR-pulsed cells showed that polynucleated cells did not replicate DNA after 96 h of treatment with 1,25D₃ or analogs. Taken together, these experiments show that at least a portion of the G2/M compartment in 1,25D₃-arrested cultures of HL60 cells represents G1 cells at a higher ploidy level, which are blocked from entering the high ploidy S phase. © 1996 Wiley-Liss, Inc.     

Characteristics of the cell cycle of matrix cells in the mouse embryo during histogenesis of telencephalon

The cell cycle of matrix cells in the telencephalon of the mouse embryo at different stages at day 10, 13, and 17 of gestation was investigated by means of ³H-thymidine autoradiography.The cell cycle time of matrix cells in the day 10 group was found to be 7.0 h, and lengthened linearly with embryonic age. The cell cycle times of day 13 and 17 groups were 15.5 and 26.0 h, respectively.The duration of G1 and S phases also lengthened linearly with embryonic age. The durations of G1 phase were 0.1, 6.8, and 13.8 h, for day 10, 13, and 17 groups, respectively, and those of S phase were 5.1, 6.9, and 10.4 h, for day 10, 13, and 17 groups, respectively. On the other hand, the durations of both G2 and M phases remained unchanged and these were 1.0 and 0.8 h, respectively, throughout the embryonic stages.It was a characteristic of the alteration of the cell cycle of the telencephalon during mouse embryonic life that not only G1 but also S phases lengthened linearly with embryonic age and both G2 and M phases remained constant.     

Late Signals from the PDGF Receptors Leading to the Activation of the p70-Kinase Are Necessary for the Transition from G1 to S phase in AKR-2B Cells

Platelet-derived growth factor AB (PDGF-AB) has to be permanently present in the culture medium to achieve full proliferation (>90%) of AKR-2B fibroblasts. Upon removal after 1 h incubation time, only a small number of cells (<20%) entered the cell cycle. Concomitantly there was no increase in RNA- and protein-synthesis. The PDGF-receptor autophosphorylation reached a maximum after 30 min incubation with PDGF-AB. Tyrosine phosphorylation was no longer detectable after 2–4 h. The clustering of receptors into coated pits, analyzed by indirect immunofluorescence using a specific antibody against PDGF-β-receptor, showed in contrast to autophosphorylation a biphasic kinetic. A first maximum was reached after 30 min, followed by a complete disappearance of coated pits, which regenerated in a second phase after 3 h and were long lasting. If PDGF-AB was removed after 1 h, the second phase was obliterated.The involvement of two different signalling pathways in these two phases was investigated in detail: (1) The ras-raf-MAP-kinase pathway and (2) the PI-3-kinase/p70^S6-kinase pathway. PDGF-AB addition caused a fast (10 min) activation of MAP-kinase, which returned to background level after 1 h without any further activation later on. In contrast PDGF-AB led to a rapid (15–30 min) activation of the p70^S6-kinase that persisted for 8–12 h just prior to the entry of the cells into S-phase. If PDGF-AB was removed after 1 h, the activation of this kinase ceased 3 h later. PDGF-AA, which is unable to promote division of AKR-2B cells, induced only a shortlasting p70^S6-kinase activation. These observations add further evidence for the involvement of the p70^S6-kinase pathway in the proliferation control of AKR-2B fibroblasts in the late G1 phase (4–8 h after growth factor addition). On the other hand, if the p70^S6-kinase activation was prevented by the addition of 10 nM rapamycin, the cell division was not inhibited but only delayed by 4 h. Similar kinetics were observed when the PI-3-kinase was inhibited by 400 nM wortmannin. It is suggested that a regulatory element exists upstream of the p70^S6-kinase and the PI-3-kinase. This regulatory element should be responsible for the transmission of late signals required for the progression through the cell cycle. This element is not involved in the immediate responses after PDGF-AB addition but must be stimulated within a second later phase of PDGF activation.     

The p70S6K Specific Inhibitor PF-4708671 Impedes Non-Small Cell Lung Cancer Growth

Background

As a serine/threonine protein kinase, p70S6K plays an important role in tumor cells. Evidence has revealed overexpression of p70S6K and phosphorylated p70S6K (p-p70S6K) in various tumor tissues, with these proteins identified as independent prognostic markers in non-small cell lung cancer (NSCLC). In this study, we explored the role of the p70S6K specific inhibitor PF-4708671 in NSCLC.

Methods

Three NSCLC cell lines (A549, SK-MES-1, and NCI-H460) were treated with PF-4708671 at five different concentrations, including 0.1μM, 0.3μM, 1μM, 3μM and 10μM, and protein levels were determined by Western-blot. Then, PF-4708671’s effects were assessed both in vitro (cell proliferation, apoptosis, cell cycle distribution, and invasion) and in vivo.

Results

The expression levels of p-p70S6K and the downstream effector S6 were significantly reduced by PF-4708671. Diametrically opposite, the downstream protein levels of BAD, Caspase3 and ERK had increased after treatment with PF-4708671. In addition, PF-4708671 drastically inhibited cell proliferation and invasion ability in A549, SK-MES-1 and NCI-H460 cells in vitro, causing cell cycle arrest in G0-G1 phase. Limited effects of PF-4708671 were observed on apoptosis in the three NSCLC cell lines assessed. Importantly, PF-4708671 could inhibit tumorigenesis in nude mice in vivo.

Conclusion

These findings demonstrated that the p70S6K specific inhibitor PF-4708671 has inhibitory effects on NSCLC tumorigenesis in vitro and in vivo. Therefore, P70S6K should be considered a new potential therapeutic target, and PF-470867 may be used as targeted drug for cancer treatment.     

The effect of cadmium on cell cycle control in suspension culture cells of soybean

Heavy metals inhibit plant growth. This proces may be directly or indirectly connected with mechanisms regulating cell division. We analyzed the effect of Cd²⁺ on cell cycle progression in partially synchronized soybean (Glycine max) cell suspension culture and followed the expression of cell cycle genes (cyclin B1 and cyclin-dependent kinase A - CDK-A). We have checked the hypothesis that Cd²⁺-induced impairment of cell division is connected with DNA damage. The [³H]-thymidine incorporation in cell cultures synchronized either with hydroxyurea (HU) or phosphate starvation have shown, that Cd²⁺ strongly affects the S phase of soybean cell cycle, by causing the earlier entry of cells into S phase and by decreasing the rate of DNA synthesis. RT-PCR analysis indicated that Cd²⁺ decreases the level of cyclin B1 mRNA and has no effect on CDK-A mRNA. The result of comet assay indicated the damaging effect of Cd²⁺ on DNA of soybean cells. We suggest that Cd²⁺ affects plant cell cycle at two major checkpoints: the G1/S — by damaging of DNA, and G2/M - by decreasing the level of cyclin B1 mRNA     

A Detailed Study of the Complex Cell Cycle of the Dinoflagellate Crypthecodinium cohnii Biecheler and Evidence for Variation in Histone H1 Kinase Activity

ABSTRACT By adding the protein synthesis inhibitor, emetine (10^-4 M) to a highly synchronized population of Crypthecodinium cohnii Biecheler 1938 at different phases of its cycle, we were able to determine: 1. The existence and the lengthening of the G2-Phase (30 min) in the first cycle (cycle with swimming G1 phase). 2. The time of the second cell cycle phases (cycle in the cyst): G1, 30 min; S, 1.5 h; G2, 2 h and M, 2 h. These results, together with the estimation of the cell volume of the two and four swimming daughter cells emerging from the cysts, allowed us to state the existence of two transition points: G1/S and G2/M, which are necessary for completion of mitosis. We completed this refined approach of the cell cycle in studying the activities of the histone H1 kinase either in dividing or in non-dividing Crypthecodinium cohnii cells with either total soluble proteins or the isolated mitotic kinase complex. The H1 kinase activity of this purified complex is noticeably higher (twice as high) in the dividing cells than in the non-dividing ones. These data are discussed in the light of the basic characteristics of the dinokaryon, and also compared with recent biochemical observations on the same organism and studies on other higher eukaryotic protists and metazoa.     

Cell cycle-dependent expression of surface antigens in murine T-lymphoma cells : II. Murine leukemia virus gp70 increases in S phase

Studies were undertaken to identify cell surface markers specific for different phases of the cell cycle. Antisera were prepared in rabbits against membrane protein preparations from synchronized BW 5147 cells, an AKR mouse T-lymphoma cell line, in the G1, S, G2 or M phases of the cell cycle. These antisera were used to precipitate radioiodinated surface proteins from synchronized cells in the different phases. The immunoprecipitates were quantitatively analyzed by sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Cells in S phase had significantly higher concentrations of proteins weighing 70 × 10³ and 165 × 10³ D than cells in G1 or G2 phase. The other major labeled surface components did not vary. These results were confirmed by quantitative absorption of the antisera with synchronized cells. Comparative analysis of the antisera showed that the 165 × 10³ D peak contained at least two antigens, one recognized by both a-G1 and a-S and the other by a-G1 only. Though cells in S phase had large quantities of the 70 × 10³ D protein, intact and SDS-solubilized membrane preparations from S phase could not elicit in rabbits any antibody against that protein. These antisera did, however, have good antibody titers to the other major protein peaks and the antisera developed against cells in G1, G2 or M had good anti-70 × 10³ activity. The results suggest a qualitative molecular change in the 70 × 10³ protein during S phase.