Effect of serum starvation and chemical inhibitors on cell cycle synchronization of canine dermal fibroblasts

The cell cycle stage of donor cells and the method of cell cycle synchronization are important factors influencing the success of somatic cell nuclear transfer. In this study, we examined the effects of serum starvation, culture to confluence, and treatment with chemical inhibitors (roscovitine, aphidicolin, and colchicine) on cell cycle characteristics of canine dermal fibroblast cells. The effect of the various methods of cell cycle synchronization was determined by flow cytometry. Short periods of serum starvation (24-72 h) increased (P<0.05) the proportion of cells at the G0/G1 phase (88.4-90.9%) as compared to the control group (73.6%). A similar increase in the percentage of G0/G1 (P<0.05) cells were obtained in the culture to confluency group (91.8%). Treatment with various concentrations of roscovitine did not increase the proportion of G0/G1 cells; conversely, at concentrations of 30 and 45 microM, it increased (P<0.05) the percentage of cells that underwent apoptosis. The use of aphidicolin led to increase percentages of cells at the S phase in a dose-dependent manner, without increasing apoptosis. Colchicine, at a concentration of 0.1 microg/mL, increased the proportion of cells at the G2/M phase (38.5%, P<0.05); conversely, it decreased the proportions of G0/G1 cells (51.4%, P<0.05). Concentrations of colchicines >0.1 microg/mL did not increase the percentage of G2/M phase cells. The effects of chemical inhibitors were fully reversible; their removal led to a rapid progression in the cell cycle. In conclusion, canine dermal fibroblasts were effectively synchronized at various stages of the cell cycle, which could have benefits for somatic cell nuclear transfer in this species.     

不同方法处理供核细胞对细胞周期和重构胚发育的影响

利用流式细胞仪和细胞染色体核型分析技术,比较奶牛的转基因体细胞和正常细胞经血清饥饿、抑制培养周期同步化处理后的G0/G1期细胞比例;并将同步化处理的核供体细胞进行核移植,然后统计囊胚发育率.结果表明,血清饥饿和抑制培养均能获得较高比例的G0/G1期细胞,两组间差异不显著(P>0.05),但均显著高于未处理对照组(P<0.05);血清饥饿组的囊胚率显著高于抑制培养组和非处理对照组(P<0.05);但细胞同步化处理6 d后细胞染色体核型异常率增加.因此,要获得正常核型的G0/G1核移植供体细胞和较高的囊胚率,同步化处理时间以不超过4 d为宜.     

Production of calves from G1 fibroblasts.

Since the landmark study of Wilmut et al. describing the birth of a cloned lamb derived from a somatic cell nucleus, there has been debate about the donor nucleus cell cycle stage required for somatic cell nuclear transfer (NT). Wilmut et al. suggested that induction of quiescence by serum starvation was critical in allowing donor somatic cells to support development of cloned embryos. In a subsequent report, Cibelli et al. proposed that G0 was unnecessary and that calves could be produced from actively dividing fibroblasts. Neither study conclusively documented the importance of donor cell cycle stage for development to term. Other laboratories have had success with NT in several species, and most have used a serum starvation treatment. Here we evaluate methods for producing G0 and G1 cell populations and compare development following NT. High confluence was more effective than serum starvation for arresting cells in G0. Pure G1 cell populations could be obtained using a "shake-off" procedure. No differences in in vitro development were observed between cells derived from the high-confluence treatment and from the "shake-off" treatment. However, when embryos from each treatment were transferred to 50 recipients, five calves were obtained from embryos derived from "shake-off" cells, whereas no embryos from confluent cells survived beyond 180 days of gestation. These results indicate that donor cell cycle stage is important for NT, particularly during late fetal development, and that actively dividing G1 cells support higher development rates than cells in G0.     

Improved efficiency of canine nucleus transfer using roscovitine-treated canine fibroblasts

The aim of this study was to investigate whether roscovitine (the cyclin-dependent kinase 2 inhibitor) effectively induces synchronization of the donor cell cycle at G0/G1 and to examine the effect of donor cell cycle synchronization protocols on canine somatic cell nucleus transfer. Canine fibroblasts were obtained from skin biopsy cultures taken from a 7-yr-old retriever. The donor cell cycle was synchronized either by culturing cells to reach confluency or by treating cells with 15 μg/mL roscovitine for 24 h. Cell cycle stages and apoptosis were analyzed by flow cytometry. After synchronization of the donor cell cycle, cells were placed with enucleated in vivo-matured dog oocytes, fused by electric stimulation, activated, and transferred into 18 naturally estrus-synchronized surrogates. There was no significant difference in cell cycle synchronization and apoptosis rates between the confluent and roscovitine groups. After transfer of reconstructed embryos, pregnancy was detected in three of nine surrogates that received cloned embryos reconstructed with roscovitine-treated cells, whereas only one of nine surrogates was pregnant after transfer of cloned embryos reconstructed with confluent cells. One pregnant female from the confluent cell group delivered one live and one dead pup, but the live one died within 5 days after birth. Three pregnant females from the roscovitine-treated cell group delivered eight live pups and one dead pup, and one of eight live pups died within 6 days after birth. In conclusion, the current results demonstrated that reconstructing embryos with roscovitine-treated cells resulted in increased efficiency of canine somatic cell nucleus transfer.     

Studies of the cell cycle of in vitro cultured skin fibroblasts in goats: work in progress

The effect of serum starvation and olomoucine treatment on the cell cycle and apoptosis of goat skin fibroblasts cultured in vitro is reported in this paper. The cells were obtained from the ear of a female goat 1.5 years of age. Analysis of cell cycle distribution by fluorescence-activated cell sorting (FACS) showed that 3.4, 60.8 and 15.1% of normally cycling cells were at G1, G0 and S phase, respectively. Serum starvation for 1, 3 and 5 days arrested 70.1, 70.2 and 83.4% cells, respectively, at G0/G1 phase. Seventy-eight percent of confluent cells were at G0/G1 stage, but in contrast to the serum starved group, this high percentage of G0/G1 cells was mainly associated with G1 cells. Of cells not deprived of serum, 73.6% were arrested at G1/G0 when treated with 100 microM olomoucine for 9 h compared to 85.5% of cells that had been starved of serum for 2 days (co-inhibition) (P<0.01). After co-inhibition, 45% of cells entered S phase when re-cultured in normal medium for 5 h, indicating that the inhibition was reversible. Under normal culture conditions, 1.2% of cells underwent apoptosis. Serum starvation for 1, 2, 3, 5 and 10 days caused apoptosis in 1.7, 3.9, 4.5, 11.7 and 90.3% of cells, respectively. Treatment with 100 microM olomoucine for 9h did not increase the number of apoptotic cells significantly (1.9%, P>0.05). When cells were co-inhibited, 4.1% of cells underwent apoptosis. In conclusion, although serum withdrawal for 5 days or more effectively arrested cells at G0/G1 stages, it increased apoptosis of cells significantly. However, co-inhibition by serum withdrawal and olomoucine treatment was found to be an appropriate treatment to obtain more healthy G0/G1 cells based on the low percentage of apoptotic cells after treatment.     

Enhanced sensitivity of G1 arrested human cancer cells suggests a novel therapeutic strategy using a combination of simvastatin and TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells over normal cells. To study the relationship between cell cycle progression and TRAIL-induced apoptosis, SW480 colon cancer and H460 lung cancer cell lines were examined for their sensitivity to TRAIL after arrest in different cell cycle phases. Cells were synchronized in G0/G1, S, and G2/M phase by serum starvation, aphidicolin, or nocodazole treatment, respectively. We found that arrest of cells in G0/G1 phase confers significantly higher susceptibility to TRAIL-induced apoptosis as compared to cells in late G1, S, or G2/M phase. To determine if cell cycle phase could be harnessed for therapeutic gain in the presence of TRAIL, we used the HMG-CoA reductase inhibitor, Simvastatin and lovastatin, to enrich a cancer cell population in G0/G1. Both simvastatin and lovastatin significantly augmented TRAIL-induced apoptosis in tumor cells, but not in normal keratinocytes. The results indicate that TRAIL, in combination with a HMG-CoA reductase inhibitor, may have therapeutic potential in the treatment of human cancer.     

Culture,characteristics and chromosome complement of Siberian tiger fibroblasts for nuclear transfer

Tiger (Panthera tigris Linnaeus, 1758) is a characteristic species of Asia, which is in severe danger. Siberian tiger (Panthera tigris altaica) is the largest one of the five existent tiger subspecies. It is extremely endangered. One new way for tiger protection and rescue is to study interspecies cloning. But there is few research data about Siberian tiger. In this study, we cultured Siberian tiger fibroblasts in vitro, analyzed their biological characteristics, chromosomes, and cell cycles, to provide not only nuclear donors with good morphology, normal biological characteristics, and chromosome quantity for tiger interspecies cloning, but also reliable data for further studying Siberian tiger. The results indicated that Siberian tiger ear fibroblasts can be successfully obtained by tissue culture either with or without overnight cold digestion, the cultured cells were typical fibroblasts with normal morphology, growth curve, and chromosome quantity; G0/G1 percentage increased and S percentage decreased with the confluence of cells. G0/G1 and S stage rate was significantly different between 40–50% and 80–90%, 95–100% confluence; there is no distinct difference between 80–90% and 95–100% confluence. The cells at the same density (80–90% confluence) were treated with or without 0.5% serum starving, GO/G1 rate of the former was higher than the latter, but the difference was not significant. GO/G1 proportion of 95–100% confluence was slightly higher than serum starving (80–90% confluence), but no significant difference. Therefore, the Siberian tiger fibroblasts we cultured in vitro can be used as donor cells, and the donor cells do not need to be treated with normal serum starvation during nuclear transfer; if we will just consider the rate of the G0/G1 stage cells, serum starvation can be replaced by confluence inhibition when cultured cells were more than 80–90% confluence.     

Synchronization of goat fibroblast cells at quiescent stage and determination of their transition from G0 to G1 by detection of cyclin D1 mRNA

A number of studies have reported that donor cells consisting of serum starved cells, which are assumed to be at quiescence (G0), or non-starved confluent cells or mitotic cells obtained by shake-off, both of which are assumed to be at G1 phase, give better results in nuclear transfer (NT) than cells at other phases of the cell cycle. Whether G0 or G1 cells function better as donor cells is yet to be determined by detailed studies. The aims of this study were to analyze the cell cycle of goat transfected fibroblasts and determine the timing of transition from G0 to G1 by detecting G1-specific marker, cyclin D1 mRNA. Fluorescent-activated cell sorting (FACS) analyses of cells after 4 days of serum starvation showed that more that 90% of cells were in G0/G1. Additionally, detection of cyclin D1 mRNA by northern blot analysis showed that 4-day serum starved quiescent cells started entering G1 a few hours after addition of 10% serum to the medium. Taken together, the data indicated that serum starved transfected primary fibroblasts of adult goats experienced the G0 to G1 transition within 5 h of serum stimulation and were at the mid-G1 stage within 10 h of serum stimulation.     

Enhanced Sensitivity of G1 Arrested Human Cancer Cells Suggests a Novel Therapeutic Strategy Using a Combination of Simvastatin and TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells over normal cells. To study the relationship between cell cycle progression and TRAIL-induced apoptosis, SW480 colon cancer and H460 lung cancer cell lines were examined for their sensitivity to TRAIL after arrest in different cell cycle phases. Cells were synchronized in G0/G1, S, and G2/M phase by serum starvation, aphidicolin, or nocodazole treatment, respectively. We found that arrest of cells in G0/G1 phase confers significantly higher susceptibility to TRAIL-induced apoptosis as compared to cells in late G1, S, or G2/M phase. To determine if cell cycle phase could be harnessed for therapeutic gain in the presence of TRAIL, we used the HMG-CoA reductase inhibitor, Simvastatin and lovastatin, to enrich a cancer cell population in G0/G1. Both simvastatin and lovastatin significantly augmented TRAIL-induced apoptosis in tumor cells, but not in normal keratinocytes. The results indicate that TRAIL, in combination with a HMG-CoA reductase inhibitor, may have therapeutic potential in the treatment of human cancer.

Key Words

TRAIL, Synchronization, Simvastatin, Cancer Therapy, Lovastatin, Cell Cycle, Apoptosis     

Viability and cell cycle analysis of equine fibroblasts cultured in vitro

This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO₂ until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and propidium iodide. The analysis of apoptosis/necrosis and cell cycle was performed using a flow cytometer (FACS Calibur BD^®) after staining the cells with annexin V and propidium iodide. Both optical microscopy and flow cytometry confirmed that cellular viability was similar for serum starvation and confluent groups (average 84%). Similarly, both methods were efficient to synchronize the cell cycle before freezing. However, after thawing, serum starvation, for more than 24 h, was superior to culture for synchronizing cells in G0/G1 (69% × 90%). The results of this experiment indicate that equine fibroblasts can be efficiently cultured after thawing.     

血清饥饿、汇合培养及放线菌酮对奶牛成纤维细胞周期的影响

在动物克隆研究中,研究者普遍认为位于细胞周期的G0+G1期的二倍体细胞对于核移植中供核细胞的重新程序化是必需的。本文探讨了血清饥饿、汇合培养及放线菌酮（CHX）处理对不同传代次数的体外培养奶牛成纤维细胞周期分布的影响。流式细胞仪分析结果显示：第3代和第13代细胞经血清饥饿处理72h后,细胞周期分布与对照差异显著(P<0.05);汇合培养可以显著增加奶牛成纤维细胞处于G0+G1期细胞数。CHX处理第3代细胞经CHX处理后处于G0+G1期的细胞数差异不显著,而第13代细胞处理后差异显著。结果表明体外培养奶牛成纤维细胞高代对血清饥饿、汇合培养及CHX处理更敏感。     

Cell cycle synchronization of porcine granulosa cells in G1 stage with mimosine

The success of somatic cell nuclear transfer depends critically on the cell cycle stage of the donor nucleus and the recipient cytoplast. Karyoplasts in the G0 or G1 stages are considered to be the most suitable for nuclear transfer. In the present study, we used a reversible cell cycle inhibitor, mimosine, to synchronize porcine granulosa cells (GCs) in G1 phase of the cell cycle. Porcine GCs were obtained from 3 to 5mm ovarian follicles of slaughtered gilts. The effect of mimosine on the proliferation, DNA synthesis and cell cycle stage of cultured cells was examined by incorporation of radiochemical 3H-thymidine, immunocytochemical detection of incorporated thymidine analogue 5-bromo-2-deoxyuridine (BrdU) and flow cytometry analyses. Mimosine treatment of pig GCs for 24h resulted in proliferation arrest in vitro. Treatment with 0.5mM mimosine significantly (P<0.05) inhibited 3H-thymidine incorporation after 24h of culture (4.6% +/- 0.1) and after 24h of culture in serum deprived medium (41.3% +/- 3.8), in comparison to controls (100%). Inhibition of DNA synthesis was further confirmed by immunocytochemical and flow cytometry analyses. Compared with controls (78.2%), mimosine treatment for 24h increased the proportion of G0/G1 cells in the culture (85.7%) more effectively than serum starvation (SS; 81.2%). Mimosine-caused G1 arrest of porcine GCs was fully reversible and cells continued to proliferate after removing the drug, especially when they were stimulated by EGF.     

Inhibition of apoptosis in serum starved porcine embryonic fibroblasts

In nuclear transplantation, serum starvation is a general method to synchronize donor cells at the quiescent stage (G(0)) of the cell cycle. However, serum starvation during culture of mammalian cells may induce cell death, especially through apoptosis, thus contributing to the low efficiency of nuclear transplantation. This study was performed to characterize apoptosis during serum starvation and to determine the effects of apoptosis inhibitors such as a protease inhibitor [alpha(2)-macroglobulin (MAC)] and antioxidants [N-acetylcysteine (NAC), glutathione (GSH)] on serum starved porcine embryonic fibroblasts (PEF). PEF, collected from day 25-30 porcine fetuses, were cultured for 5 days in media containing 0.5% FBS to induce quiescence. Serum starved PEF showed typical morphology of apoptotic cells and stained for DNA fragmentation by TUNEL assay (26.7%). All apoptosis inhibitors tested in this study significantly (P < 0.05) reduced apoptosis of serum starved PEF, with antioxidants having better results (MAC: 7.4% vs. NAC: 1.0%, and GSH: 0.8%). Equally and importantly, the treatment with apoptosis inhibitors did not change the proportion of G(0)/G(1) stage cells. Therefore, the addition of MAC and antioxidants during serum starvation of PEF reduces apoptosis of quiescent fibroblasts and may contribute to increasing the efficiency of nuclear transplantation by improving the quality of donor nuclei.     

Cell confluency is as efficient as serum starvation for inducing arrest in the G0/G1 phase of the cell cycle in granulosa and fibroblast cells of cattle

The cell cycle stage of donor cells is an important factor influencing developmental ability of nuclear transfer embryos. In the present experiment, cumulus and fibroblast cells of cattle were subjected to flow cytometric cell cycle analysis before being used in somatic cloning experiments. The following experimental groups were analyzed for each cell type: (1) actively dividing cells, (2) cells confluent for 4 days, (3) cells starved for 1, 2, 3 or 5 days. Using the propidium iodide flow cytometric assay, there were no significant differences (P > or = 0.05) in the percentage of cells in G0/G1 regardless of origin and type of cell, after confluency or serum starvation. Differences with the growing cells were found (P < or = 0.01). To determine what subset of cells in G0/G1 were in the G0 subphase of the cell cycle, an immunofluorescence analysis was conducted using monoclonal anti-PCNA antibodies in a FACS assay. There were not statistically significant differences in the percentage of cells that enter G0, between confluent and any starved group for either type of cells. Bovine fibroblast cells, confluent or serum starved for 3 days, were used in nuclear transfer experiments. A slight trend for a more desirable fusion rate in starved cells was detected, and embryo cleavage was greater in starved cells, however, in vitro development to blastocysts was similar between groups. Data indicate that prolonged culture of cells in the absence of serum does not imply a shift in the percentage of cells that enter G0/G1 or G0 alone, and that confluency is sufficient to induce quiescence. This finding can be beneficial in nuclear transfer programs, because there are negative effects such as apoptosis, associated with serum starvation.     

Transition of G1 to early S phase may be required for zinnia mesophyll cells to trans-differentiate to tracheary elements

We have used the Zinnia elegans mesophyll cell system, in which single isolated leaf mesophyll cells can be induced to trans-differentiate into tracheary elements in vitro, to study the relationship between the cell division cycle and cell differentiation. Almost all cells go through several rounds of division before characteristic features of tracheary element formation are observed. The addition of aphidicolin, a DNA synthesis inhibitor, blocks cell division but not cell differentiation in the zinnia system. Low concentrations of aphidicolin, which possibly delay cells in the early S phase, can significantly enhance levels of tracheary element formation. In contrast, roscovitine, an inhibitor of cyclin-dependent kinase activity, decelerates the cell division cycle and inhibits tracheary element formation with similar dose responses. Cells blocked in S phase and then transferred to roscovitine-containing medium can divide once, indicating that roscovitine may target the G1/S transition, but do not differentiate. Cells inhibited in G1/S in roscovitine-containing medium that are subsequently blocked in S phase by transfer to aphidicolin-containing medium, do not divide but do differentiate. Taken together, our results indicate that cells may be required to transit the G1/S checkpoint and enter early S phase to acquire competence to trans-differentiate to tracheary elements.     

Flow cytometric cell cycle analysis of somatic cells primary cultures established for bovine cloning

An important factor governing developmental rates of somatic cloned embryos is the phase of the cell cycle of donor nuclei. The aim of this experiment was to investigate the distribution of cell cycle phases in bovine cumulus and fibroblast cells cultured using routine treatment, and under cell cycle-arresting treatments. The highest percentages of cumulus cells in the G0 + G1 stage were observed in uncultured, frozen/thawed cells originating from immature oocytes (79.8 +/- 2.2%), fresh and frozen/thawed cells from in vitro matured oocytes (84.1 +/- 6.2 and 77.8 +/- 5.7%, respectively), and in cycling cells (72.7 +/- 16.3 and 78.4 +/- 11.2%, respectively for cumulus cells from immature and in vitro matured oocytes). Serum starvation of cumulus cultures markedly decreased percentages of cells in G0 + G1, and prolonged starvation significantly increased (P < 0.05) percentages of cells in G2 + M phase. Culture of cumulus cells to confluency did not increase percentages of cells in G0 + G1. Contrary to findings in cumulus cells, significantly higher percentages of cells in G0 + G1 were apparent when fibroblast cells were cultured to confluency or serum starved, and significantly increased (P < 0.01) as the starvation period was prolonged. It is concluded that for particular cell types specific strategies should be used to attain improvements in the efficiency of cloning procedures.     

The effects of in vitro age and culture state on histone variant synthesis in human diploid fibroblasts

Histone variant synthesis patterns from human diploid fibroblast-like cells of different in vitro ages were determined during exponential growth, at confluence, and during low serum arrest. The results are reported as the ratios of H2A variant synthesis (H2A.1 and H2A.2/H2A.x and H2A.z) and H3 variant synthesis (H3.1 and H3.2/H3.3) that have been used to characterize individual cell cycle states. Hydroxyurea was employed in some experiments to reduce S phase cells. The results indicate that high population doubling level (PDL) cells move through the G1 phase of the division cycle during exponential growth and exist in the G0 cell cycle state at confluence and during low serum arrest. Low PDL cells, however, exist in the G1 cell cycle state at confluence and revert to a G0 state only after maintenance as quiescent populations. This would suggest that when stimulated high PDL cells cannot enter into S phase, they revert to a GO cell cycle state.     

Cell-cycle synchronization of fibroblasts derived from transgenic cloned cattle ear skin: effects of serum starvation, roscovitine and contact inhibition

The purpose of the present study was to evaluate the effects of serum-starvation, contact-inhibition and roscovitine treatments on cell-cycle synchronization at the G0/G1 stage of ear skin fibroblasts isolated from transgenic cloned cattle. The developmental competence of re-cloned embryos was also examined. Our results showed that the proportion of G0/G1 cells from the serum-starved group at 3, 4 or 5 days was significantly higher compared with 1 or 2 days only (91.5, 91.7 and 93.5% versus 90.1 and 88.8%, respectively, p < 0.05); whilst there was no statistical difference among cells at 3, 4 or 5 days. For roscovitine-treated cells, the proportion of G0/G1 cells at 2, 3, 4 or 5 days was significantly higher than those treated for 1 day only (91.1, 90.1, 89.4 and 91.3% versus 86.51%, respectively, p < 0.05). The proportion of contact-inhibited G0/G1 cells rose significantly with treatment time, but was similar at 3, 4 and 5 days (89.4, 90.4, 91.4, 91.6 and 92.1%, respectively, p < 0.05). The efficiency of obtaining G0/G1 phase cells was lower when roscovitine treatment was employed to synchronize the cell cycle compared with the serum-starvation and contact-inhibition methods (89.7 versus 91.1% and 91.0%, p < 0.05). Moreover, obvious differences were observed in the rate of fused couplets and blastocysts (89.88 +/- 2.70 versus 87.40 +/- 5.13; 44.10 +/- 8.62 versus 58.38 +/- 13.28, respectively, p < 0.05), when nuclear transfer embryos were reconstructed using donors cells that had been serum starved or contact inhibited for 3 days. Our data indicate that 3 day treatment is feasible for harvesting sufficient G0/G1 cells to produce re-cloned transgenic bovine embryos, regardless of whether serum-starvation, contact-inhibition or roscovitine treatments are used as the synchronization methods.