Visualization of radiation-induced cell cycle-associated events in tumor cells expressing the fusion protein of Azami Green and the destruction box of human Geminin

Ionizing radiation (IR) influences cell cycle-associated events in tumor cells. We expressed the fusion protein of Azami Green (AG) and the destruction box plus nuclear localization signal of human Geminin, an inhibitor of DNA replication licensing factor, in oral tumor cells. This approach allowed us to visualize G2 arrest in living cells following irradiation. The combination of time-lapse imaging analysis allowed us to observe the nuclear envelope break down (NEBD) at early M phase, and disappearance of fluorescence (DF) at the end of M phase. The duration from NEBD to DF was not much affected in irradiated cells; however, most of daughter cells harbored double-strand breaks. Complete DF was also observed in cells exhibiting abnormal mitosis or cytokinesis. We conclude that the fluorescent Geminin probe could function as a stable cell cycle indicator irrespective of genome integrity.     

蛋白激酶C抑制剂staurosporine对HeLa细胞周期的影响

蛋白激酶Ｃ抑制剂ｓｔａｕｒｏｓｐｏｒｉｎｅ对ＨｅＬａ细胞周期的影响石法武＊任洪波张兆山＊＊王会信周廷冲（军事医学科学院基础医学研究所,＊＊生物工程研究所,北京１００８５０ＴｈｅｅｆｅｃｔｏｆｓｔａｕｒｏｓｐｏｒｉｎｅｏｎｔｈｅｃｅｌｃｙｃｌｅｏｆＨｅ...     

Visualizing the effect of hypoxia on fluorescence kinetics in living HeLa cells using the fluorescent ubiquitination-based cell cycle indicator (Fucci)

Fluorescent proteins are widely used for the direct visualization of events such as gene expression and subcellular localization in mammalian cells. It is well established that oxygen is required for formation of functional chromophore; however, the effect of hypoxia on fluorescence emission has rarely been studied. For this purpose, under hypoxic conditions, we investigated the kinetics of red and green fluorescence in HeLa cells from two fluorescent proteins, monomeric Kusabira Orange 2 (mKO2) and monomeric Azami Green (mAG), respectively, using the fluorescent ubiquitination-based cell cycle indicator (Fucci). In this system, cells in G1 or other phases emit red or green fluorescence, respectively. We found that hypoxia abrogated both red and green fluorescence about ~10h after the treatment, although their protein levels were almost maintained. The treatment did not significantly affect fluorescence in cells constitutively expressing the same fluorescent proteins lacking the ubiquitin ligase-binding domains. The abrogation of fluorescence resulted from a combination of ubiquitination-dependent degradation of pre-existing functional proteins during specific cell cycle phases, and the expression of newly synthesized non-fluorescent proteins containing non-oxidized chromophore during hypoxic treatment. Indeed, non-fluorescent cells after hypoxic treatment gradually developed fluorescence after reoxygenation in the presence of cycloheximide; kinetics of recovery were much faster for mAG than for mKO2. Using the Fucci system, we could clearly visualize for the first time the effect of hypoxia on the fluorescence kinetics of proteins expressed in living mammalian cells.     

Visualizing and quantifying the differential cleavages of the eukaryotic translation initiation factors eIF4GI and eIF4GII in the enterovirus‐infected cell

Enterovirus (EV) infection has been shown to cause a marked shutoff of host protein synthesis, an event mainly achieved through the cleavages of eukaryotic translation initiation factors eIF4GI and eIF4GII that are mediated by viral 2A protease (2A^pro). Using fluorescence resonance energy transfer (FRET), we developed genetically encoded and FRET‐based biosensors to visualize and quantify the specific proteolytic process in intact cells. This was accomplished by stable expression of a fusion substrate construct composed of the green fluorescent protein 2 (GFP²) and red fluorescent protein 2 (DsRed2), with a cleavage motif on eIF4GI or eIF4GII connected in between. The FRET biosensor showed a real‐time and quantifiable impairment of FRET upon EV infection. Levels of the reduced FRET closely correlated with the cleavage kinetics of the endogenous eIF4Gs isoforms. The FRET impairments were solely attributed to 2A^pro catalytic activity, irrespective of other viral‐encoded protease, the activated caspases or general inhibition of protein synthesis in the EV‐infected cells. The FRET biosensors appeared to be a universal platform for several related EVs. The spatiotemporal and quantitative imaging enabled by FRET can shed light on the protease–substrate behaviors in their normal milieu, permitting investigation into the molecular mechanism underlying virus‐induced host translation inhibition. Biotechnol. Bioeng. 2009; 104: 1142–1152. © 2009 Wiley Periodicals, Inc.     

蛋白激酶C抑制剂Staurosporine对HeLa细胞周期的影响

用蛋白激酶C的抑制剂Staurosporine(10nmol/L)处理HeLa细胞,明显抑制HeLa细胞的增殖。这种抑制作用不是由于引起细胞死亡,而是因为细胞被阻断在G2期。这种阻断作用伴随着HeLa细胞多倍体的形成,提示Staurosporine抑制了HeLa细胞蛋白激酶C活性后引起的细胞阻滞,对细胞核的周期运转没有影响。进一步的探讨发现这种抑制作用可能是通过干扰细胞骨架的正确分布形成的,表明蛋白激酶C对于HeLa细胞由G2到M期正确过渡起重要作用。     

Fatty acid degradation plays an essential role in proliferation of mouse female primordial germ cells via the p53-dependent cell cycle regulation

Primordial germ cells (PGCs) are embryonic founders of germ cells that ultimately differentiate into oocytes and spermatogonia. Embryonic proliferation of PGCs starting from E11.5 ensures the presence of germ cells in adulthood, especially in female mammals whose total number of oocytes declines after this initial proliferation period. To better understand mechanisms underlying PGC proliferation in female mice, we constructed a proteome profile of female mouse gonads at E11.5. Subsequent KEGG pathway analysis of the 3,662 proteins profiled showed significant enrichment of pathways involved in fatty acid degradation. Further, the number of PGCs found in in vitro cultured fetal gonads significantly decreased with application of etomoxir, an inhibitor of the key rate-limiting enzyme of fatty acid degradation carnitine acyltransferase I (CPT1). Decrease in PGCs was further determined to be the result of reduced proliferation rather than apoptosis. The inhibition of fatty acid degradation by etomoxir has the potential to activate the Ca²⁺/CamKII/5′-adenosine monophosphate-activated protein kinase (AMPK) pathway; while as an upstream activator, activated AMPK can function as activator of p53 to induce cell cycle arrest. Thus, we detected the expressional level of AMPK, phosphorylated AMPK (P-AMPK), phosphorylated p53 (P-p53) and cyclin-dependent kinase inhibitor 1 (p21) by Western blots, the results showed increased expression of them after treatment with etomoxir, suggested the activation of p53 pathway was the reason for reduced proliferation of PGCs. Finally, the involvement of p53-dependent G1 cell cycle arrest in defective proliferation of PGCs was verified by rescue experiments. Our results demonstrate that fatty acid degradation plays an important role in proliferation of female PGCs via the p53-dependent cell cycle regulation.     

Curative potential of GM-CSF-secreting tumor cell vaccines on established orthotopic liver tumors: Mechanisms for the superior antitumor activity of live tumor cell vaccines

In preclinical studies, tumor cells genetically engineered to secrete cytokines, hereafter referred to as tumor cell vaccines, can often generate systemic antitumor immunity. This study investigated the therapeutic effects of live or irradiated tumor cell vaccines that secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) on established orthotopic liver tumors. Experimental results indicated that two doses (3 × 10⁷ cells per dose) of irradiated tumor cell vaccines were therapeutically ineffective, whereas one dose (3 × 10⁶ cells) of live tumor cell vaccines caused complete tumor regression. In vivo depletion of CD8+ T cells, but not natural killer cells, restored tumor formation in the live vaccine-treated animals. Additionally, the treatment of cells with live vaccine induced markedly higher levels of cytotoxic T lymphocyte activity than the irradiated vaccines in the draining lymph nodes. The higher levels of cytokine and antigen loads could partly explain the superior antitumor activity of live tumor cell vaccines, but other unidentified mechanisms could also play a role in the early T cell activation in the lymph nodes. A protocol using multiple and higher dosages of irradiated tumor cell vaccines also caused significant regression of liver tumors. These results suggest that the GM-CSF-secreting tumor cell vaccines are highly promising for orthotopic liver tumors if higher levels of immune responses are elicited during early tumor development.     

Nutlin-3 induces apoptosis,disrupts viral latency and inhibits expression of angiopoietin-2 in Kaposi sarcoma tumor cells

Kaposi sarcoma (KS) tumors often contain a wild-type p53. However, the function of this tumor suppressor in KS tumor cells is inhibited by both MDM2 and latent nuclear antigen (LANA) of Kaposi sarcoma-associated herpes virus (KSHV). Here, we report that MDM2 antagonist Nutlin-3 efficiently reactivates p53 in telomerase-immortalized human umbilical vein endothelial cells (TIVE) that had been malignantly transformed by KSHV as well as in KS tumor cells. Reactivation of p53 results in a G₁ cell cycle arrest, leading to inhibition of proliferation and apoptosis. Nutlin-3 inhibits the growth of “KS-like” tumors resulting from xenografted TIVE-KSHV cells in nude mice. In addition, Nutlin-3 strongly inhibits expression of the pro-angiogenic and pro-inflammatory cytokine angiopoietin-2 (Ang-2). It also disrupts viral latency by inducing expression of KSHV lytic genes. These results suggest that Nutlin-3 might serve as a novel therapy for KS.     

PARP-1-induced cell death through inhibition of the MEK/ERK pathway in MNNG-treated HeLa cells

Poly(ADP-ribose) polymerase-1 (PARP-1) hyper-activation promotes cell death but the signaling events downstream of PARP-1 activation are not fully identified. To gain further information on the implication of PARP-1 activation and PAR synthesis on signaling pathways influencing cell death, we exposed HeLa cells to the DNA alkylating agent N-methyl-N′-methyl-nitro-N-nitrosoguanidine (MNNG). We found that massive PAR synthesis leads to down-regulation of ERK1/2 phosphorylation, Bax translocation to the mitochondria, release of cytochrome c and AIF and subsequently cell death. Inhibition of massive PAR synthesis following MNNG exposure with the PARP inhibitor PJ34 prevented those events leading to cell survival, whereas inhibition of ERK1/2 phosphorylation by inhibiting MEK counteracted the cytoprotective effect of PJ34. Together, our results provide evidence that PARP-1-induced cell death by MNNG exposure in HeLa cells is mediated in part through inhibition of the MEK/ERK signaling pathway and that inhibition of massive PAR synthesis by PJ34, which promotes sustained activation of ERK1/2, leads to cytoprotection.     

Melatonin effect on the ultrastructure of Ehrlich ascites tumor cells,lifetime and histopathology in Swiss mice

Aims

One of the models used for studying cancer is the Ehrlich ascites tumor (EAT) due to its ability to grow in liquid suspension, allowing a standard number of cells to be inoculated, growth quantification and regression of tumor mass. Among the oncostatic substances, melatonin has shown effectiveness in limiting the tumor cell proliferation. However, studies have shown contradictory effects of melatonin on the EAT. This study has investigated the melatonin effect on tumor growth, time and survival percentage, ultrastructure and metastasis of EAT cells in mice submitted or not to pinealectomy.

Main methods

Animals were inoculated with 5 × 10⁶ cells/mL and treated or not with exogenous melatonin with doses of at 150 and 300 μg/30 g animal weight for 12 days. Melatonin significantly reduced the abdominal circumference, volume of ascites liquid and EAT-cell viability, raising rates of time and mice survival percentage.

Key findings

Ultrastructurally, the melatonin treatment revealed changes in the shape of cells, the cell surface showed numerous projections, some bifurcated, cytoplasmic vacuolation, mitochondrial degeneration and nuclear fragmentation, peculiar characteristics of apoptosis. Histopathology revealed no metastasis in the liver, small intestine and large intestine in any of the animals in the experimental groups; however this process was evident in the lungs and kidneys, being inhibited by melatonin administration.

Significance

Thus, we can conclude that doses of 150 and 300 μg/30 g of melatonin for 12 consecutive days have a very effective oncostatic and cytotoxic activity on EAT cells in mice.     

High temperature causes arrest of cell cycle in G2 phase in BmN cells derived from the silkworm, Bombyx mori

Abstract.  The influence of temperature on the insect cell line, BmN, derived from the silkworm, Bombyx mori is investigated. These cells proliferate at an accelerated pace as the temperature increases from 22 to 30 °C, but the growth rate slows at 34 °C, and proliferation stops at 38 °C. At high temperatures, abnormal cellular morphology is observed. Cells treated at 38 °C have cytoplasmic bilateral protrusions and they gradually aggregate and float in the medium. BmN cells without proliferation at 38 °C are viable but have reduced DNA synthesis. At high temperatures, the cell cycle of BmN cells halts at the G₂ phase. After heat treatment of the larvae, an accumulation of larval haemocytes with high DNA content is found, which suggests that the cell cycle arrest at G₂ also occurs in the silkworm at high temperatures.     

A molecular understanding of d‐homoestrone‐induced G2/M cell cycle arrest in HeLa human cervical carcinoma cells

2‐Methoxyestradiol (ME), one of the most widely investigated A‐ring‐modified metabolites of estrone, exerts significant anticancer activity on numerous cancer cell lines. Its pharmacological actions, including cell cycle arrest, microtubule disruption and pro‐apoptotic activity, have already been described in detail. The currently tested d ‐ring‐modified analogue of estrone, d ‐homoestrone, selectively inhibits cervical cancer cell proliferation and induces a G2/M phase cell cycle blockade, resulting in the development of apoptosis. The question arose of whether the difference in the chemical structures of these analogues can influence the mechanism of anticancer action. The aim of the present study was therefore to elucidate the molecular contributors of intracellular processes induced by d ‐homoestrone in HeLa cells. Apoptosis triggered by d ‐homoestrone develops through activation of the intrinsic pathway, as demonstrated by determination of the activities of caspase‐8 and ‐9. It was revealed that d ‐homoestrone‐treated HeLa cells are not able to enter mitosis because the cyclin‐dependent kinase 1‐cyclin B complex loses its activity, resulting in the decreased inactivation of stathmin and a concomitant disturbance of microtubule formation. However, unlike 2‐ME, d ‐homoestrone does not exert a direct effect on tubulin polymerization. These results led to the conclusion that the d ‐homoestrone‐triggered intracellular processes resulting in a cell cycle arrest and apoptosis in HeLa cells differ from those in the case of 2‐ME. This may be regarded as an alternative mechanism of action among steroidal anticancer compounds.     

Inhibitory effect of phycocyanin from Spirulina platensis on the growth of human leukemia K562 cells

The effect of phycocyanin from Spirulinaplatensis on the growth of human chronic myelogenousleukemia-blast crisis K562 cells was studied bysemi-solid agar assay and cell viability measurement. Phycocyanin significantly inhibited the growth of K562cells in a dose-dependent manner. The IC50 value ofthe phycocyanin was 72.5 mg L^-1. After the K562cells were cultured with phycocyanin for 6 days, flowcytometric assays showed that more K562 cells wereblocked to progress through S-phase and arrested at G1phase. DNA fragmentation assay indicated that therewas no ladder of DNA fragments of approximately 200-bpmultiples, indicating that apoptosis had not occurred. Western blot analysis showed that Bcl-2 protein wasexpressed, but its level remained unchanged, whereasthe expression level of c-myc increased. Thesefindings suggest that phycocyanin may be able toinhibit the growth of K562 cells by pathways otherthan apoptosis, and that changed a expression patternof the c-myc protein may be involved in such inhibition.     

Impact of the tumor microenvironment on host infiltrating cells and the efficacy of flt3-ligand combination immunotherapy evaluated in a treatment model of mouse prostate cancer

We have previously reported that Fms-like tyrosine kinase-3 ligand (flt3-L) induced tumor stabilization and regression of palpable ectopic prostate tumors (TRAMP-C1). Although some mice remained "tumor free" for several months following termination of therapy, tumors invariably reappeared and grew progressively in all animals. The lack of a curative response suggests that TRAMP-C1 tumors may inhibit the development of a flt3-L-induced anti-tumor immune response. Consistent with this view, we demonstrate herein that TRAMP-C1 tumors isolated from flt3-L treated animals contained a marked dendritic cell (DC) infiltrate that was temporally correlated with tumor regression. However, tumor-associated DCs, especially in a flt3-L setting, progressively lost MHC class II antigen expression during tumor growth. Treatment with the DC maturation factor trimeric CD40 ligand (CD40-L) either alone or in combination with fl3-L neither prevented loss of DC class II antigens nor disease relapse. Because loss of class II antigens would prevent CD4+ helper T (Th) cell development, we treated tumor-bearing mice with agonistic anti-4-1BB antibody (Ab), which can promote cytotoxic T lymphocyte (CTL) development independent of Th cell function. However, anti-4-1BB Ab alone did not alter TRAMP-C1 growth kinetics, and, when used in combination, was no more effective than flt3-L alone. The inability of the 4-1BB co-stimulatory signal to promote tumor regression may have been related to two additional features of TRAMP-C1 tumors. First, tumor-associated T cells, but not splenic T cells from tumor-bearing animals, were profoundly deficient in expression of CD3-epsilon (CD3) and T cell receptor-beta chain (TCR). Second, CTLs required 24 h to efficiently kill TRAMP-C1 target cells even after up-regulation of MHC class I antigens by interferon-. This rate of tumor cell destruction by CTLs may not be sufficient to prevent tumor progression. Taken together, these data reveal several important immunosuppressive characteristics of the prostate tumor microenvironment (TME) that immunotherapeutic interventions must first overcome to achieve long-term cures. These data also highlight the importance of utilizing treatment versus vaccination models in the evaluation of immunotherapeutic modalities.     

Erythromycin inhibition of cell proliferation and in vitro mitochondrial protein synthesis in human HeLa cells is pH dependent

The growth of HeLa cells in Hepes-buffered medium was significantly more sensitive to the inhibitory effects of erythromycin than in medium buffered by the more conventional bicarbonate-CO₂ system. Since growth inhibition by erythromycin became more pronounced as the pH of the medium was increased the difference in erythromycin sensitivity between the Hepes-buffered medium vs. the bicarbonate-CO₂-buffered medium is most likely due to pH effects. The relative growth sensitivity to erythromycin of ERY2301, an erythromycin-resistant mutant of HeLa, was also affected by elevated pH of the growth medium. However, ERY2301 cells were able to proliferate to a greater extent in the presence of erythromycin than HeLa cells grown under the same conditions. The selective growth advantage of ERY2301 (in the presence of erythromycin) is best seen in medium of pH 7.4, or in the Hepes-buffered medium. In vitro protein synthesis by intact mitochondria isolated from HeLa cells was relatively insensitive to erythromycin inhibition at pH 7.4 and 7.6, but at high pH values was inhibited approx. 50%. Although the erythromycin sensitivity of ERY2301 mitochondrial protein synthesis was also affected by increasing the pH, the incorporation of [³H]leucine was more resistant to erythromycin than that observed for HeLa mitochondria over the pH range tested. Increasing the concentration of erythromycin at a given pH did not result in a further increase in the inhibition of either HeLa or ERY2301 mitochondrial protein synthesis. When the mitochondrial membranes were disrupted by Triton X-100, erythromycin inhibition of HeLa mitochondrial protein synthesis was pH dependent and, at the lower pH values tested, greater inhibition was observed as the erythromycin concentration was increased. ERY2301 mitochondrial protein synthesis under the same conditions displayed a high level of erythromycin-resistant activity independent of both pH and erythromycin concentration. It is suggested that, as has been proposed for bacterial systems, only the non-protonated molecule of erythromycin is effective in inhibiting mitochondrial protein synthesis. The ability of erythromycin to permeate the mitochondrial membranes and the plasma membres may also be facilitated by a higher pH.     

Value of the cell cycle arrest biomarkers in the diagnosis of pregnancy-related acute kidney injury

Background: Pregnancy-related acute kidney injury (PRAKI) is still a common serious problem in developing countries. Insulin-like growth factor-binding protein 7 (IGFBP7) and tissue inhibitor metalloproteinases-2 (TIMP-2) can identify critically ill patients at risk for the development of severe AKI. Aim: To identify main causes and timing of PRAKI and to study the G1 cell cycle arrest biomarkers in cases diagnosed with (PRAKI) as a diagnostic tool. Methods: 80 pregnant women diagnosed with PRAKI were recruited from a single hospital as well as 30 age-matched pregnant women with normal pregnancy participated in the present study. A urine specimen was collected from all study participants with established AKI within 24 h of ICU admission to measure [TIMP-2]*[IGFBP7]. Results: The incidence of PRAKI was 1.1%. The most common cause of PRAKI is pre-eclampsia/eclampsia spectrum (61%). Most of the cases occur in the third trimester (60%) and postpartum period (23%). At a cutoff 0.33 ng/ml, the estimated sensitivity and specificity of urinary [TIMP-2]*[IGFBP7] in predicting PRAKI is 100% (95% CI) with NPV and PPV are 100%. Conclusion: Urinary [TIMP-2]*[IGFBP7] serves as a sensitive and specific biomarker in the diagnosis of PRAKI.     

Inhibition of polyamine biosynthesis by alpha-difluoromethyl ornithine potentiates the cytotoxic effects of arabinosyl cytosine in HeLa cells

The object of this study was to examine the effect of inhibition of polyamine biosynthesis on the cell cycle traverse of HeLa cells using α-difluoromethyl ornithine (DFMO), a catalytic irreversible inhibitor of ornithine decarboxylase. The results of this study indicate that DFMO inhibits HeLa cell growth by causing a decrease in the intracellular levels of putrescine and spermidine without any significant effect on concentration of spermine. The inhibition is readily reversible by exogenous supply of putrescine to the medium. The DFMO treatment also results in an accumulation of cells in S phase. Further, the use of an S phase-specific drug like Ara-C following DFMO treatment results in a synergistic killing of the tumor cells as revealed by the inhibition of cell growth. These observations suggest that exploitation of regulation of the cell cycle by the depletion of polyamines with the use of inhibitors like DFMO might help in designing better therapeutic regimes in combination with other cytotoxic drugs.     

A pro-apoptotic effect of the CDK inhibitor p57(Kip2) on staurosporine-induced apoptosis in HeLa cells

Apoptosis, or programmed cell death, is involved in many biological events, including tumorigenesis. Recently, it has been reported that two members of the Cip/Kip family of CDK inhibitors, p21(Cip1) and p27(Kip1), are involved in the regulation of apoptosis. Here, we report that selective expression of the third member in this family, p57(Kip2), potentiated staurosporine-induced apoptosis in HeLa cells. This pro-apoptotic effect was associated with an increased caspase-3 activity. In contrast, glucocorticoid treatment, despite inducing p57(Kip2) expression in HeLa cells, was found to have an inhibitory effect on staurosporine-induced apoptosis. This anti-apoptotic effect of glucocorticoids could be explained by a concomitant increase in Bcl-x(L) expression. The results presented in this study show that p57(Kip2) has a stimulatory effect on apoptosis induced by staurosporine, suggesting a role for p57(Kip2) in the response of tumor cells to cytotoxic drugs.