Growth regulatory effects of cyclic AMP and polyamine depletion are dissociable in cultured mouse lymphoma cells

Treatment of mouse lymphoma S49 cells with D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, depleted cellular polyamine levels and stopped cell growth. The cells were arrested predominantly in G1. Thus, polyamine depletion may lead to a regulatory growth arrest in S49 cells. We tested two hypotheses regarding the relationship of growth arrest mediated by polyamine limitation to that mediated by cyclic AMP (cAMP). The hypothesis that cAMP-induced arrest results from polyamine depletion is not tenable, because the arrest could not be reversed by addition of exogenous polyamines, and because cellular polyamine levels do not drop in dibuturyl cyclic AMP (Bt2cAMP)-arrested cells. The hypothesis that polyamine-mediated growth arrest is effected via modulation of cAMP levels or cAMP-dependent protein kinase activity was also shown to be incorrect, because a S49 variant deficient in cAMP-dependent protein kinase was arrested by DFMO. The activities of the polyamine-synthesizing enzymes ornithine decarboxylase (ODC) and S-adenosyl methionine decarboxylase (SAMD) are both reduced in Bt2cAMP-treated cells to about 10% of that in control populations, as shown previously. DFMO diminishes ODC activity and augments SAMD activity in both untreated and Bt2cAMP-treated cells, leading to polyamine depletion in both cases.     

鸟氨酸脱羧酶基因反义RNA对前列腺癌细胞生长的抑制作用

为研究鸟氨酸脱羧酶 (ODC)基因反义RNA对前列腺癌细胞的生长抑制作用 ,将表达ODC第 3外显子反义RNA的重组腺病毒rAd ODC Ex3as分别感染前列腺癌细胞株PC 3和LNCap .通过MTT法观察其对前列腺癌细胞增殖的影响 ,并确定不同细胞合适的感染滴度 ,再采用Western印迹和流式细胞术检测rAd ODC Ex3as对细胞中ODC表达的抑制作用、对细胞周期和凋亡的影响以及与CDK抑制物p2 1的关系 .实验显示 ,rAd ODC Ex3as分别以 5 0MOI、2 5MOI感染PC 3和LNCap细胞可明显抑制其生长增殖 ,而不引起细胞毒性作用 ;其对两种细胞中ODC表达的抑制作用分别为4 5 %和 5 9% .流式细胞DNA含量分析证实 ,rAd ODC Ex3as可引起PC 3和LNCap细胞周期G1期阻滞 ,但并未引起凋亡 .通过Western印迹发现 ,细胞中ODC表达的降低可诱导p2 1蛋白的过表达 .结果表明 ,rAd ODC Ex3as在体外能有效地干扰ODC基因的表达 ,并通过诱导p2 1的过表达使其细胞周期停于G1期 ,从而抑制前列腺癌细胞PC 3和LNCap的增殖 ,为其进一步基因治疗的研究打下基础 .     

Polyamine depletion arrests cell cycle and induces inhibitors p21Waf1/Cip1, p27Kip1, and p53 in IEC-6 cells

The polyamines spermidine and spermine and their precursorputrescine are intimately involved in and are required for cell growthand proliferation. This study examines the mechanism by whichpolyamines modulate cell growth, cell cycle progression, and signaltransduction cascades. IEC-6 cells were grown in the presence orabsence ofDL--difluoromethylornithine(DFMO), a specific inhibitor of ornithine decarboxylase, which is thefirst rate-limiting enzyme for polyamine synthesis. Depletion ofpolyamines inhibited growth and arrested cells in theG₁ phase of the cell cycle. Cellcycle arrest was accompanied by an increase in the level of p53 proteinand other cell cycle inhibitors, including p21^Waf1/Cip1 andp27^Kip1. Induction of cell cycleinhibitors and p53 did not induce apoptosis in IEC-6 cells, unlike manyother cell lines. Although polyamine depletion decreased the expressionof extracellular signal-regulated kinase (ERK)-2 protein, a sustainedincrease in ERK-2 isoform activity was observed. The ERK-1 proteinlevel did not change, but ERK-1 activity was increased inpolyamine-depleted cells. In addition, polyamine depletion induced thestress-activated proteinkinase/c-JunNH₂-terminal kinase (JNK) type ofmitogen-activated protein kinase (MAPK). Activation of JNK-1 was theearliest event; within 5 h after DFMO treatment, JNK activity wasincreased by 150%. The above results indicate that polyamine depletioncauses cell cycle arrest and upregulates cell cycle inhibitors andsuggest that MAPK and JNK may be involved in the regulation of theactivity of these molecules.     

多胺生物合成的抑制对转化细胞c－Ha－ras癌基因表达的调控

抗多胺代谢剂──二氟甲基鸟氨酸（ＤＦＭＯ）作用于经含点突变的Ｈａ－ｒａｓ基因片段转染的转化细胞（ＨＲ－１细胞）引起细胞生长的抑制,其抑制率随ＤＦＭＯ浓度的增加而增大,此时细胞多停滞于Ｇ_１期;多胺合成的关键酶鸟氨酸脱羧酶（ＯＤＣ）活性显著下降;Ｈａ－ｒａｓ癌基因ｍＲＮＡ及ｒａｓＰ~（２１）蛋白的表达受到抑制;而外源性腐胺与ＤＦＭＯ的同时加入可防止上述一系列改变的发生,说明ＤＦＭＯ使ＨＲ－１细胞某些表型向亲本细胞逆转的作用是与细胞多胺生物合成的抑制直接相关。     

Inhibition of ornithine decarboxylase by retinoic acid and difluoromethylornithine in relation to their effects on differentiation and proliferation

Murine embryonal carcinoma F9 cells can be induced to differentiate by 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC). The differentiated phenotype is similar to that of retinoic acid (RA)-treated F9 cells. In contrast to F9 cells the differentiated cells secrete plasminogen activator and express keratin intermediate filaments. Both DFMO and RA reduce ornithine decarboxylase activity, polyamine levels and inhibit cell proliferation of F9 cells. These compounds also reduce ODC, polyamine levels and proliferation of mouse BALB/c 3T6 fibroblasts. RA inhibits the induction of ODC by insulin, serum and to a lesser extent that of epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The action of DFMO and RA can be distinguished by their response to putrescine. The induction of differentiation and the inhibition of cell proliferation by DFMO can be totally abolished upon the addition of putrescine, whereas the actions of RA are not affected at all. These results suggest that the inhibition of ODC and reduction of polyamines are not causal in the induction of differentiation and the inhibition of proliferation by RA.     

The role of p21(CIP1/WAF1) in growth of epithelial cells exposed to hyperoxia

Previous studies have shown that hyperoxia inhibits proliferation and increases the expression of the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(CIP1/WAF1), which inhibits proliferation in the G1 phase of the cell cycle. To determine whether growth arrest was mediated through activation of the p21-dependent G1 checkpoint, the kinetics of cell cycle movement during exposure to 95% O2 were assessed in the Mv1Lu and A549 pulmonary adenocarcinoma cell lines. Cell counts, 5-bromo-2'-deoxyuridine incorporation, and cell cycle analyses revealed that growth arrest of both cell lines occurred in S phase, with A549 cells also showing evidence of a G1 arrest. Hyperoxia increased p21 in A549 but not in Mv1Lu cells, consistent with the activation of the p21-dependent G1 checkpoint. The ability of p21 to exert the G1 arrest was confirmed by showing that hyperoxia inhibited proliferation of HCT 116 colon carcinoma cells predominantly in G1, whereas an isogenic line lacking p21 arrested in S phase. The cell cycle arrest in S phase appears to be a p21-independent process caused by a gradual reduction in the rate of DNA strand elongation. Our data reveal that hyperoxia inhibits proliferation in G1 and S phase and demonstrate that p53 and p21 retain their ability to affect G1 checkpoint control during exposure to elevated O2 levels.     

Ehrlich ascites tumour cells become refractory to alpha-difluoromethylornithine at a certain stage of growth

When Ehrlich ascites tumour cells are induced to proliferate by serum stimulation, the ornithine decarboxylase (ODC) activity increases rapidly and reaches two to three peaks during the first 24 h. Inhibition of the first peak in ODC activity (occurring at 4 h) by adding alpha-difluoromethylornithine (DFMO) within 2 h of serum stimulation, results in maximal growth inhibition. Under these conditions, similar degrees of polyamine depletion are achieved. When DFMO is added 3 h after seeding, however, enough polyamines have already accumulated during the initial burst in ODC activity to reduce the antiproliferative effect of the drug. The antiproliferative effect is further reduced when DFMO is added 6 h after seeding. When DFMO is added 23 h after seeding, i.e. after maximal accumulation of polyamines, there is no inhibition of cell proliferation. These findings are important to consider both when designing experimental as well as clinical regimens for this drug.     

Intracellular chloride regulates cell proliferation through the activation of stress‐activated protein kinases in MKN28 human gastric cancer cells

Recently, we reported that reduction of intracellular Cl^? concentration ([Cl^?]_i) inhibited proliferation of MKN28 gastric cancer cells by diminishing the transition rate from G₁ to S cell‐cycle phase through upregulation of p21, cyclin‐dependent kinase inhibitor, in a p53‐independent manner. However, it is still unknown how intracellular Cl^? regulates p21 expression level. In this study, we demonstrate that mitogen‐activated protein kinases (MAPKs) are involved in the p21 upregulation and cell‐cycle arrest induced by reduction of [Cl^?]_i. Culture of MKN28 cells in a low Cl^? medium significantly induced phosphorylation (activation) of MAPKs (ERK, p38, and JNK) and G₁/S cell‐cycle arrest. To clarify the involvement of MAPKs in p21 upregulation and cell growth inhibition in the low Cl^? medium, we studied effects of specific MAPKs inhibitors on p21 upregulation and G₁/S cell‐cycle arrest in MKN28 cells. Treatment with an inhibitor of p38 or JNK significantly suppressed p21 upregulation caused by culture in a low Cl^? medium and rescued MKN28 cells from the low Cl^?‐induced G₁ cell‐cycle arrest, whereas treatment with an ERK inhibitor had no significant effect on p21 expression or the growth of MKN28 cells in the low Cl^? medium. These results strongly suggest that the intracellular Cl^? affects the cell proliferation via activation of p38 and/or JNK cascades through upregulation of the cyclin‐dependent kinase inhibitor (p21) in a p53‐independent manner in MKN28 cells. J. Cell. Physiol. 223:764–770, 2010. © 2010 Wiley‐Liss, Inc.     

Tumor cytokinetic effects of acute starvation versus polyamine depletion in tumor-bearing mice.

Previous investigations in our laboratory have demonstrated that both acute host starvation and polyamine depletion by means of the irreversible ODC-inhibitor (ODC = ornithine-decarboxylase) fluoro-methylornithine (DFMO) lead to pronounced growth retardation of rapidly proliferating tumors. The aim of this investigation was to elucidate how these different interventions affect cell kinetics and cell cycle phases in vivo. Adult nongrowing mice (C57Bl/J) bearing a poorly differentiated rapidly growing methylcholanthrene induced sarcoma were used. Combined measurements of bromodeoxyuridine incorporation into DNA and flow cytometric techniques were used. Starvation and DFMO treatment resulted in a prolonged cell cycle transit compared to freely fed animals. Tumor cells from DFMO-treated mice demonstrated an increased time for DNA synthesis and a relatively larger accumulation of cells in the G2M phase, whereas tumor cells from starved animals were accumulated in the G0G1 phase. The fractional cell loss of tumor cell during proliferation was calculated to be around 18% higher in DFMO-treated animals compared to starved and freely fed tumor-bearing mice. This study demonstrates that different mechanisms are involved in tumor growth suppression from substrate deficiency (starvation) and from inhibition of polyamine synthesis.     

Helicobacter pylori inhibits gastric cell cycle progression

Helicobacter pylori infection of the gastric mucosa is associated with changes in gastric epithelial cell proliferation. In vitro studies have shown that exposure to H. pylori inhibits proliferation of gastric cells. This study sought to investigate the cell cycle progression of gastric epithelial cell lines in the presence and absence of H. pylori. Unsynchronized and synchronized gastric epithelial cell lines AGS and KatoIII were exposed to H. pylori over a 24-h period. Cell cycle progression was determined by flow cytometry using propidium iodide (PI), and by analysis of cyclin E, p21, and p53 protein expression using Western blots. In the absence of H. pylori 40, 45, and 15% of unsynchronized AGS cells were in G(0)-G(1), S, and G(2)-M phases, respectively, by flow cytometry analysis. When AGS cells were cultured in the presence of H. pylori, the S phase decreased 10% and the G(0)-G(1) phase increased 17% after 24 h compared with the controls. KatoIII cells, which have a deleted p53 gene, showed little or no response to H. pylori. When G1/S synchronized AGS cells were incubated with media containing H. pylori, the G(1) phase increased significantly (25%, P < 0.05) compared with controls after 24 h. In contrast, the control cells were able to pass through S phase. The inhibitory effects of H. pylori on the cell cycle of AGS cells were associated with a significant increase in p53 and p21 expression after 24 h. The expression of cyclin E was downregulated in AGS cells following exposure of AGS cells to H. pylori for 24 h. This study shows that H. pylori-induced growth inhibition in vitro is predominantly at the G(0)-G(1) checkpoint. Our results suggest that p53 may be important in H. pylori-induced cell cycle arrest. These results support a role for cyclin-dependent kinase inhibitors in the G(1) cell cycle arrest exerted by H. pylori and its involvement in changing the regulatory proteins, p53, p21, and cyclin E in the cell cycle.     

Biological effects of G1 phase arrest compound,sesquicillin, in human breast cancer cell lines

Sesquicillin, isolated from fungal fermentation broth, strongly induced G1 phase arrest in human breast cancer cells. During G1 phase arrest, the expression level of cyclin D1, cyclin A, and cyclin E was decreased, and the expression of CDK (cyclin-dependent-kinase) inhibitor, protein p21(Waf1/Cip1), was increased in a time-dependent manner in a breast cancer cell MCF-7. Interestingly, the G1 phase arrest induced by sesquicillin also occurred independently of the tumor suppressor protein, p53. Sesquicillin inhibits the proliferation of MCF-7 via G1 phase arrest in association with the induction of CDK inhibitor protein, p21(Waf1/Cip1), and the reduction of G1 phase related-cyclin proteins.     

Induction of F9 embryonal carcinoma cell differentiation by inhibition of polyamine synthesis

alpha-Difluoromethylornithine (DFMO), a highly selective inhibitor of ornithine decarboxylase (ODC), induced terminal differentiation of F9 mouse embryonal carcinoma cells in culture. Differentiation was assessed using morphological criteria and the level of plasminogen activator activity. The observed phenotypic changes and the fact that the cells did not synthesize alpha-fetoprotein, indicate that they were parietal endoderm cells. The putrescine, spermidine and spermine content of untreated control cells increased during exponential growth and then decreased gradually with continued time in culture. The increases in putrescine and spermidine contents were prevented by DFMO treatment. In fact, the putrescine and spermidine content decreased below the limits of detection after only one day of treatment. The addition of putrescine to the culture medium at any time within 4 days of DFMO treatment, prevented the DFMO-induced differentiation, suggesting that the effects observed were indeed caused by polyamine depletion. The phenotypic changes induced by DFMO were similar to those induced by retinoic acid, a very potent inducer of embryonal carcinoma differentiation. Although retinoic acid can inhibit ODC activity and putrescine accumulation, it is unlikely that this mechanism of action is responsible for retinoic acid-induced F9 cell differentiation, inasmuch as putrescine addition did not prevent the expression of the differentiated phenotype. Undifferentiated F9 embryonal carcinoma cells exhibited a very short G1 phase, and in this respect they are similar to the cells of the preimplantation mouse embryo. In control (exponentially growing) cultures a majority of the F9 cells were in the S phase, but in DFMO-treated cultures they accumulated in the G1 phase and showed no further proliferative potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of cell proliferation and polyamine metabolism in rat liver cell cultures by the iron chelator O-trensox

The antiproliferative effects of the iron chelator O-trensox and the ornithine-decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO) were characterized in the rat hepatoma cell line FAO, the rat liver epithelial cell line (RLEC) and the primary rat hepatocyte cultures stimulated by EGF. We observed that O-trensox and DFMO decreased cell viabilty and DNA replication in the three culture models. The cytostatic effect of O-trensox was correlated to a cytotoxicity, higher than for DFMO, and to a cell cycle arrest in G0/G1 or S phases. Moreover, O-trensox and DFMO decreased the intracellular concentration of spermidine in the three models without changing significantly the spermine level. We concluded that iron, but also polyamine depletion, decrease cell growth. However, the drop in cell proliferation obtained with O-trensox was stronger compared to DFMO effect. Altogether, our data provide insights that, in the three rat liver cell culture models, the cytostatic effect of the iron chelator O-trensox may be the addition of two mechanisms: iron and polyamine depletion.     

Knock-down of methylenetetrahydrofolate reductase reduces gastric cancer cell survival: an in vitro study

The present study aimed to investigate the effect of knocking-down methylenetetrahydrofolate reductase (MTHFR) on the survival of the human gastric cancer cell line MKN45. Antisense and small interfering RNA (siRNA) plasmids were used to target MTHFR in MKN45. Meanwhile, we also constructed a wild-type MTHFR plasmid to assess the effect of over-expression of this protein on cell viability. The knock-down of MTHFR decreased cell survival by approximately 30% compared to the control and resulted in cell cycle arrest at the G2 phase. These cells also had lower levels of c-myc compared to control cells, while over-expression of MTHFR increased cell proliferation and induced the down-regulation of p21WAF1 and hMLH1. Inhibiting MTHFR with either antisense or siRNA decreases the viability of methionine-dependent transformed gastric cancer cells and suggests that MTHFR inhibition may be a novel anticancer approach.     

Ornithine decarboxylase activity and cell cycle regulation in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the specific activity of the enzyme ornithine decarboxylase (ODC) was correlated with overall growth status. The activity of ODC was highest in actively growing cells, whereas the specific activity was lower in slow-growing cultures limited for nitrogen or inhibited by low concentrations of cycloheximide. Specific activities of ODC were also low in cultures arrested in the stationary phase (in the G1 portion of the cell cycle) by starvation for required nutrients. Although correlated with overall growth, ODC activity was not required for growth or cell cycle regulation. Cells continued to grow in the presence of the polyamine spermidine or spermine, which markedly reduced ODC specific activities. Thus, high levels of ODC activity were not necessary for growth, nor were decreased ODC specific activities sufficient to cause cells to arrest in G1. Conversely, one agent (o-phenanthroline) which causes growing cells to arrest in G1 did so with no effect on ODC specific activity. Therefore, ODC specific activity changes are not necessary for cell cycle regulation but simply reflect the normal growth status of cells.     

Different cell cycle mechanisms between UV-induced and X-ray-induced apoptosis in WiDr colorectal carcinoma cells

Although DNA-damaging agents such as ultraviolet (UV) and X-ray can induce apoptosis, the difference in the apoptotic mechanism is not clearly understood. In the present study, we investigated the effects of these two genotoxic agents on the induction of DNA damage and subsequent apoptotic cell death from the viewpoint of cell cycle regulation by using WiDr cells. Transient G1 arrest was observed after UV exposure, whereas G2 but not G1 arrest was induced after X-ray irradiation. UV-exposure could induce G1 arrest in both mutant-type (mt-p53) and wild-type p53 (wt-p53) cells, but obvious G1 arrest was not observed in the cells lacking in p53 expression. An increase in the DNA fragmentation was observed at S phase in UV-irradiated cells and at G2 phase in X-irradiated cells, respectively. UV-irradiated cells showed an increase production of p53 protein and accumulation of p21 protein. On the contrary, both p53 and p21 proteins remained at a low level in X-irradiated cells. Treatment with aphidicolin, an S phase blocking agent, prolonged cell cycle arrest and reduced the rate of apoptotic cell death in both UV-irradiated and X-irradiated cells. From these results, it is suggested that UV-induced apoptosis occurs mainly at S phase and is regulated by increased production of p53 and p21 proteins, while X-ray-induced apoptosis occurs after G2 blockade and may be independent of p53.