Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner

The synchronization effects of the plant amino acid mimosine on proliferating higher eukaryotic cells are still controversial. Here, I show that 0.5 mM mimosine can induce a cell cycle arrest of human somatic cells in late G1 phase, before establishment of active DNA replication forks. The DNA content of nuclei isolated from mimosine-treated cells was determined by flow cytometry. The presence or absence of DNA replication forks in these isolated nuclei was then detected by DNA replication run-on assays in vitro. Treatment of asynchronously proliferating HeLa or EJ30 cells for 24 h with 0.5 mM mimosine resulted in a population synchronized in late G1 phase. S phase entry was inhibited by 0.5 mM mimosine in cells released from a block in mitosis or from quiescence. When added to early S phase cells, 0.5 mM mimosine did not prevent S phase transit, but delayed progression through late stages of S phase after a lag of 4 h, eventually resulting in a G1 phase population by preventing entry into the subsequent S phase. In contrast, lower concentrations of mimosine (0.1-0.2 mM) failed to prevent S phase entry, resulting in cells containing active DNA replication foci. The G1 phase arrest by 0.5 mM mimosine was reversible upon mimosine withdrawal. This synchronization protocol using 0.5 mM mimosine can be exploited for studying the initiation of human DNA replication in vitro.     

Inhibition of ultraviolet B (UVB) induced apoptosis in A431 cells by mimosine is not dependent on cell cycle arrest

Ultraviolet (UV) radiation is a strong apoptotic trigger in many cell types. We have previously reported that a plant amino acid, mimosine (beta [N-(3-hydroxy-4-pyridone)]-alpha-aminopropionic acid), with a well-known reversible G1 cell cycle arrest activity can inhibit apoptosis induced by UV irradiation and RNA polymerase II blockage in human A431 cells. Here, apoptosis was measured with a fluorimetric caspase activation assay. Interestingly, the protective state was effective up to 24 h following removal of mimosine from the culture medium while cells were progressing in the cell cycle. Our results demonstrate that the protective effect of mimosine against UV-induced apoptosis can be dissociated from its G1 cell-cycle arrest activity.     

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     

Mitotic UV Irradiation Induces a DNA Replication-Licensing Defect that Potentiates G1 Arrest Response

Cdt1 begins to accumulate in M phase and has a key role in establishing replication licensing at the end of mitosis or in early G1 phase. Treatments that damage the DNA of cells, such as UV irradiation, induce Cdt1 degradation through PCNA-dependent CRL4-Cdt2 ubiquitin ligase. How Cdt1 degradation is linked to cell cycle progression, however, remains unclear. In G1 phase, when licensing is established, UV irradiation leads to Cdt1 degradation, but has little effect on the licensing state. In M phase, however, UV irradiation does not induce Cdt1 degradation. When mitotic UV-irradiated cells were released into G1 phase, Cdt1 was degraded before licensing was established. Thus, these cells exhibited both defective licensing and G1 cell cycle arrest. The frequency of G1 arrest increased in cells expressing extra copies of Cdt2, and thus in cells in which Cdt1 degradation was enhanced, whereas the frequency of G1 arrest was reduced in cell expressing an extra copy of Cdt1. The G1 arrest response of cells irradiated in mitosis was important for cell survival by preventing the induction of apoptosis. Based on these observations, we propose that mammalian cells have a DNA replication-licensing checkpoint response to DNA damage induced during mitosis.     

Screening of five specific cell cycle inhibitors using a T Cell lymphoma cell line synchrony/release assay

To obtain different cell populations at specific cell cycle stages, we used a cell culture synchronization protocol. Effects of five different cell cycle inhibitors acting throughout the cell cycle were examined by DNA flow cytometric analysis of a synchrony/release lymphoma cell line (CEM). The screening synchronized protocol showed that staurosporine, mimosine and aphidicolin are reversible G1 phase inhibitors that act at different times. Staurosporine acted in early G1, exhibited the strongest cytotoxic effect, and induced apoptosis. Mimosine and aphidicolin acted in late G1 and at the G1/S boundary, respectively. Hydroxyurea arrested CEM cells in early S phase, but later than the aphidicolin arrest point. Nocodazole synchronized CEM cells in M phase. All the inhibitors examined in this study can be used to synchronize cells at different phases of the cell cycle and were reversible with little toxicity except for staurosporine which is highly toxic. Because the regulatory mechanism of the cell cycle is disrupted by their effects on protein synthesis, however, these drugs must be used with caution.     

Apoptosis and cell cycle progression in an acidic environment after irradiation

Apoptosis and cell cycle progression in HL60 cells irradiated in an acidic environment were investigated. Apoptosis was determined by TUNEL staining, PARP cleavage, DNA fragmentation, and flow cytometry. The majority of the apoptosis that occurred in HL60 cells after 4 Gy irradiation took place after G(2)/M-phase arrest. When irradiated with 12 Gy, a fraction of the cells underwent apoptosis in G(1) and S phases while the rest of the cells underwent apoptosis in G(2)/M phase. The apoptosis caused by 4 and 12 Gy irradiation was transiently suppressed in medium at pH 7.1 or lower. An acidic environment was found to perturb progression of irradiated cells through the cell cycle, including progression through G(2)/ M phase. Thus it was concluded that the suppression of apoptosis in the cells after 4-12 Gy irradiation in acidic medium was due at least in part to a delay in cell cycle progression, particularly the prolongation of G(2)/M-phase arrest. Irradiation with 20 Gy indiscriminately caused apoptosis in all cell cycle phases, i.e. G(1), S and G(2)/M phases, rapidly in neutral pH medium and relatively slowly in acidic pH medium. The delay in apoptosis in acidic medium after 20 Gy irradiation appeared to result from mechanisms other than prolonged G(2)/ M-phase arrest.     

A reversible arrest point in the late G1 phase of the mammalian cell cycle

The effects of two different cell cycle inhibitors on the proliferation of human lymphoblastoid cells have been analyzed by flow cytometric techniques. Mimosine, a plant amino acid, reversibly blocks the cell cycle at a point which occurs roughly 2 h before the arrest mediated by aphidicolin, an inhibitor of DNA polymerase alpha activity, which defines the G1/S phase boundary. The levels of thymidine kinase mRNA, which increase at the onset of S phase, are higher in cells blocked with aphidicolin than in cells treated with mimosine whereas the opposite results are obtained in the case of p53 mRNA levels, which are known to be maximal in the late G1 phase. These results indicate that mimosine inhibits cell cycle traverse in the late G1 phase prior to the onset of DNA synthesis and identifies a previously undefined reversible cell cycle arrest point.     

Role of G1 phase in the UV-induced apoptosis of EL-4 mouse lymphoma cells

Although UV is known to induce apoptotic cell death to various animal cells, relationship between cell cycle and UV-induced apoptosis is still unclear. In this study, we investigated the role of G1 phase in UV-induced apoptosis by using EL-4 mouse lymphoma cells which have wild type p53. After 500 J/m UV irradiation, an increase of apoptotic fraction was accompanied by cell cycle accumulation in the G1 phase. Apoptotic fraction after UV-exposure was remarkably augmented by treatment with 2-AP, a G1 checkpoint inhibitor. In contrast, aphidicolin, an inhibitor of DNA polymerase , suppressed the rate of apoptotic fraction.These results suggest that mandatory cell cycle progression from G1 to S leaves the damaged DNA unrepaired and may increase the apoptotic fraction. To investigate the precise mechanism in the G1 phase, UV was exposed to the G1-synchronized cells and apoptotic fraction was serially observed. Synchronized EL-4 cells passed through the G1 phase in 8 h. Within the G1 phase, late-G1 cells (6 h after M) were more sensitive to UV-induced apoptosis than early-G1 cells (2 h after M) (49.7 ± 9.0% vs. 41.5 ± 8.5%, p < 0.05). In HL-60 cells, lacking in p53 expression, such a difference was not observed. Western blot analysis revealed that expression of p53 in synchronized EL-4 cells was increasingly enhanced during G1 phase. After UV-exposure, p53 expression gradually decreased in early-G1 cells, but it was kept at almost the same level in late-G1 cells. In addition, bcl-2 expression in early-G1 cells showed a more rapid and larger increase than that in late-G1 cells. These results suggest that susceptibility of the G1 cells to UV-induced apoptosis depends on their position within the G1 phase, and late-G1 is more sensitive than early-G1. Sensitivity to UV-induced apoptosis is closely related to the expression level of p53 and bcl-2 proteins. Early-G1 cells may be able to take enough time to repair damaged DNA until they reach the G1 checkpoint compared to the late-G1 cells.     

Ras-dependent cell cycle commitment during G2 phase

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

Effect of UV irradiation on the mitotic cycle of Chinese hamster cells with varying UV sensitivity. I. The time ratios after irradiation of the cells with UV light

The distribution of cells through the phases of the cell cycle by DNA flow cytofluorimetry was analysed to investigate the effects of UV irradiation on cell cycle progression in asynchronous Chinese hamster cells with different UV-sensitivity: cell line V79 (UV-resistant cells), and UV-sensitive clones: B6, CHS1, CHS2 and XII. The UV-irradiated cultures show a large accumulation of cells in S phase, the effect increasing with UV dose increase, which may point to an inhibition of the DNA chain elongation. UV-sensitive clones show a larger and more prolongated increase in the proportion of cells in S phase after irradiation with smaller dose than UV-resistant cells. Besides, the UV-sensitive clone XII shows an inhibition of movement of irradiated cells from G1 into S phase, that may testify to an inhibition of replicon initiation. These results suggest that there is a correlation in UV-irradiated Chinese hamster cells between alteration in cell cycle progression and UV-sensitivity of cells.     

The Gcn2 Kinase as a Cell Cycle Regulator

Cell cycle progression through G1 phase is of particular importance because this is the phase where the decision to embark on another cell cycle is made. An aberrant G1/S transition often leads to cell cycle deregulation and cancer development. Therefore, there is a complex regulatory network to ensure timely entry into S phase, coordinating initiation of DNA replication with growth and stress signals. We have studied the response of fission yeast cells to ultraviolet (UV) irradiation in G1 phase and identified a Gcn2-dependent checkpoint that delays entry into S phase. UV irradiation activates Gcn2 which, in turn, phosphorylates the translation initiation factor eIF2α and depresses translation. Phosphorylation of eIF2α is a well-known response to various forms of stress, but whether or how this response is causing the specific cell cycle effects is not known. Here we discuss the relationships between Gcn2 activity, eIF2α phosphorylation, translation downregulation and cell cycle delay.     

Cell cycle progression in the presence of irreparable DNA damage is controlled by a Mec1- and Rad53-dependent checkpoint in budding yeast.

We studied the response of nucleotide excision repair (NER)-defective rad14Delta cells to UV irradiation in G(1) followed by release into the cell cycle. Only a subset of checkpoint proteins appears to mediate cell cycle arrest and regulate the timely activation of replication origins in the presence of unrepaired UV-induced lesions. In fact, Mec1 and Rad53, but not Rad9 and the Rad24 group of checkpoint proteins, are required to delay cell cycle progression in rad14Delta cells after UV damage in G(1). Consistently, Mec1-dependent Rad53 phosphorylation after UV irradiation takes place in rad14Delta cells also in the absence of Rad9, Rad17, Rad24, Mec3 and Ddc1, and correlates with entry into S phase. Two-dimensional gel analysis indicates that late replication origins are not fired in rad14Delta cells UV-irradiated in G(1) and released into the cell cycle, which instead initiate DNA replication from early origins and accumulate replication and recombination intermediates. Progression through S phase of UV-treated NER-deficient mec1 and rad53 mutants correlates with late origin firing, suggesting that unregulated DNA replication in the presence of irreparable UV-induced lesions might result from a failure to prevent initiation at late origins.     

Mimosine reversibly arrests cell cycle progression at the G1-S phase border

It has previously been demonstrated that the compound mimosine inhibits cell cycle traverse in late G1 phase prior to the onset of DNA synthesis (Hoffman BD, Hanauske-Abel HM, Flint A, Lalande M: Cytometry 12:26-32, 1991; Lalande M: Exp Cell Res 186:332-339, 1990). These results were obtained by using flow cytometric analysis of DNA content to compare the effects of mimosine on cell cycle traverse with those of aphidicolin, an inhibitor of DNA polymerase alpha activity. We have now measured the incorporation of bromodeoxyuridine into lymphoblastoid cells by flow cytometry to determine precisely where the two inhibitors act relative to the initiation of DNA synthesis. It is demonstrated here that mimosine arrests cell cycle progression at the G1-S phase border. The onset of DNA replication occurs within 15 min of releasing the cells from the mimosine block. In contrast, treatment with aphidicolin results in the accumulation of cells in early S phase. These results indicate that mimosine is a suitable compound for affecting the synchronous release of cells from G1 into S phase and for analyzing the biochemical events associated with this cell cycle phase transition.     

The plant amino acid mimosine may inhibit initiation at origins of replication in Chinese hamster cells.

An understanding of replication initiation in mammalian cells has been hampered by the lack of mutations and/or inhibitors that arrest cells just prior to entry into the S period. The plant amino acid mimosine has recently been suggested to inhibit cells at a regulatory step in late G1. We have examined the effects of mimosine on cell cycle traverse in the mimosine [corrected]-resistant CHO cell line CHOC 400. When administered to cultures for 14 h after reversal of a G0 block, the drug appears to arrest the population at the G1/S boundary, and upon its removal cells enter the S phase in a synchronous wave. However, when methotrexate is administered to an actively dividing asynchronous culture, cells are arrested not only at the G1/S interface but also in early and middle S phase. Most interestingly, two-dimensional gel analysis of replication intermediates in the initiation locus of the amplified dihydrofolate reductase domain suggests that mimosine may actually inhibit initiation. Thus, this drug represents a new class of inhibitors that may open a window on regulatory events occurring at individual origins of replication.     

Diarsenic and tetraarsenic oxide inhibit cell cycle progression and bFGF- and VEGF-induced proliferation of human endothelial cells

Arsenic trioxide (As2O3, diarsenic oxide) has recently been reported to induce apoptosis and inhibit the proliferation of various human cancer cells derived from solid tumors as well as hematopoietic malignancies. In this study, the in vitro effects of As2O3 and tetraasrsenic oxide (As4O6) on cell cycle regulation and basic fibroblast growth factor (bFGF)- or vascular endothelial growth factor (VEGF)-stimulated cell proliferation of human umbilical vein endothelial cells (HUVEC) were investigated. Significant dose-dependent inhibition of cell proliferation was observed when HUVEC were treated with either arsenical compound for 48 h, and flow cytometric analysis revealed that these two arsenical compounds induced cell cycle arrest at the G1 and G2/M phases--the increases in cell population at the G1 and G2/M phase were dominantly observed in As2O3- and As4O6-treated cells, respectively. In both arsenical compounds-treated cells, the protein levels of cyclin A and CDC25C were significantly reduced in a dose-dependent manner, concomitant to the reduced activities of CDK2- and CDC2-associated kinase. In G1-synchronized HUVEC, the arsenical compounds prevented the cell cycle progression from G1 to S phase, which was stimulated by bFGF or VEGF, through the inhibition of growth factor-dependent signaling. These results suggest that arsenical compounds inhibit the proliferation of HUVEC via G1 and G2/M phase arrest of the cell cycle. In addition, these inhibitory effects on bFGF- or VEGF-stimulated cell proliferation suggest antiangiogenic potential of these arsenical compounds.     

Protein tyrosine nitration in the cell cycle

Nitration of tyrosine residues in proteins is associated with cell response to oxidative/nitrosative stress. Tyrosine nitration is relatively low abundant post-translational modification that may affect protein functions. Little is known about the extent of protein tyrosine nitration in cells during progression through the cell cycle. Here we report identification of proteins enriched for tyrosine nitration in cells synchronized in G0/G1, S or G2/M phases of the cell cycle. We identified 27 proteins in cells synchronized in G0/G1 phase, 37 proteins in S phase synchronized cells, and 12 proteins related to G2/M phase. Nineteen of the identified proteins were previously described as regulators of cell proliferation. Thus, our data indicate which tyrosine nitrated proteins may affect regulation of the cell cycle.