A lymphocyte blastogenesis inhibitory factor (LBIF) reversibly arrests a human melanoma cell line, A375, at G1 and G2 phases of cell cycle

A lymphocyte blastogenesis inhibitory factor, LBIF, has been found in the culture supernatant of a human macrophage-like cell line, U937. The factor has been purified by fast protein liquid chromatography. Partial amino acid sequencing analysis showed that LBIF was a novel immunoregulatory factor. Recent study has demonstrated that LBIF possesses a remarkable tumor growth inhibitory activity. In this study, the cell growth inhibitory activity of LBIF was characterized on the proliferation of a human melanoma cell line A375 in vitro. LBIF strongly inhibits the proliferation of A375 cells. The inhibitory activity was cytostatic and reversible by Day 5 although the lethal effect became apparent at Day 7. Cell cycle analysis by flow cytometry showed that LBIF arrested A375 cells at both G₁ and G₂/M phases. Mitotic index analysis indicated that A375 cells were arrested in G₁ and G₂ phases. LBIF function was not attributed to the elevation of intracytoplasmic cyclic-AMP levels. Thus, these results suggest that LBIF plays an important role in controlling cell cycle and there is a similarity between the mechanisms of G₁ and G₂ arrests in eukaryotic cell proliferation. LBIF-induced reversible cell-cycle arrest of A375 cells can be a useful system to analyze the signal transduction for cell proliferation and cell-cycle arrest.     

cAMP-mediated growth inhibition of a B-lymphoid precursor cell line Reh is associated with an early transient delay in G2/M, followed by an accumulation of cells in G1

This study was undertaken to gain more insight into the effects of cyclic adenosine monophosphate (cAMP) on cell-cycle progression in the B-lymphoid precursor cell line Reh. The adenylate cyclase activator forskolin reduced the proliferation of asynchronously growing Reh cells by 50% after 72 hr culture. Growth inhibition was associated with an accumulation of cells in G1. Furthermore, we demonstrated that forskolin provoked a delay of cells for approximately 10 hr in G2/M prior to the G1 arrest. Two different methods were applied to elucidate how cells in different phases of the cell cycle were affected by an elevated cAMP level. One method was based on centrifugal elutriation, whereby synchronous cell populations from the different phases of the cell cycle were isolated. By the other method, S-phase cells were selectively stained by pulsing asynchronously growing cells with bromo-deoxyuridine (BrdU). The data demonstrate that the position of a cell in the cell cycle is critical in determining how the cell will respond to an elevated cAMP level. Thus cells in G1 at the time forskolin is added are not delayed in G2/M, but they will subsequently accumulate in G1 after 48 hr. Cells given forskolin in G2/m, however, are delayed for 10 hr in G2/M, but they do not accumulate in G1. Cells given forskolin in the S phase are delayed in G2/M as well as arrested in G1. The results suggest that cAMP inhibits growth of the Reh cells by preventing the cells from passing important restriction points located in the G1 and G2 phases of the cell cycle.     

How the change from FLM to FACS affected our understanding of the G1-phase of the cell cycle

The frequency of labeled mitoses (FLM) method for analyzing cell-cycle phases necessitates a determination of cell-cycle interdivision times and the absolute lengths of the cell-cycle phases. The change to flow sorting (FACS) analysis, a simpler, less labor intensive, and more rapid method, eliminated determinations of absolute phase times, yielding only percents of cells exhibiting particular DMA contents. Without an interdivision time value, conversion of these fractions into absolute phase lengths is not possible. This change in methodology has led to an alteration in how the cell cycle is viewed. The FLM method allowed the conclusion that G1 phase variability resulted from constancy of S and G2 phase lengths. In contrast, with FACS analysis, slow growing cells exhibiting a large fraction of cells with a G1-phase amount of DMA appeared to be "arrested in G1 phase". The loss of absolute phase length determinations has therefore led to the proposals of G1-phase arrest, G1-phase controls, restriction points, and G0 phase. It is suggested that these G1-phase controls and phenomena require a critical reevaluation in the light of an alternative cell-cycle model that does not require or postulate such G1-phase controls.     

A novel peptide sansalvamide analogue inhibits pancreatic cancer cell growth through G0/G1 cell-cycle arrest

Patients with pancreatic cancer have little hope for cure because no effective therapies are available. Sansalvamide A is a cyclic depsipeptide produced by a marine fungus. We investigated the effect of a novel sansalvamide A analogue on growth, cell-cycle phases, and induction of apoptosis in human pancreatic cancer cells in vitro. The sansalvamide analogue caused marked time- and concentration-dependent inhibition of DNA synthesis and cell proliferation of two human pancreatic cancer cell lines (AsPC-1 and S2-013). The analogue induced G0/G1 phase cell-cycle arrest and morphological changes suggesting induction of apoptosis. Apoptosis was confirmed by annexin V binding. This novel sansalvamide analogue inhibits growth of pancreatic cancer cells through G0/G1 arrest and induces apoptosis. Sansalvamide analogues may be valuable for the treatment of pancreatic cancer.     

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.     

Phosphoinositide 3-kinase signaling overrides a G2 phase arrest checkpoint and promotes aberrant cell cycling and death of hematopoietic cells after DNA damage

DNA damage activates arrest checkpoints to halt cell cycle progression in G1 and G2 phases. These checkpoints can be overridden in hematopoietic cells by cytokines, such as erythropoietin, through the activation of a phosphoinositide 3-kinase (PI3K) signaling pathway. Here, we show that PI3K activity specifically overrides delayed mechanisms effecting permanent G1 and G2 phase arrests, but does not affect transient checkpoints arresting cells up to 10 hours after gamma-irradiation. Assessing the status of cell cycle regulators in hematopoietic cells arrested after gamma-irradiation, we show that Cdk2 activity is completely inhibited in both G1 and G2 arrested cells. Despite the absence of Cdk2 activity, cells arrested in G2 phase did retain detectable levels of Cdk1 activity in the absence of PI3K signaling. However, reactivation of PI3K promoted robust increases in both Cdk1 and Cdk2 activity in G2-arrested cells. Reactivation of Cdks was accompanied by a resumption of cell cycling, but with strikingly different effectiveness in G1 and G2 phase arrested cells. Specifically, G1-arrested cells resumed normal cell cycle progression with little loss in viability when PI3K was activated after gamma-irradiation. Conversely, PI3K activation in G2-arrested cells promoted endoreduplication and death of the entire population. These observations show that cytokine-induced PI3K signaling pathways promote Cdk activation and override permanent cell cycle arrest checkpoints in hematopoietic cells. While this activity can rescue irradiated cells from permanent G1 phase arrest, it results in aberrant cell cycling and death when activated in hematopoietic cells arrested at the G2 phase DNA damage checkpoint.     

Insulin-like factor binding protein-3 promotes the G1 cell cycle arrest in several cancer cell lines

Insulin-like growth factor binding protein-3 (IGFBP-3) is a multi-functional protein known to induce apoptosis of various cancer cells in an insulin-like growth factor (IGF)-dependent and IGF-independent manner. In our previous study, we found that IGFBP-3 induced apoptosis through the activation of caspases in 786-O cells. In this study, we further examined that whether IGFBP-3 induced apoptosis through the induction of cell cycle arrest in 786-O, A549 and MCF-7 cells. Our results showed that overexpressed IGFBP-3 resulted in typical apoptotic ultrastructures in A549 cells under transmission electron microscope. The result of flow cytometry analysis indicated that IGFBP-3 arrested the cell cycle at G1-S phase in 786-O, A549 and MCF-7 cells. In A549 cells, quantitative real-time PCR and Western blot analysis showed a significant change in the expression of cell cycle-regulated proteins—a decrease in cyclin E1 expression, an increase in p21 expression. These results indicate a possible mechanism for G1 cell cycle arrest by IGFBP-3. Taken together, cyclin E1 and p21 may play important roles in the IGFBP-3-inducing G1 cell cycle arrest and apoptosis in several human cancer cells.     

G2 cell cycle arrest induced by glycopeptides isolated from the bovine cerebral cortex

The ability of glycopeptides, isolated from bovine cerebral cortex, to alter cell division was studied by cell-cycle analyses. The results showed that glycopeptides arrested baby hamster kidney (BHK)-21 cells and Chinese hamster ovary (CHO) cells in the G2 phase of the cell cycle. Upon removal of the growth inhibition from arrested BHK-21 cells, the mitotic index in colchicine-treated cultures increased from 5 to 40% within 6 h and the increase in mitotic activity was accompanied by a complete doubling of all arrested cells within this 6- h time period. Determination of DNA content in growth-arrested BHK-21 cells showed that growth-arrested cells contained about twice the DNA of control cell cultures. Although CHO cells treated in a like manner with growth inhibitor could not be arrested for the same length of time as BHK-21 cells (18 h vs. 72 h before initiation of escape) and to the same degree (60% of the cell population vs. 99% of BHK-21 cells), the escape kinetics of CHO cells did indicate a G2 arrest. Approximately 3.5 h after escape began, CHO cell numbers in treated cultures attained the cell numbers found in control cultures. This rapid growth phase occurring in less than 4 h indicated that the growth inhibitor induced a G2 arrest-point in CHO cells that was not lethal since the entire arrested cell population divided.     

Growth arrest in G1 protects against oxygen-induced DNA damage and cell death

Although oxygen is required for normal aerobic respiration, hyperoxia (95% O(2)/5% CO(2)) damages DNA, inhibits proliferation in G1, S and G2 phases of the cell cycle, and induces necrosis. The current study examines whether growth arrest in G1 protects pulmonary epithelial cells from oxidative DNA damage and cell death. Mv1Lu pulmonary adenocarcinoma cells were chosen for studies because hyperoxia inhibits their proliferation in S and G2 phase, while they can be induced to arrest in G1 by altering culture conditions. Hyperoxia inhibited proliferation, increased intracellular redox, and rapidly reduced clonogenic survival. In contrast, Mv1Lu cells treated with transforming growth factor (TGF)-beta1, deprived of serum or grown to confluency, arrested and remained predominantly in G1 even during exposure. Growth arrest in G1 significantly enhanced clonogenic survival by 10-50-fold. Enhanced survival was not due to reduction in the intracellular redox-state of the cells, but instead was associated with reduced DNA strand breaks and p53 expression. Our findings suggest that the protective effects of G1 is mediated not simply by a reduction in intracellular ROS, but rather through an enhanced ability to limit or rapidly recognize and repair damaged DNA.     

Involvement of p21 in the PKC-induced regulation of the G2/M cell cycle transition

Activation of protein kinase C (PKC) inhibits cell cycle progression at the G1/S and G2/M transitions. We found that phorbol 12-myristate 13-acetate (PMA) induced upregulation of p21, not only in MCF-7 cells arrested in the G1 phase as previously shown, but also in cells delayed in the G2 phase. This increase in p21 in cells accumulated in the G1 and G2/M phases of the cell cycle after PMA treatment was inhibited by the PKC inhibitor GF109203X. This indicates that PKC activity is required for PMA-induced p21 upregulation and cell cycle arrest in the G1 and G2/M phases of the cell cycle. To further assess the role of p21 in the PKC-induced G2/M cell cycle arrest independently of its G1 arrest, we used aphidicolin-synchronised MCF-7 cells. Our results show that, in parallel with the inhibition of cdc2 activity, PMA addition enhanced the associations between p21 and either cyclin B or cdc2. Furthermore, we found that after PMA treatment p21 was able to associate with the active Tyr-15 dephosphorylated form of cdc2, but this complex was devoid of kinase activity indicating that p21 may play a role in inhibition of cdc2 induced by PMA. Taken together, these observations provide evidence that p21 is involved in integrating the PKC signaling pathway to the cell cycle machinery at the G2/M cell cycle checkpoint.     

How the Change from FLM to FACS Affected Our Understanding of the G1-Phase of the Cell Cycle

The frequency of labeled mitoses (FLM) method for analyzing cell-cycle phases necessitates a determination of cell-cycle interdivision times and the absolute lengths of the cell-cycle phases. The change to flow sorting (FACS) analysis, a simpler, less labor intensive, and more rapid method, eliminated determinations of absolute phase times, yielding only percents of cells exhibiting particular DNA contents. Without an interdivision time value, conversion of these fractions into absolute phase lengths is not possible. This change in methodology has led to an alteration in how the cell cycle is viewed. The FLM method allowed the conclusion that G1-phase variability resulted from constancy of S and G2 phase lengths. In contrast, with FACS analysis, slow growing cells exhibiting a large fraction of cells with a G1-phase amount of DNA appeared to be “arrested in G1 phase”. The loss of absolute phase length determinations has therefore led to the proposals of G1-phase arrest, G1-phase controls, restriction points, and G0 phase. It is suggested that these G1-phase controls and phenomena require a critical reevaluation in the light of an alternative cell-cycle model that does not require or postulate such G1-phase controls.     

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.     

Roscovitine, a novel cyclin-dependent kinase inhibitor, characterizes restriction point and G2/M transition in tobacco BY-2 cell suspension

Although the developmental programs of plants and animals differ, key regulatory components of their cell cycle have been conserved. Particular attention has been paid to the role of the complexes between highly conserved cyclin and cyclin-dependent kinases in regulating progression through the cell cycle. The recent demonstration that roscovitine is a potent and selective inhibitor of the animal cyclin-dependent kinases cdc2 (CDK1), CDK2 and CDK5 prompted an investigation into its effects on progression through the plant cell cycle. Roscovitine induced arrests both in late G1 and late G2 phase in BY-2 tobacco cell suspensions. Both blocks were fully reversible when roscovitine was used at concentrations similar to those used in the animal system. Stationary-phase cells subcultured in the presence of roscovitine were arrested at a 2C DNA content. This arrest was more efficient without exogenous addition of plant growth regulator. Roscovitine induced a block in G1 earlier than that induced by aphidicolin. S-phase synchronized cells treated with roscovitine were arrested at a 4C DNA content at the G2/ M transition. The expression analysis of a mitotic cyclin (NTCYC1) indicated that the roscovitine-induced G2 block probably occurs in late G2. Finally, cells in metaphase were insensitive to roscovitine. The purified CDK/cyclin kinase activities of late G1 and early M arrested cells were inhibited in vitro by roscovitine. The implications of these experimental observations for the requirement for CDK activity during progression through the plant cell cycle are discussed.     

Reversible G1 arrest of a human lung epithelial cell line by staurosporine.

Staurosporine, a microbial-derived protein kinase inhibitor, reversibly blocked non-synchronized, replicating cultures of the human lung epithelial cell line EKVX in the G1 phase of cell cycle and inhibited DNA synthesis and cell replication. The mechanism of this cell-cycle arrest in EKVX cells by staurosporine was likely due to inhibition of protein kinase C (PKC) because: 1) dose-dependent inhibition of DNA synthesis occurred at levels of staurosporine that inhibit phosphorylation of PKC substrate, 2) inhibition of DNA synthesis was also seen after treatment with another PKC inhibitor H7, but not by the chemically similar HA1004, which has a relative inhibitory specificity for cAMP-dependent protein kinase, and 3) the DNA synthesis was not inhibited by specific tyrosine kinase inhibitors Genistein and Lavendustin A at concentrations that inhibit tyrosine kinase activity. Removal of staurosporine from cell culture media resulted in a rebound in PKC activity and synchronized DNA synthesis in EKVX cultures. The reversibility of the inhibition was noted even after 5 days of treatment with staurosporine, and DNA synthesis remained synchronized for at least two rounds of cell replication after removal of staurosporine. Flow cytometric analysis confirmed that more than 90% of the cell population was blocked in the G1 phase after cells were treated with staurosporine for 24 h. Agents such as staurosporine may be useful for synchronizing cell populations to study cell-cycle specific biochemical events important for the regulation of cell replication in the EKVX cell line.     

Human immunodeficiency virus type 1 Vpr modifies cell proliferation via multiple pathways.

Vpr, one of the accessory molecules of HIV-1, has been demonstrated to arrest the cell cycle at the G2 phase. This Vpr-mediated cell cycle arrest is implicated to have an important role in the viral life cycle. In the present study, we quantitate the extent of Vpr-mediated cell cycle arrest with the use of a bicistronic vector consisting of a vpr gene and a green fluorescence protein sequence. Using this system, we examined the effect of several Vprs on cell cycle progression and growth of cells from different species quantitatively. We found that Vpr from the T-cell line-adapted HIV-1SF2 strain (Vpr2) could not significantly induce G2 arrest in HeLa cells but was able to induce it in 293T cells. However, strong inhibition of cell proliferation in HeLa cells as well as in 293T cells was observed by Vpr2. This ability of Vpr2 to inhibit cell proliferation without G2 arrest was also observed when expressed in monkey cell line. Analyses of chimeric Vprs revealed that this species-non-specific growth inhibitory activity of Vpr was not mediated solely by the C-terminal region of Vpr. These results indicated that the growth inhibitory activity of Vpr is independent of its G2 arresting activity. In addition, the species-non-specific nature of this activity suggests that Vpr has a novel mechanism to retard cell proliferation by influencing basic cellular functions.     

MCPIP1 overexpression in human neuroblastoma cell lines causes cell-cycle arrest by G1/S checkpoint block

Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) has a multidomain structure, which assures its pleiotropic activity. The physiological functions of this protein include repression of inflammatory processes and the prevention of immune disorders. The influence of MCPIP1 on the cell cycle of cancer cells has not been sufficiently elucidated. A previous study by our group reported that overexpression of MCPIP1 affects the cell viability, inhibits the activation of the phosphoinositide-3 kinase/mammalian target of rapamycin signalling pathway, and reduces the stability of the MYCN oncogene in neuroblastoma (NB) cells. Furthermore, a decrease in expression and phosphorylation levels of cyclin-dependent kinase (CDK) 1, which has a key role in the M phase of the cell cycle, was observed. On the basis of these previous results, the purpose of our present study was to elucidate the influence of MCPIP1 on the cell cycle of NB cells. It was confirmed that ectopic overexpression of MCPIP1 in two human NB cell lines, KELLY and BE(2)-C, inhibited cell proliferation. Furthermore, flow cytometric analyses and imaging of the cell cycle with a fluorescence ubiquitination cell-cycle indicator test, demonstrated that overexpression of MCPIP1 causes an accumulation of NB cells in the G1 phase of the cell cycle, while the possibility of an increase in G0 phase due to induction of quiescence or senescence was excluded. Additional assessment of the molecular machinery responsible for the transition between the cell-cycle phases confirmed that MCPIP1 overexpression reduced the expression of cyclins A2, B1, D1, D3, E1, and E2 and decreased the phosphorylation of CDK2 and CDK4, as well as retinoblastoma protein. In conclusion, the present results indicated a relevant impact of overexpression of MCPIP1 on the cell cycle, namely a block of the G1/S cell-cycle checkpoint, resulting in arrest of NB cells in the G1 phase.     

Activation of two distinct anti-proliferative pathways, apoptosis and p38 MAP kinase-dependent cell cycle arrest, by tumor necrosis factor in human melanoma cell line A375

The proliferation of human melanoma cell line A375-6 cells is inhibited by several cytokines, including interleukin-1 (IL-1). A375-R8 cells, a subclone of A375-6, are resistant to IL-1-induced growth inhibition. The proliferation of both cell lines is inhibitable by tumor necrosis factor (TNF). In this study, we characterized the mechanisms of TNF-induced growth inhibition. TNF-induced growth inhibition in both cell lines was partially suppressed by a selective p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), whereas a combination of SB203580 and Z-VAD-fmk, an inhibitor for a wide range of caspases, completely blocked TNF-induced growth inhibition, indicating that TNF-induced growth inhibition is mediated by both p38 MAPK and caspases. However, Z-VAD-fmk alone suppressed TNF-induced growth inhibition in A375-R8, but not A375-6, cells, suggesting that there may exist a TNF-induced anti-apoptotic mechanism in A375-6 cells which is lost or mutated in A375-R8 cells. Evidence in support of this notion includes (1) TNF-induced apoptosis only in A375-R8, but not A375-6 cells; (2) cycloheximide enabled TNF to induce apoptosis even in A375-6 cells; and (3) somatic hybrid cells between A375-6 and A375-R8 cells are resistant to TNF-induced apoptosis. Since TNF-induced NF-kappa B activation, cell cycle arrest, RB dephosphorylation, and E2F downregulation are indistinguishable in both cell lines, none of these factors is likely to be involved in the TNF-induced anti-apoptotic mechanism in A375-6 cells. Our results indicate that TNF activates two distinct anti-proliferative pathways including p38 MAPK-dependent cell cycle arrest and caspase-mediated apoptosis, as well as an anti-apoptotic mechanism in melanoma cells.