No influence of magnetic fields on cell cycle progression using conditions relevant for patients during MRI

The purpose of this study was to examine whether exposure to magnetic fields (MFs) relevant for magnetic resonance imaging (MRI) in clinical routine influences cell cycle progression in two tumor cell lines in vitro. HL60 and EA2 cells were exposed to four types of MFs: (i) static MF of 1.5 and 7.05 T, (ii) extremely low frequency magnetic gradient fields (ELFMGFs) with +/- 10 mT/m and 100 Hz, as well as +/- 100 mT/m and 100 Hz, (iii) pulsed high frequency MF in the radiofrequency (RF) range (63.6 MHz, 5.8 microT), and (iv) a combination of (i-iii). Exposure periods ranged from 1 to 24 h. Cell cycle distribution (G(0)/G(1), S, and G(2)/M phases) was analyzed by flow cytometry. Cell cycle analysis did not reveal differences between the exposed and the control cells. As expected, positive controls with irradiated (8 Gy) HL60 and EA2 cells showed a strong G(2)/M arrest. Using conditions that are relevant for patients during MRI, no influence of MFs on cell cycle progression was observed in these cell lines. Care was taken to control secondary parameters of influence, such as vibration by the MR scanner or temperature to avoid false positive results.     

PA200, a nuclear proteasome activator involved in DNA repair

We have identified a novel 200 kDa nuclear protein that activates the proteasome. The protein, which we call PA200, has been purified to homogeneity from bovine testis and has been shown to activate proteasomal hydrolysis of peptides, but not proteins. Following gamma-irradiation of HeLa cells the uniform nuclear distribution of PA200 changes to a strikingly punctate pattern, a behavior characteristic of many DNA repair proteins. Homologs of PA200 are present in worms, plants and yeast. Others have shown that mutation of yeast PA200 results in hypersensitivity to bleomycin, and exposure of yeast to DNA damaging agents induces the PA200 message. Taken together, these findings implicate PA200 in DNA repair, possibly by recruiting proteasomes to double strand breaks.     

Delphinidin,a dietary anthocyanidin in pigmented fruits and vegetables: A new weapon to blunt prostate cancer growth

In a recent publication, we have shown that delphinidin, an anthocyanidin induces apoptosis and cell cycle arrest in highly metastatic human prostate cancer (PCa) PC3 cells. Extending these studies, we provide additional evidence that delphinidin induces apoptosis and cell cycle arrest in androgen refractory human PCa 22Rn1 cells and that these effects are concomitant with inhibition of NF-kB. We observed that delphinidin treatment to 22Rn1 cells resulted in a dose-dependent (i) G2/M phase cell cycle arrest, (ii) induction of apoptosis (iii) and inhibition of NF-kB signaling. The induction of apoptosis by delphinidin was mediated via activation of caspases since a general caspase inhibitor Z-VAD-FMK significantly reversed this effect. Delphinidin treatment to cells resulted in a dose-dependent decrease in (i) phosphorylation of IKKgamma (NEMO), (ii) phosphorylation of NF-kB inhibitory protein, (iii) phosphorylation of NF-kB/p65 at Ser536 and NF-kB/p50 at Ser529, (iv) NF-kB/p65 nuclear translocation, and (v) NF-kB DNA binding activity. Taken together, our data show that delphinidin induces apoptosis of both androgen independent and androgen refractory human PCa cells via activation of caspases and in addition, this effect might be due to inhibition of NF-kB signaling. We suggest that delphinidin could be developed as a novel agent against PCa.     

Two Molecularly Distinct G2/M Checkpoints Are Induced by Ionizing Irradiation

Cell cycle checkpoints are among the multiple mechanisms that eukaryotic cells possess to maintain genomic integrity and minimize tumorigenesis. Ionizing irradiation (IR) induces measurable arrests in the G(1), S, and G(2) phases of the mammalian cell cycle, and the ATM (ataxia telangiectasia mutated) protein plays a role in initiating checkpoint pathways in all three of these cell cycle phases. However, cells lacking ATM function exhibit both a defective G(2) checkpoint and a prolonged G(2) arrest after IR, suggesting the existence of different types of G(2) arrest. Two molecularly distinct G(2)/M checkpoints were identified, and the critical importance of the choice of G(2)/M checkpoint assay was demonstrated. The first of these G(2)/M checkpoints occurs early after IR, is very transient, is ATM dependent and dose independent (between 1 and 10 Gy), and represents the failure of cells which had been in G(2) at the time of irradiation to progress into mitosis. Cell cycle assays that can distinguish mitotic cells from G(2) cells must be used to assess this arrest. In contrast, G(2)/M accumulation, typically assessed by propidium iodide staining, begins to be measurable only several hours after IR, is ATM independent, is dose dependent, and represents the accumulation of cells that had been in earlier phases of the cell cycle at the time of exposure to radiation. G(2)/M accumulation after IR is not affected by the early G(2)/M checkpoint and is enhanced in cells lacking the IR-induced S-phase checkpoint, such as those lacking Nbs1 or Brca1 function, because of a prolonged G(2) arrest of cells that had been in S phase at the time of irradiation. Finally, neither the S-phase checkpoint nor the G(2) checkpoints appear to affect survival following irradiation. Thus, two different G(2) arrest mechanisms are present in mammalian cells, and the type of cell cycle checkpoint assay to be used in experimental investigation must be thoughtfully selected.     

The proteasome activator PA200 regulates tumor cell responsiveness to glutamine and resistance to ionizing radiation

The cellular response to ionizing radiation (IR) involves a variety of mechanisms to repair damage and maintain cell survival. We previously reported that the proteasome activator PA200 promotes long-term cell survival after IR exposure. The molecular function of PA200 is to enhance proteasome-mediated cleavage after glutamate; however, it is not known how this molecular function promotes survival after IR exposure. Here, we report that upon IR exposure, cellular demand for exogenous glutamine is increased. Cells containing PA200 are capable of surviving this IR-induced glutamine demand, whereas PA200-deficient cells show impaired long-term survival. Additional glutamine supplementation reverses the radiosensitivity of PA200-knockdown cells suggesting impaired glutamine homeostasis in these cells. Indeed, PA200-knockdown cells are unable to maintain intracellular glutamine levels. Furthermore, when extracellular glutamine is limiting, cells that contain PA200 respond by slowing growth, but PA200-knockdown cells and cells in which post-glutamyl proteasome activity is inhibited are nonresponsive and continue rapid growth. This cellular unresponsiveness to nutrient depletion is also reflected at the level of the mTOR substrate ribosomal S6 kinase (S6K). Thus, inability to restrict growth causes PA200-deficient cells to continue growing and eventually die due to lack of available glutamine. Together, these data indicate an important role for PA200 and post-glutamyl proteasome activity in maintaining glutamine homeostasis, which appears to be especially important for long-term survival of tumor cells after radiation exposure.     

DNA replication is required To elicit cellular responses to psoralen-induced DNA interstrand cross-links

Following introduction of DNA interstrand cross-links (ICLs), mammalian cells display chromosome breakage or cell cycle delay with a 4N DNA content. To further understand the nature of the delay, previously described as a G(2)/M arrest, we developed a protocol to generate ICLs during specific intervals of the cell cycle. Synchronous populations of G(1), S, and G(2) cells were treated with photoactivated 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and scored for normal passage into mitosis. In contrast to what was found for ionizing radiation, ICLs introduced during G(2) did not result in a G(2)/M arrest, mitotic arrest, or chromosome breakage. Rather, subsequent passage through S phase was required to trigger both chromosome breakage and arrest in the next cell cycle. Similarly, ICLs introduced during G(1) did not cause a G(1)/S arrest. We conclude that DNA replication is required to elicit the cellular responses of cell cycle arrest and genomic instability after psoralen-induced ICLs. In primary human fibroblasts, the 4N DNA content cell cycle arrest triggered by ICLs was long lasting but reversible. Kinetic analysis suggested that these cells could remove up to approximately 2,500 ICLs/genome at an average rate of 11 ICLs/genome/h.     

Prolonged cell cycle arrest in irradiated human diploid skin fibroblasts: the role of nutrient deprivation

Ionizing radiation has been reported to cause an irreversible cell cycle arrest in normal human diploid fibroblasts. However, colony survival assays show that even at high doses of gamma radiation, human diploid fibroblasts do not irreversibly arrest, and that a dose-dependent fraction is capable of continued cycling. In this study, we resolve the apparent discrepancy between colony survival assays and the observed radiation-induced prolonged arrest. Using flow cytometry analysis, we have confirmed that human diploid fibroblasts do exhibit a prolonged cell cycle arrest in both G(1) and G(2)/M phases of the cell cycle. However, a single replacement of fresh growth medium stimulated a fraction of the arrested population of cells to transiently re-enter the cell cycle. Daily medium changes stimulated these irradiated human diploid fibroblasts to continue cycling until they were contact-inhibited. Thus the fraction of human diploid fibroblasts which survive radiation exposure and are capable of cycling appears to permanently arrest as a result of nutrient insufficiency. Western blot analysis demonstrated a radiation-induced elevation in TP53 (formerly known as p53) protein levels within 2 h postirradiation, followed by a decrease to levels comparable to those in unirradiated controls. The TP53 and CDKN1A (formerly known as p21) protein levels were indistinguishable after 24 h and remained elevated for a 6-day period of observation in both control and irradiated cultures. Our studies indicate that human diploid fibroblasts are capable of re-entering the cell cycle after exposure to ionizing radiation and that this re-entry is dependent on a constant supply of nutrients provided by fresh medium changes. The fraction of cells capable of resuming cell cycling is consistent with the surviving fraction of cells in colony assays.     

Effect of treatment schedule on the interaction of Cisplatin and radiation in human lung cancer cells

This study was designed to determine the effects of the treatment schedule on the interaction between cisplatin and radiation. Cells of a human squamous cell lung cancer cell line were treated with cisplatin and radiation using three treatment protocols: 1-h exposure to cisplatin immediately followed by irradiation (A), 4-day continuous exposure to cisplatin immediately followed by irradiation (B), and 1-h exposure to cisplatin followed by irradiation after a 4-day interval (C). The interactions were assessed by isobologram, cell cycle distribution and apoptosis. The combination resulted in a additive effect in every protocol. Cell cycle accumulation at G(2)/M phase before irradiation was observed in Protocols B and C, whereas no cell cycle shift in the limited time course was noted in Protocol A. Although a 4-day continuous exposure to cisplatin and a 1-h exposure to cisplatin followed by a 4-day interval before irradiation caused significantly increased apoptosis, an additional increase in apoptosis after irradiation was not observed in Protocols B and C, whereas Protocol A showed an additional increase. Despite a cell cycle shift favoring radiation sensitivity, the drug-radiation interactions in Protocols B and C were additive, possibly because of negative effects including induction of a durable G(2)/M-phase arrest and suppression of apoptosis by cisplatin.     

Distribution of SH-SY5Y human neuroblastoma cells in the cell cycle following exposure to organophosphorus compounds

The current study investigated the relationship of the cell cycle phase (as G(0)/G(1), S, and G(2)/M) and cytotoxicity (as sub-G(1) DNA) to determine whether alterations in cell replication were associated with organophosphate (OP) compound induced cytotoxicity. Results demonstrated that, overall, OP compound--induced cell cycle changes were variable and depended on the OP compound, exposure concentration, and temporal relationship to cytotoxicity. Noncytotoxic OP compound treatments substantially decreased the percentage of cells in S phase of the cell cycle when compared to controls. A corresponding increase was seen in the percent of cells in G(0)/G(1) phase of the cell cycle. In the precytotoxic interval of exposure, most cytotoxic OP compound treatments substantially decreased the percentage of cells in G(2)/M phase of the cell cycle. Corresponding increases were seen primarily in G(0)/G(1) phase cells. Effects on cells in S stage of the cell cycle varied with the OP compound. In the during cytotoxic interval of exposure, most cytotoxic OP compound treatments substantially increased the percentage of cells in S phase of the cell cycle. A corresponding decrease in the percent of cells in G(0)/G(1) stage of the cell cycle was observed. Furthermore, treatments either increased or decreased the percentage of cells in G(2)/M phase of the cell cycle when compared to controls, with decreases more likely with the most cytotoxic OP compound exposures. Overall, the in vitro data suggest that exposure to OP compounds can alter the cell cycle status of SH-SY5Y neuroblastoma cells depending on compound, concentration, and interval from initial exposure. Changes in cell cycle, however, did not differentiate between OP compounds that are known for their ability to acutely inhibit acetylcholinesterase versus those inducing type I and type II delayed neurotoxicity.     

Cell cycle dysregulation by green tea polyphenol epigallocatechin-3-gallate

Epidemiological, in vitro cell culture, and in vivo animal studies have shown that green tea or its constituent polyphenols, particularly its major polyphenol epigallocatechin-3-gallate (EGCG) may protect against many cancer types. In earlier studies, we showed that green tea polyphenol EGCG causes a G0/G1-phase cell cycle arrest and apoptosis of human epidermoid carcinoma (A431) cells. We also demonstrated that these effects of EGCG may be mediated through the inhibition of nuclear factor kappa B that has been associated with cell cycle regulation and cancer. In this study, employing A431 cells, we provide evidence for the involvement of cyclin kinase inhibitor (cki)-cyclin-cyclin-dependent kinase (cdk) machinery during cell cycle deregulation by EGCG. As shown by immunoblot analysis, EGCG treatment of the cells resulted in significant dose- and time-dependent (i) upregulation of the protein expression of WAF1/p21, KIP1/p27, p16 and p18, (ii) downmodulation of the protein expression of cyclin D1, cdk4 and cdk6, but not of cyclin E and cdk2, (iii) inhibition of the kinase activities associated with cyclin E, cyclin D1, cdk2, cdk4 and cdk6. Taken together, our study suggests that EGCG causes an induction of G1-phase ckis, which inhibit the cyclin-cdk complexes operative in G0/G1 phase of the cell cycle thereby causing a G0/G1-phase arrest of the cell cycle, which is an irreversible process ultimately resulting in an apoptotic cell death. We suggest that the naturally occurring agents such as green tea polyphenols which may inhibit cell cycle progression could be developed as potent anticancer agents for the management of cancer.     

Human immunodeficiency virus type 1 Vpr induces cell cycle arrest at the G(1) phase and apoptosis via disruption of mitochondrial function in rodent cells

Vpr of human immunodeficiency virus type 1 causes cell cycle arrest at the G(2)/M phase and induces apoptosis after G(2)/M arrest in primate cells. We have reported previously that Vpr also induces apoptosis independently of G(2)/M arrest in human HeLa cells. By contrast, Vpr does not induce G(2)/M arrest in rodent cells, but it retards cell growth. To clarify the relationship between cell cycle arrest and apoptosis, we expressed Vpr endogenously in rodent cells and investigated cell cycle profiles and apoptosis. We show here that Vpr induces cell cycle arrest at the G(1) phase and apoptosis in rodent cells. Vpr increased the activity of caspase-3 and caspase-9, but not of caspase-8. Moreover, Vpr-induced apoptosis could be inhibited by inhibitors of caspase-3 and caspase-9, but not by inhibitor of caspase-8. We also showed that Vpr induces the release of cytochrome c from mitochondria into the cytosol and disrupts the mitochondrial transmembrane potential. Finally, we showed that apoptosis occurred in HeLa cells through an identical pathway. These results suggest that disruption of mitochondrial functions by Vpr induces apoptosis via cell cycle arrest at G(1), but that apoptosis is independent of G(2)/M arrest. Furthermore, it appears that Vpr acts species-specifically with respect to induction of cell cycle arrest but not of apoptosis.     

From START to FINISH: The Influence of Osmotic Stress on the Cell Cycle

The cell cycle is a sequence of biochemical events that are controlled by complex but robust molecular machinery. This enables cells to achieve accurate self-reproduction under a broad range of different conditions. Environmental changes are transmitted by molecular signalling networks, which coordinate their action with the cell cycle. The cell cycle process and its responses to environmental stresses arise from intertwined nonlinear interactions among large numbers of simpler components. Yet, understanding of how these pieces fit together into a coherent whole requires a systems biology approach. Here, we present a novel mathematical model that describes the influence of osmotic stress on the entire cell cycle of S. cerevisiae for the first time. Our model incorporates all recently known and several proposed interactions between the osmotic stress response pathway and the cell cycle. This model unveils the mechanisms that emerge as a consequence of the interaction between the cell cycle and stress response networks. Furthermore, it characterises the role of individual components. Moreover, it predicts different phenotypical responses for cells depending on the phase of cells at the onset of the stress. The key predictions of the model are: (i) exposure of cells to osmotic stress during the late S and the early G2/M phase can induce DNA re-replication before cell division occurs, (ii) cells stressed at the late G2/M phase display accelerated exit from mitosis and arrest in the next cell cycle, (iii) osmotic stress delays the G1-to-S and G2-to-M transitions in a dose dependent manner, whereas it accelerates the M-to-G1 transition independently of the stress dose and (iv) the Hog MAPK network compensates the role of the MEN network during cell division of MEN mutant cells. These model predictions are supported by independent experiments in S. cerevisiae and, moreover, have recently been observed in other eukaryotes.     

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.     

Cannabinoid receptor agonist-induced apoptosis of human prostate cancer cells LNCaP proceeds through sustained activation of ERK1/2 leading to G1 cell cycle arrest

We have recently shown that the expression levels of both cannabinoid receptors CB(1) and CB(2) are higher in human prostate cancer cells than in normal prostate epithelial cells, and treatment of LNCaP cells with WIN-55,212-2 (a mixed CB(1)/CB(2) agonist) resulted in inhibition of cell growth and induction of apoptosis (Sarfaraz, S., Afaq, F., Adhami, V. M., and Mukhtar, H. (2005) Cancer Res. 65, 1635-1641). This study was conducted to understand the mechanistic basis of these effects. Treatment of LNCaP cells with WIN-55,212-2 (1-10 microm; 24 h) resulted in: (i) an arrest of the cells in the G(0)/G(1) phase of the cell cycle; (ii) an induction of p53 and p27/KIP1; (iii) down-regulation of cyclins D1, D2, E; (iii) decrease in the expression of cdk-2, -4, and -6; (iv) decrease in protein expression of pRb; (v) down-regulation of E2F (1-4); and (vi) decrease in the protein expression of DP1 and DP2. Similar effects were also observed when androgen-independent PC3 cells were treated with WIN-55,212-2 (5-30 microm). We further observed sustained up-regulation of ERK1/2 and inhibition of PI3k/Akt pathways in WIN-55,212-2-treated cells. Inhibition of ERK1/2 abrogated WIN-55,212-2-indued cell death suggesting that sustained activation of ERK1/2 leads to cell cycle dysregulation and arrest of cells in G(0)/G(1) phase subsequently leading to an induction of apoptosis. Further, WIN-55,212-2 treatment of cells resulted in a dose-dependent increase in Bax/Bcl-2 ratio in such a way that favors apoptosis. The induction of apoptosis proceeded through down-regulation of caspases 3, 6, 7, and 9 and cleavage of poly (ADP-ribose) polymerases. Based on these data we suggest that cannabinoid receptor agonists should be considered as novel agents for the management of prostate cancer.     

Mornings with Art, lessons learned: feedback regulation, restriction threshold biology, and redundancy govern molecular stress responses

Work from the laboratory of Dr. Arthur B. Pardee has highlighted basic principles that govern cellular and molecular biological processes in living cells. Among the most important governing principles in cellular and molecular responses are: (i) threshold "restriction" responses, wherein a level of response is reached and a "point of no return" is achieved; (ii) feedback regulation; and (iii) redundancy. Lessons learned from the molecular biology of cellular stress responses in mammalian cancer versus normal cells after ionizing radiation (IR) or chemotherapeutic agent exposures reveal similar instances of these guiding principles in mammalian cells. Among these are the: (i) induction of cell death responses by beta-lapachone (beta-lap), a naphthoquinone anti-tumor agent that kills cancer cells via an NQO1 (i.e., X-ray-inducible protein-3, xip3)-dependent mechanism; (ii) induction of secretory clusterin (sCLU) in response to TGF-beta1 exposure, and the ability of induced sCLU protein to down-regulate TGF-beta1 signaling; and (iii) induction of DNA mismatch repair-dependent G(2) cell cycle checkpoint responses after exposure to alkylating agents. We have learned these lessons and now adopted strategies to exploit them for improved therapy. These examples will be discussed and compared to the pioneering findings of researchers in the Pardee laboratory over the years.     

Radiation-induced apoptosis and cell cycle progression in Jurkat T cells.

The purpose of this study was to explore the connection between radiation-induced apoptosis and progression of cells through the phases of the cell cycle. Cells of the human T-cell line Jurkat were separated by centrifugal elutriation into populations enriched in G(1)-, S- and G(2)/M-phase cells before irradiation. After a dose of 20 Gy, the onset of massive apoptosis occurred at about 6 h in all populations regardless of the phase of the cell cycle in which they were irradiated. In contrast, after 2 Gy, cells died at various times after a pronounced G(2)/M-phase arrest. These results indicate that radiation-induced apoptosis can occur independently of cell cycle arrest and that the time for onset of apoptosis may be dependent on the radiation dose.     

Study of the cytolethal distending toxin-induced cell cycle arrest in HeLa cells: involvement of the CDC25 phosphatase

HeLa cells exposed to Escherichia coli cytolethal distending toxins (CDT) arrest their cell cycle at the G2/M transition. We have shown previously that in these cells the CDK1/cyclin B complex is inactive and can be reactivated in vitro using recombinant CDC25 phosphatase. Here we have investigated in vivo the effects of CDC25 on this cell cycle checkpoint. We report that overexpression of CDC25B or CDC25C overrides an established CDT-induced G2 cell cycle arrest and leads the cells to accumulate in an abnormal mitotic stage with condensed chromatin and high CDK1 activity. This effect can be counteracted by coexpression of the WEE1 kinase. In contrast, overexpression of CDC25B or C prior to CDT treatment prevents G2 arrest and allows most of the cells to progress through mitosis with only a low percentage of cells arrested in abnormal mitosis. The implications of these results on the biochemical nature of the CDT-induced cell cycle arrest are discussed.