G2 Delay Research Articles

Cdc123 and Checkpoint Forkhead Associated with RING Proteins Control the Cell Cycle by Controlling eIF2γ Abundance

Eukaryotic initiation factor 2 (eIF2) is a central regulator of translational initiation in times of growth and times of stress. Here we discovered three new conserved regulators of eIF2 in Saccharomyces cerevisiae. cdc123, homolog of mammalian D123, is a new cell division cycle mutant with a G2 delay at permissive temperature and a terminal, mating-proficient G1 arrest point. Cdc123 protein is regulated by nutrient availability. CHF1 and CHF2, homologs of mammalian checkpoint forkhead associated with RING genes, are required for G2 delay and G1 arrest of cdc123-4 and promote G1 delay when over-expressed. Cell cycle delaying activity and the natural instability of Chf1 and Chf2 depend on the integrity of both domains and association with Cdc123. Genetic analysis maps the Chf1 forkhead associated domain-binding site to the conserved Thr-274 of Cdc123, suggesting that mammalian D123 is a key target of Chfr. Gcd11, the gamma subunit of eIF2, is an additional Cdc123-interacting protein that is an essential target of the Cdc123 cell cycle promoting and Chf cell cycle arresting activity whose abundance is regulated by Cdc123, Chf1, and Chf2. Loss of cdc123 activity promotes Chf1 and Chf2 accumulation and Gcd11 depletion, accounting for the essentiality of Cdc123. The data establish the Cdc123-Chf-Gcd11 axis as an essential pathway for nutritional control of START that runs parallel to the Tor-Gcn2-Sui2 system of translational control.

Modifications in cell cycle kinetics and in expression of G1 phase-regulating proteins in human amniotic cells after exposure to electromagnetic fields and ionizing radiation.

Low-frequency electromagnetic fields are suspected of being involved in carcinogenesis, particularly in processes that could be related to cancer promotion. Because development of cancer is associated with deregulated cell growth and we previously observed a magnetic field-induced decrease in DNA synthesis [Lange et al. (2002) Alterations in the cell cycle and in the protein level of cyclin D1p, 21CIP1, and p16INK4a after exposure to 50 HZ. MF in human cells. Radiat. Environ. Biophys.41, 131], this study aims to document the influence of 50 Hz, 1 mT magnetic fields (MF), with or without initial gamma-ionizing radiation (IR), on the following cell proliferation-relevant parameters in human amniotic fluid cells (AFC): cell cycle distribution, expression of the G1 phase-regulating proteins Cdk4, cyclin D1, p21CIP1 and p16INK4a, and Cdk4 activity. While IR induced a G1 delay and a dose-dependent G2 arrest, no discernible changes in cell cycle kinetics were observed due to MF exposure. However, a significant decrease in the protein expression of cyclin D1 and an increase in p21CIP1- and p16INK4a-expression could be detected after exposure to MF alone. IR-exposure caused an augmentation of p21CIP1- and p16INK4a- levels as well, but did not alter cyclin D1 expression. A slight diminution of Cdk4 activity was noticed after MF exposure only, indicating that Cdk4 appears not to act as a mediator of MF- or IR-induced changes in the cell cycle of AFC cells. Co-exposure to MF/IR affected neither cell cycle distribution nor protein expression or kinase activity additionally or synergistically, and therefore MF seems not to modify the mutagenic potency of IR.

Filamentous growth in Saccharomyces cerevisiae

Fungal dimorphism is a complex phenomenon triggered by a large variety of environmental factors and consists of a reversible alternating pattern of growth between different elliptical and filamentous forms of cells. Understanding the mechanisms that regulate these events is of major interest because of their implications in fungal pathogenesis, cell differentiation and industry. Diploid cells of Saccharomyces cerevisiae transform from budding yeast to pseudohyphae when starved for nitrogen, giving the cells an advantage in food foraging, which is sensed by at least two signal transduction pathways: the MAP kinase (MAPK) and the PKA (cAMP-dependent protein kinase A) pathways. The output of these signalling pathways is the expression of pseudohypha-specific genes, whose expression profiles change and is accompanied by a G2 delay in the cell cycle and a prolonged period of polarized growth. Haploid yeast strains show a similar growth type after prolonged incubation on rich medium plates. The cells form chains and invade the agar on the edge of the colony, but they do not become elongated. This growth type is referred to as haploid invasive growth. Alcohols can also induce filamentous growth in S. cerevisiae, promoting aberrant and elongated morphology. The three forms of filamentous growth are revised in this article and also the pathways involved in sensing, signaling and signal transduction during filamentous growth.

Topoisomerase II and histone deacetylase inhibitors delay the G2/M transition by triggering the p38 MAPK checkpoint pathway.

When early prophase PtK1 or Indian muntjac cells are exposed to topoisomerase II (topo II) inhibitors that induce little if any DNA damage, they are delayed from entering mitosis. We show that this delay is overridden by inhibiting the p38, but not the ATM, kinase. Treating early prophase cells with hyperosmotic medium or a histone deacetylase inhibitor similarly delays entry into mitosis, and this delay can also be prevented by inhibiting p38. Together, these results reveal that agents or stresses that induce global changes in chromatin topology during G2 delay entry into mitosis, independent of the ATM-mediated DNA damage checkpoint, by activating the p38 MAPK checkpoint. The presence of this pathway obviates the necessity of postulating the existence of multiple “chromatin modification” checkpoints during G2. Lastly, cells that enter mitosis in the presence of topo II inhibitors form metaphase spindles that are delayed in entering anaphase via the spindle assembly, and not the p38, checkpoint.

Unique natural antioxidants (NAOs) and derived purified components inhibit cell cycle progression by downregulation of ppRb and E2F in human PC3 prostate cancer cells

Prostate cancer (PCA) is the leading cause of cancer mortality among older men in Western countries. Epidemiological studies have shown correlation between a lower risk of PCA and a higher consumption of antioxidants. However, the mechanism by which antioxidants exert their effects is still unknown. In the present study, we explored the signaling mechanism through which unique natural antioxidant derived from spinach extract (NAO) exerts their beneficial effects in the chemoprevention of PCA using human PC3 cells. Probing into the effect of NAO and its derived polyphenols on cell-cycle G1 arrest, we found that they cause cell-cycle prolongation. NAO and its two derived purified components exhibited a significant increase in the level of p21 cip1 expression after 36 h of starvation, followed by 18 h of treatment with NAO in the presence of serum. In addition, under similar conditions, the expressed level of Cyclin A and CDK-2 in the PC3 cells was significantly reduced after treatment with NAO or its purified components. Immunoblot analysis demonstrated a significant increase in the hypophosphorylated form of pRb and a decrease in ppRb. NAO and its purified derived components were found to downregulate the protein expression of another member of the pRb family, p107, as well as that of E2F-1. These results suggest that NAO-induced G1 delay and cell cycle prolongation are caused by downregulation of the protein expression of ppRb and E2F in the human PCA cell line PC3.

The Clp1p/Flp1p phosphatase ensures completion of cytokinesis in response to minor perturbation of the cell division machinery in Schizosaccharomyces pombe.

Fission yeast mutants defective in actomyosin ring formation and function exhibit a prolonged G2 delay following cytokinesis failure. This G2 delay depends on the SIN, a signaling network essential for cytokinesis, and the non-essential Cdc14p family phosphatase, Clp1p/Flp1p and has been proposed to signify a cytokinesis checkpoint mechanism. However, the physiological relevance of this proposed Clp1p/Flp1p-dependent checkpoint is unclear because all previous studies were carried out using mutations in essential actomyosin ring components under fully restrictive conditions and thus these cells would have died regardless of the presence of the checkpoint. Here we show that delays in cytokinesis caused by minor perturbations to different components of the cytokinetic machinery, which normally cause only mild defects, become lethal when Clp1p/Flp1p is inactivated. In addition, we show that Clp1p/Flp1p does not function simply to inhibit further rounds of nuclear division, but also allows damaged actomyosin rings to be maintained to facilitate completion of cell division. Ectopic activation of the SIN significantly bypasses the requirement of Clp1p/Flp1p for G2 delay as well as for completion of cytokinesis. We conclude that the Clp1p/Flp1p-dependent cytokinesis checkpoint provides a previously unrecognized cell survival advantage when the cell division apparatus is mildly perturbed.

Gamma radiation-induced apoptosis, G2 delay, and the risk of salivary and thyroid carcinomas--a preliminary report.

While radiation has been the only well-established risk factor for salivary and thyroid cancers, the exact mechanisms remain unknown. We hypothesized that individuals with altered apoptotic response to gamma irradiation may be susceptible to salivary and thyroid cancers. We tested our hypothesis in a pilot case-control study of 29 patients with neoplasms of the salivary and thyroid glands and 29 cancer-free control subjects. Patients and control subjects were matched on age, sex, and ethnicity. In vitro gamma radiation-induced apoptosis in lymphocytes was quantified utilizing the TUNEL assay and flow cytometry. The mean apoptotic capacity was 13.55 +/- 10.54 for control subjects, 5.75 +/- 4.96 for patients with salivary gland carcinomas (p =.003), and 6.87 +/- 4.45 for patients with thyroid carcinomas (p =.006). These differences were associated with a 10-fold increased risk of salivary gland carcinoma (odds ratio [OR] = 10.71; 95% confidence interval [CI], 1.21-94.86) and a four-fold increased risk of thyroid carcinoma (OR = 3.93; 95% CI, 0.90-17.08). Our data suggests that gamma radiation-induced apoptosis may serve as a biomarker of genetic susceptibility to salivary and thyroid carcinoma, and further confirmatory studies with larger sample size are warranted.

Molecular interactions of fission yeast Skp1 and its role in the DNA damage checkpoint

Skp1 is a central component of the E3 ubiquitin ligase SCF (Skp1-Cullin-1-F-box). It forms an adapter bridge between Cullin-1 and the substrate-determining component, the F-box protein. In order to establish the role of Skp1, a temperature sensitive (ts) screen was carried out using mutagenic PCR (polymerase chain reaction) and 9 independent ts mutants were isolated. Mapping the mutated residues on the 3-D structure of human Skp1 suggested that the mutants would be compromised in binding to F-box proteins but not Cullin-1 (Pcu1). In order to assess the binding properties of ts Skp1, 12 F-box proteins and Pcu1 were epitope-tagged, and co-immunoprecipitation performed. This systematic analysis showed that ts Skp1 retains binding to Pcu1. However, binding to three specific F-box proteins, essential Pof1, Pof3 involved in maintaining genome integrity, and nonessential Pof10, was reduced. skp1ts cells exhibit a G2 cell cycle delay, which is attributable to activation of the DNA damage checkpoint. Intriguingly, contrary to pof3 mutants, in which this checkpoint is required for survival, checkpoint abrogation in skp1(ts) suppresses a G2 delay and furthermore almost rescues the ts phenotype. The activation mechanism of the DNA damage checkpoint therefore differs between pof3Delta and skp1(ts), implicating a novel role for Skp1 in the checkpoint-signalling cascade.

CENP-B Interacts with CENP-C Domains Containing Mif2 Regions Responsible for Centromere Localization

Recently, human artificial chromosomes featuring functional centromeres have been generated efficiently from naked synthetic alphoid DNA containing CENP-B boxes as a de novo mechanism in a human cultured cell line, but not from the synthetic alphoid DNA only containing mutations within CENP-B boxes, indicating that CENP-B has some functions in assembling centromere/kinetochore components on alphoid DNA. To investigate whether any interactions exist between CENP-B and the other centromere proteins, we screened a cDNA library by yeast two-hybrid analysis. An interaction between CENP-B and CENP-C was detected, and the CENP-C domains required were determined to overlap with three Mif2 homologous regions, which were also revealed to be involved in the CENP-C assembly of centromeres by expression of truncated polypeptides in cultured cells. Overproduction of truncated CENP-B containing no CENP-C interaction domains caused abnormal duplication of CENP-C domains at G2 and cell cycle delay at metaphase. These results suggest that the interaction between CENP-B and CENP-C may be involved in the correct assembly of CENP-C on alphoid DNA. In other words, a possible molecular linkage may exist between one of the kinetochore components and human centromere DNA through CENP-B/CENP-B box interaction.

Germinating fission yeast spores delay in G1 in response to UV irradiation

BackgroundCheckpoint mechanisms prevent cell cycle transitions until previous events have been completed or damaged DNA has been repaired. In fission yeast, checkpoint mechanisms are known to regulate entry into mitosis, but so far no checkpoint inhibiting S phase entry has been identified.ResultsWe have studied the response of germinating Schizosaccharomyces pombe spores to UV irradiation in G1. When germinating spores are irradiated in early G1 phase, entry into S phase is delayed. We argue that the observed delay is caused by two separate mechanisms. The first takes place before entry into S phase, does not depend on the checkpoint proteins Rad3, Cds1 and Chk1 and is independent of Cdc2 phosphorylation. Furthermore, it is not dependent upon inhibiting the Cdc10-dependent transcription required for S phase entry, unlike a G1/S checkpoint described in budding yeast. We show that expression of Cdt1, a protein essential for initiation of DNA replication, is delayed upon UV irradiation. The second part of the delay occurs after entry into S phase and depends on Rad3 and Cds1 and is probably due to the intra-S checkpoint. If the germinating spores are irradiated in late G1, they enter S phase without delay and arrest in S phase, suggesting that the delay we observe upon UV irradiation in early G1 is not caused by nonspecific effects of UV irradiation.ConclusionsWe have studied the response of germinating S. pombe spores to UV irradiation in G1 and shown that S phase entry is delayed by a mechanism that is different from classical checkpoint responses. Our results point to a mechanism delaying expression of proteins required for S phase entry.

Role of 2′-2′ difluorodeoxycytidine (gemcitabine)-induced cell cycle dysregulation in radio-enhancement of human head and neck squamous cell carcinomas

Background and purpose To try to get a better insight on the interaction between dFdC and ionizing radiation at the cellular level, we examined in vitro the effect of dFdC on the cell cycle of two human head and neck squamous cell carcinoma cell lines (SQD9 and SCC61). Patients and methods Experimental conditions yielding radio-enhancement were used. Confluent cells were incubated with dFdC (5 μM) for different incubation times, washed, pulse-labeled with BrdUrd (10 μM), fixed and then processed for flow cytometry analysis. Alternatively, cells preincubated or not with dFdC were irradiated (5 Gy) in drug-free medium, incubated at 37 °C for various times and then processed for flow cytometry analysis. Results In both cell lines, dFdC incubated between 1 and 6 h induced a DNA synthesis inhibition with accumulation of cells in the G1–S boundary followed, when DNA reinitiated, by a synchronous progression of cells throughout the cycle. A slightly different kinetics was observed in the two cell lines. A weak correlation between dFdC radio-enhancement and distribution of cells in the cell cycle was observed. It was also observed that for longer dFdC incubation times, DNA synthesis could reinitiate while cells were still incubated with dFdC. This reinitiation could be correlated with a decrease in the intracellular dFdCTP pool to non-inhibitory levels. Finally in both cell lines, dFdC modified neither the importance nor the kinetics of the radiation-induced G1 delay. Conclusions This study provides evidence that gemcitabine used at radio-enhancing concentration induces alteration of cell kinetics and cell redistribution throughout the cell cycle. This effect is cell line-dependent. However, the weak correlation between dFdC radio-enhancement and cell cycle distribution suggests that the cell cycle effect does not constitute the most important mechanism of interaction with ionizing radiation. Our study also indicated that in the two cell lines studied, a modulation of the G1–S checkpoint was not implicated in enhancement of radiation response by dFdC.

Caffeine and human DNA metabolism: the magic and the mystery

The ability of caffeine to reverse cell cycle checkpoint function and enhance genotoxicity after DNA damage was examined in telomerase-expressing human fibroblasts. Caffeine reversed the ATM-dependent S and G2 checkpoint responses to DNA damage induced by ionizing radiation (IR), as well as the ATR- and Chk1-dependent S checkpoint response to ultraviolet radiation (UVC). Remarkably, under conditions in which IR-induced G2 delay was reversed by caffeine, IR-induced G1 arrest was not. Incubation in caffeine did not increase the percentage of cells entering the S phase 6–8 h after irradiation; ATM-dependent phosphorylation of p53 and transactivation of p21 Cip1/Waf1 post-IR were resistant to caffeine. Caffeine alone induced a concentration- and time-dependent inhibition of DNA synthesis. It inhibited the entry of human fibroblasts into S phase by 70–80% regardless of the presence or absence of wildtype ATM or p53. Caffeine also enhanced the inhibition of cell proliferation induced by UVC in XP variant fibroblasts. This effect was reversed by expression of DNA polymerase η, indicating that translesion synthesis of UVC-induced pyrimidine dimers by DNA pol η protects human fibroblasts against UVC genotoxic effects even when other DNA repair functions are compromised by caffeine.

Single cell tracking reveals that Msh2 is a key component of an early-acting DNA damage-activated G2 checkpoint.

Dysfunction of cell-cycle checkpoints in DNA mismatch repair (MMR)-deficient cells in response to DNA damage has implications for anticancer therapy and genetic instability. We have studied the cell-cycle effects of MMR deficiency (Msh2(-/-)) in primary mouse embryonic fibroblasts (MEFs) exposed to cisplatin (10 microM x 1 h) using time-lapse microscopy. Kinetic responses of MEFs from different embryos and passage ages varied, but we report a consistent drug-induced inhibition of mitotic entry (approx. 50%). There was a loss of an early-acting (<5 h) delay in G2 to M transition in Msh2(-/-) cells, although a later-acting G2 arrest was apparently normal. This suggests that Msh2 primarily acts to delay mitotic entry of cells already in G2, that is, DNA damage incurred during G2 does not influence the cell once committed to mitotic traverse. Irrespective of Msh2 status, cisplatin treatment and the incurred DNA damage did not effect mitotic traverse or show any evidence for early (within 24 h) cell death. The results indicate that Msh2(-/-) status can result in the premature commitment to mitosis of a cell subpopulation, determined by the fraction residing in G2 at the time of damage induction. The findings suggest a new route to MMR-driven genetic instability that does not rely primarily on the integrity of the late-acting checkpoint.

The Cyclin-dependent Kinase Inhibitor p21CIP/WAF Is a Positive Regulator of Insulin-like Growth Factor I-induced Cell Proliferation in MCF-7 Human Breast Cancer Cells

To study the role of IGF-I receptor signaling on cell cycle events we utilized MCF-7 breast cancer cells. IGF-I at physiological concentrations increased the level of p21CIP/WAF mRNA after 4has well as protein after 8hby 10- and 6-fold, respectively, in MCF-7 cells. This IGF-1 effect was reduced by 50% in MCF-7-derived cells (SX13), which exhibit a 50% reduction in IGF-1R expression, demonstrating that IGF-1 receptor activation was involved in this process. Preincubation with the ERK1/2 inhibitor U0126 significantly reduced the IGF-1 effect on the amount of p21CIP/WAF protein in MCF-7 cells. These results were confirmed by the expression of a dominant negative construct for MEK-1 suggesting that the increase of the abundance of p21CIP/WAF in response to IGF-1 occurs via the ERK1/2 mitogen-activated protein kinase pathway. Using an antisense strategy, we demonstrated that abolition of p21CIP/WAF expression decreased by 2-fold the IGF-1 effect on cell proliferation in MCF-7. This latter result is explained by a delay in G1 to S cell cycle progression due partly to a reduction in the activation of some components of cell cycle including the induction of cyclin D1 expression in response to IGF-1. MCF-7 cells transiently overexpressing p21 showed increased basal and IGF-I-induced thymidine incorporation. Taken together, these results define p21CIP/WAF as a positive regulator in the cell proliferation induced by IGF-1 in MCF-7 cells.

Phospholipid Hydroperoxide Glutathione Peroxidase Induces a Delay in G1 of the Cell Cycle

Phospholipid hydroperoxide glutathione peroxidase (PhGPx) is an antioxidant enzyme that reduces cellular phospholipid hydroperoxides (PLOOHs) to alcohols. Cellular peroxide tone has been implicated in cell growth and differentiation. By reducing the PLOOH level in the cell membrane, PhGPx regulates the peroxide tone and thereby might be involved in cell growth. We hypothesized that overexpression of PhGPx in human breast cancer cells would decrease their growth rate. We stably transfected MCF-7 cells (Wt) with L-PhGPx and measured cell doubling time, plating efficiency, and cell cycle phase transit times. P-4 cells (8-fold increase in PhGPx activity) showed a 2-fold increase in doubling time; doubling time increased directly with PhGPx activity (r=0.95). The higher the PhGPx activity, the lower the plating efficiency (r=-0.86). The profile of other antioxidant enzymes was unchanged. Overexpression of PhGPx lowered the steady-state level of PLOOH (by >60%). Results from bromodeoxyuridine pulse-chase experiments and flow cytometry indicate that PhGPx induced a delay in MCF-7 proliferation that was primarily due to a slower progression from G1 to S. These results support the hypothesis that PhGPx plays a regulatory role in the progression of MCF-7 cells from G1 to S possibly by regulating the steady-state levels of PLOOH. These data suggest that PhGPx can lower the peroxide tone, which might change the cellular redox environment resulting in a delay in G1 transit. Thus, PhGPx could be an important factor in cell growth.

Histone deacetylase 4 interacts with 53BP1 to mediate the DNA damage response.

Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage–induced foci remain unclear. Here we show in a variety of human cell lines that histone deacetylase (HDAC) 4 is recruited to foci with kinetics similar to, and colocalizes with, 53BP1 after exposure to agents causing double-stranded DNA breaks. HDAC4 foci gradually disappeared in repair-proficient cells but persisted in repair-deficient cell lines or cells irradiated with a lethal dose, suggesting that resolution of HDAC4 foci is linked to repair. Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage–induced G2 delay, and radiosensitized HeLa cells. Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint.

Human CENP-I specifies localization of CENP-F, MAD1 and MAD2 to kinetochores and is essential for mitosis.

The kinetochore, a macromolecular complex located at the centromere of chromosomes, provides essential functions for accurate chromosome segregation. Kinetochores contain checkpoint proteins that monitor attachments between the kinetochore and microtubules to ensure that cells do not exit mitosis in the presence of unaligned chromosomes. Here we report that human CENP-I, a constitutive protein of the kinetochore that shares limited similarity with Mis6 of Schizosaccharomyces pombe, is required for the localization of CENP-F and the checkpoint proteins MAD1 and MAD2 to kinetochores. Depletion of CENP-I from kinetochores causes the cell cycle to delay in G2. Although monopolar chromosomes in CENP-I-depleted cells fail to establish bipolar connections, the cells are unable to arrest in mitosis. These cells are transiently delayed in mitosis in a MAD2-dependent manner, even though their kinetochores are depleted of MAD2. The delay is extended considerably when the number of unattached kinetochores is increased. This suggests that no single unattached kinetochore in CENP-I-depleted cells can arrest mitosis. The collective output from many unattached kinetochores is required to reach a threshold signal of 'wait for anaphase' to sustain a prolonged mitotic arrest.

Increased manganese superoxide dismutase (SOD-2) is part of the mechanism for prostate tumor suppression by Mac25/insulin-like growth factor binding-protein-related protein-1.

Increased expression of mac25/insulin-like growth factor binding-protein related protein-1 (IGFBP-rP1) in human breast and prostate epithelial cell lines results in the suppression of tumor growth. CDNA expression array analysis revealed increased manganese superoxide dismutase (SOD-2) expression in the mac25/IGFBP-rP1-transfected M12 human prostate cancer cell line compared to M12 control cells. SOD-2 has been postulated to be a tumor suppressor. SOD-2 was also increased in LNCaP cells stably transfected with mac25/IGFBP-rP1, but not in mac25/IGFBP-rP1-transfected PC-3 cells. Mac25 LNCaP cells had a marked decrease in tumor growth in nude mice compared to controls, but there was no difference in tumor growth in mac25 PC-3 cells compared to control. Phosphorylated Erk and Akt were increased in the M12 and LNCaP transfected mac25/IGFBP-rP1 cells but not PC-3 mac25. Inhibition of PI-3 kinase results in a marked decrease in viability of the M12-mac25 cells compared to M12 controls. Cells treated with H(2)O(2) result in an increase in phospho-ERK. Transfection of SOD-2 in M12 cells markedly decreased tumor growth, apoptosis, G1 delay in the cell cycle, and expression of senescence associated beta-galactosidase. These results suggest that one of the downstream mediators of the senescence-associated tumor suppression effect of mac25/IGFBP-rP1 is SOD-2.

Immunomodulatory and cytoprotective role of RP-1 in gamma-irradiated mice.

RP-1 has been reported to provide protection against lethal gamma-irradiation in mice. The present study was undertaken to understand its mechanism of action, especially with respect to modulation of radiation-induced changes in immune cell function, plasma antioxidant potential, cell cycle perturbations, apoptosis in mouse bone marrow cells, and micronuclei frequency in mice reticulocytes. 2 Gy reduced mitogenic response of splenic lymphocytes significantly at 48 h. Pre-irradiation RP-1 treatment significantly countered the radiation-induced loss of splenocyte proliferation. RP-1 treatment, with or without radiation, suppressed macrophage activation as compared to control. Irradiation decreased plasma antioxidant status significantly (p < 0.05) at 1 and 2 h (4.8 +/- 0.224 and 4.9 +/- 0.057 mM Fe2+) as compared to control (6.29 +/- 0.733 mM Fe2+) that was countered by RP-1 pre-treatment significantly (p < 0.05). RP-1 and irradiation individually caused G2 delay in bone marrow cells. RP-1 pre-treatment augmented radiation-induced G2 delay and elicited significant (p < 0.05) recovery in S-phase fraction at 48 h in comparison to irradiated group. Radiation-induced apoptosis (3%) was significantly higher than the control. RP-1 pre-treatment further enhanced apoptosis frequency (7.2%) in bone marrow cells. RP-1 pre-treatment significantly (p < 0.05) reduced (1.23%) the radiation-induced MN frequency (2.9%) observed at 48 h post-irradiation interval. Since the radioprotective manifestation of RP-1 is mediated through multiple mechanisms, needs further investigation.

HIV-1 Tat targets microtubules to induce apoptosis, a process promoted by the pro-apoptotic Bcl-2 relative Bim.

Depletion of CD4(+) T cells is the hallmark of HIV infection and AIDS progression. In addition to the direct killing of the viral-infected cells, HIV infection also leads to increased apoptosis of predominantly uninfected bystander cells. This is mediated in part through the HIV-1 Tat protein, which is secreted by the infected cells and taken up by uninfected cells. Using an affinity-purification approach, a specific and direct interaction of Tat with tubulin and polymerized microtubules has been detected. This interaction does not affect the secretion and uptake of Tat, but is critical for Tat to induce apoptosis. Tat binds tubulin/microtubules through a four-amino-acid subdomain of its conserved core region, leading to the alteration of microtubule dynamics and activation of a mitochondria-dependent apoptotic pathway. Bim, a pro-apoptotic Bcl-2 relative and a transducer of death signals initiated by perturbation of microtubule dynamics, facilitates the Tat-induced apoptosis. Our findings reveal a strategy by which Tat induces apoptosis by targeting the microtubule network. Thus HIV-1 Tat joins a growing list of pathogen-derived proteins that target the cytoskeleton of host cells.