G2 arrest primes hematopoietic stem cells for megakaryopoiesis.

In contrast to most hematopoietic lineages, megakaryocytes (MKs) can derive rapidly and directly from hematopoietic stem cells (HSCs). The underlying mechanism is unclear, however. Here, we show that DNA damage induces MK markers in HSCs and that G2 arrest, an integral part of the DNA damage response, suffices for MK priming followed by irreversible MK differentiation in HSCs, but not in progenitors. We also show that replication stress causes DNA damage in HSCs and is at least in part due to uracil misincorporation in vitro and in vivo. Consistent with this notion, thymidine attenuated DNA damage, improved HSC maintenance, and reduced the generation of CD41+ MK-committed HSCs. Replication stress and concomitant MK differentiation is therefore one of the barriers to HSC maintenance. DNA damage-induced MK priming may allow rapid generation of a lineage essential to immediate organismal survival, while also removing damaged cells from the HSC pool.

PEMF Potentiates Doxorubicin-induced Late G2 Arrest in MDA-MB-231 Breast Cancer Cells.

BACKGROUND/AIM: Pulsed electromagnetic field (PEMF) stimulation enhances the efficacy of several anticancer drugs. Doxorubicin is an anticancer drug used to treat various types of cancer, including breast cancer. However, the effect of PEMF stimulation on the efficacy of doxorubicin and the underlying mechanisms remain unclear. Thus, this study aimed to investigate the effect of PEMF stimulation on the anticancer activity of doxorubicin in MDA-MB-231 human breast cancer cells. MATERIALS AND METHODS: MDA-MB-231 cells were seeded and allowed to incubate for 48 h. The cells were treated with doxorubicin, cisplatin, 5-fluorouracil, or paclitaxel for 48 h. Subsequently, the cells were stimulated with a 60-min PEMF session thrice a day (with an interval of 4 h between each session) for 24 or 48 h. Cell viability was assessed by trypan blue dye exclusion assay and cell-cycle analysis was analyzed by flow cytometry. Molecular mechanisms involved in late G2 arrest were confirmed by a western blot assay and confocal microscopy. RESULTS: MDA-MB-231 cells treated with a combination of doxorubicin and PEMF had remarkably lower viability than those treated with doxorubicin alone. PEMF stimulation increased doxorubicin-induced cell-cycle arrest in the late G2 phase by suppressing cyclin-dependent kinase 1 (CDK1) activity through the enhancement of myelin transcription factor 1 (MYT1) expression, cell division cycle 25C (CDC25C) phosphorylation, and stratifin (14-3-3σ) expression. PEMF also increased doxorubicin-induced DNA damage by inhibiting DNA topoisomerase II alpha (TOP2A). CONCLUSION: These findings support the use of PEMF stimulation as an adjuvant to strengthen the antiproliferative effect of doxorubicin on breast cancer cells.

Differential endothelial cell cycle status in postnatal retinal vessels revealed using a novel PIP-FUCCI reporter and zonation analysis.

Cell cycle regulation is critical to blood vessel formation and function, but how the endothelial cell cycle integrates with vascular regulation is not well-understood, and available dynamic cell cycle reporters do not precisely distinguish all cell cycle stage transitions in vivo. Here we characterized a recently developed improved cell cycle reporter (PIP-FUCCI) that precisely delineates S phase and the S/G2 transition. Live image analysis of primary endothelial cells revealed predicted temporal changes and well-defined stage transitions. A new inducible mouse cell cycle reporter allele was selectively expressed in postnatal retinal endothelial cells upon Cre-mediated activation and predicted endothelial cell cycle status. We developed a semi-automated zonation program to define endothelial cell cycle status in spatially defined and developmentally distinct retinal areas and found predicted cell cycle stage differences in arteries, veins, and remodeled and angiogenic capillaries. Surprisingly, the predicted dearth of S-phase proliferative tip cells relative to stalk cells at the vascular front was accompanied by an unexpected enrichment for endothelial tip and stalk cells in G2, suggesting G2 stalling as a contribution to tip-cell arrest and dynamics at the front. Thus, this improved reporter precisely defines endothelial cell cycle status in vivo and reveals novel G2 regulation that may contribute to unique aspects of blood vessel network expansion.

Host-Pathogen Interactions and Correlated Factors That Are Affected in Replicative-Aged Cryptococcus neoformans.

Cryptococcus neoformans is a facultative intracellular fungal pathogen. Ten-generation-old (10GEN) C. neoformans cells are more resistant to phagocytosis and killing by macrophages than younger daughter cells. However, mechanisms that mediate this resistance and intracellular parasitism are poorly understood. Here, we identified important factors for the intracellular survival of 10GEN C. neoformans, such as urease activity, capsule synthesis, and DNA content using flow cytometry and fluorescent microscopy techniques. The real-time visualization of time-lapse imaging was applied to determine the phagosomal acidity, membrane permeability, and vomocytosis (non-lytic exocytosis) rate in J774 macrophages that phagocytosed C. neoformans of different generational ages. Our results showed that old C. neoformans exhibited higher urease activity and enhanced Golgi activity. In addition, old C. neoformans were more likely to be arrested in the G2 phase, resulting in the occasional formation of aberrant trimera-like cells. To finish, the advanced generational age of the yeast cells slightly reduced vomocytosis events within host cells, which might be associated with increased phagolysosome pH and membrane permeability. Altogether, our results suggest that old C. neoformans prevail within acidic phagolysosomes and can manipulate the phagosome pH. These strategies may be used by old C. neoformans to resist phagosomal killing and drive cryptococcosis pathogenesis. The comprehension of these essential host-pathogen interactions could further shed light on mechanisms that bring new insights for novel antifungal therapeutic design.

HIV-1 Vpr Functions in Primary CD4+ T Cells.

HIV-1 encodes four accesory proteins in addition to its structural and regulatory genes. Uniquely amongst them, Vpr is abundantly present within virions, meaning it is poised to exert various biological effects on the host cell upon delivery. In this way, Vpr contributes towards the establishment of a successful infection, as evidenced by the extent to which HIV-1 depends on this factor to achieve full pathogenicity in vivo. Although HIV infects various cell types in the host organism, CD4+ T cells are preferentially targeted since they are highly permissive towards productive infection, concomitantly bringing about the hallmark immune dysfunction that accompanies HIV-1 spread. The last several decades have seen unprecedented progress in unraveling the activities Vpr possesses in the host cell at the molecular scale, increasingly underscoring the importance of this viral component. Nevertheless, it remains controversial whether some of these advances bear in vivo relevance, since commonly employed cellular models significantly differ from primary T lymphocytes. One prominent example is the "established" ability of Vpr to induce G2 cell cycle arrest, with enigmatic physiological relevance in infected primary T lymphocytes. The objective of this review is to present these discoveries in their biological context to illustrate the mechanisms whereby Vpr supports HIV-1 infection in CD4+ T cells, whilst identifying findings that require validation in physiologically relevant models.

Differential endothelial cell cycle status in postnatal retinal vessels revealed using a novel PIP-FUCCI reporter and zonation analysis.

Cell cycle regulation is critical to blood vessel formation and function, but how the endothelial cell cycle integrates with vascular regulation is not well-understood, and available dynamic cell cycle reporters do not precisely distinguish all cell cycle stage transitions in vivo. Here we characterized a recently developed improved cell cycle reporter (PIP-FUCCI) that precisely delineates S phase and the S/G2 transition. Live image analysis of primary endothelial cells revealed predicted temporal changes and well-defined stage transitions. A new inducible mouse cell cycle reporter allele was selectively expressed in postnatal retinal endothelial cells upon Cre-mediated activation and predicted endothelial cell cycle status. We developed a semi-automated zonation program to define endothelial cell cycle status in spatially defined and developmentally distinct retinal areas and found predicted cell cycle stage differences in arteries, veins, and remodeled and angiogenic capillaries. Surprisingly, the predicted dearth of proliferative tip cells at the vascular front was accompanied by an unexpected enrichment for endothelial tip cells in G2, suggesting G2 stalling as a contribution to tip-cell arrest. Thus, this improved reporter precisely defines endothelial cell cycle status in vivo and reveals novel G2 regulation that may contribute to unique aspects of blood vessel network expansion.

DON induced DNA damage triggers absence of p53-mediated G2 arrest and apoptosis in IPEC-1 cells.

Deoxynivalenol (DON) stands among the prevalent mycotoxins, and usually contaminates cereal foods and animal feed, leading to human and animal clinical poisoning symptoms such as abdominal pain, diarrhea, and vomiting. To date, the mechanism of toxicity of DON in different mammalian cells is not fully elucidated. In this study, we explored the detrimental impacts of DON on porcine intestinal epithelial cells (IPEC-1), serving as a representative model for porcine intestinal epithelial cells. After treating cells with DON for 24 h, DON can significantly inhibit the activity of cells, induce the production of reactive oxygen species (ROS), significantly reduce the content of glutathione and the activity of catalase, and increase the activity of superoxide dismutase and malondialdehyde, leading to an imbalance in intracellular redox status. In addition, DON can induce DNA double-strand breaks, and decrease mitochondrial membrane potential. Furthermore, DON can promote the release of Cyt C through changes in mitochondrial permeability through inhibit the expression of B-cell lymphoma 2 (Bcl-2) proteins, leading to apoptosis through the mitochondrial pathway. On the other hand, we found that DON can cause IPEC-1 cells G2 phase cycle arrest. Different with our pervious study, DON induces cell cycle arrest in the G2 phase only by activating the ATM-Chk2-Cdc 25 C pathway, but cannot regulate the cell cycle arrest via the ATM-p53 pathway. These results indicate that DON can induce the same toxic phenotype in different cells, but its toxic mechanism is different. All these provide a rationale for revealing DON induced cytotoxicity and intestinal diseases.

Aurora A Kinase Plays a Key Role in Mitosis Skip during Senescence Induced by Ionizing Radiation.

Objective: To investigate the fate and underlying mechanisms of G2 phase arrest in cancer cells elicited by ionizing radiation (IR). Methods: Human melanoma A375 and 92-1 cells were treated with X-rays radiation or Aurora A inhibitor MLN8237 (MLN) and/or p21 depletion by small interfering RNA (siRNA). Cell cycle distribution was determined using flow cytometry and a fluorescent ubiquitin-based cell cycle indicator (FUCCI) system combined with histone H3 phosphorylation at Ser10 (pS10 H3) detection. Senescence was assessed using senescence-associated-ß-galactosidase (SA-ß-Gal), Ki67, and γH2AX staining. Protein expression levels were determined using western blotting. Results: Tumor cells suffered severe DNA damage and underwent G2 arrest after IR treatment. The damaged cells did not successfully enter M phase nor were they stably blocked at G2 phase but underwent mitotic skipping and entered G1 phase as tetraploid cells, ultimately leading to senescence in G1. During this process, the p53/p21 pathway is hyperactivated. Accompanying p21 accumulation, Aurora A kinase levels declined sharply. MLN treatment confirmed that Aurora A kinase activity is essential for mitosis skipping and senescence induction. Conclusion: Persistent p21 activation during IR-induced G2 phase blockade drives Aurora A kinase degradation, leading to senescence via mitotic skipping.

Chemosensitivity to doxorubicin in primary cells derived from tumor of FVB/N-Trp53tm1Hw1 with TALEN-mediated Trp53 mutant gene.

BACKGROUND: To evaluate the chemosensitivity to doxorubicin (DOX) in two primary cells derived from a tumor of FVB/N-Trp53tm1Hw1 knockout (KO) mice with TALEN-mediated Trp53 mutant gene, we evaluated the cell survivability, cell cycle distribution, apoptotic cell numbers and apoptotic protein expression in solid tumor cells and ascetic tumor cells treated with DOX. RESULTS: The primary tumor cells showed a significant (P < 0.05) defect for UV-induced upregulation of the Trp53 protein, and consisted of different ratios of leukocytes, fibroblasts, epithelial cells and mesenchymal cells. The IC50 level to DOX was lower in both primary cells (IC50 = 0.12 µM and 0.20 µM) as compared to the CT26 cells (IC50 = 0.32 µM), although the solid tumor was more sensitive. Also, the number of cells arrested at the G0/G1 stage was significantly decreased (24.7-23.1% in primary tumor cells treated with DOX, P < 0.05) while arrest at the G2 stage was enhanced to 296.8-254.3% in DOX-treated primary tumor cells compared with DOX-treated CT26 cells. Furthermore, apoptotic cells of early and late stage were greatly increased in the two primary cell-lines treated with DOX when compared to same conditions for CT26 cells. However, the Bax/Bcl-2 expression level was maintained constant in the primary tumor and CT26 cells. CONCLUSIONS: To the best of our knowledge, these results are the first to successfully detect an alteration in chemosensitivity to DOX in solid tumor cells and ascetic tumor cells derived from tumor of FVB/N-Trp53tm1Hw1 mice TALEN-mediated Trp53 mutant gene.

Mechanistic study of dual-function inhibitors targeting topoisomerase II and Rad51-mediated DNA repair pathway against castration-resistant prostate cancer.

BACKGROUND: Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment and the therapeutic options are continuously advancing. This study aims to discover the anti-CRPC effects and underlying mechanisms of small-molecule compounds targeting topoisomerase (TOP) II and cellular components of DNA damage repair. METHODS: Cell proliferation was determined in CRPC PC-3 and DU-145 cells using anchorage-dependent colony formation, sulforhodamine B assay and flow cytometric analysis of CFSE staining. Flow cytometric analyses of propidium iodide staining and JC-1 staining were used to examine the population of cell-cycle phases and mitochondrial membrane potential, respectively. Nuclear extraction was performed to detect the nuclear localization of cellular components in DNA repair pathways. Protein expressions were determined using Western blot analysis. RESULTS: A series of azathioxanthone-based derivatives were synthesized and examined for bioactivities in which WC-A13, WC-A14, WC-A15, and WC-A16 displayed potent anti-CRPC activities in both PC-3 and DU-145 cell models. These WC-A compounds selectively downregulated both TOP IIα and TOP IIß but not TOP I protein expression. WC-A13, WC-A14, and WC-A15 were more potent than WC-A16 on TOP II inhibition, mitochondrial dysfunction, and induction of caspase cascades indicating the key role of amine-containing side chain of the compounds in determining anti-CRPC activities. Furthermore, WC-A compounds induced an increase of γH2AX and activated ATR-Chk1 and ATM-Chk2 signaling pathways. P21 protein expression was also upregulated by WC-A compounds in which WC-A16 showed the least activity. Notably, WC-A compounds exhibited different regulation on Rad51, a major protein in homologous recombination of DNA in double-stranded break repair. WC-A13, WC-A14, and WC-A15 inhibited, whereas WC-A16 induced, the nuclear translocation of Rad51. CONCLUSION: The data suggest that WC-A compounds exhibit anti-CRPC effects through the inhibition of TOP II activities, leading to mitochondrial stress-involved caspase activation and apoptosis. Moreover, WC-A13, WC-A14, and WC-A15 but not WC-A16 display inhibitory activities of Rad51-mediated DNA repair pathway which may increase apoptotic effect of CRPC cells.

Exploring the Synergy: Chinese Female Tennis Players and Chansu-Medicated Serum's Potential in Inhibiting Breast Cancer Cell Proliferation Through Apoptosis and G2 Arrest

This study delves into the synergy between Chinese female tennis players and the potential of Chansu-Medicated Serum in inhibiting breast cancer cell proliferation through apoptosis and G2 arrest. Chinese female tennis players have garnered international recognition for their achievements on the court and their philanthropic endeavors off it. This research investigates the intersection of their impact and the advancement of breast cancer research. Chansu-Medicated Serum, a traditional Chinese medicine derivative, presents promising mechanisms for inhibiting breast cancer cell proliferation, including apoptosis induction and G2 cell cycle arrest. By exploring this conjunction, we aim to shed light on the possible contributions of these athletes and traditional medicine to the ongoing battle against breast cancer. (AU)

DAP3-mediated cell cycle regulation and its association with radioresistance in human lung adenocarcinoma cell lines.

Mitochondria play important roles in the cellular response to various types of stress, including that triggered by ionizing radiation. We have previously reported that the mitochondrial ribosomal protein death-associated protein 3 (DAP3) regulates the radioresistance of human lung adenocarcinoma (LUAD) cell lines A549 and H1299. However, the underlying mechanism of this regulation remains to be elucidated. To this end, we have herein investigated the role of DAP3 in the cell cycle regulation after irradiation. Notably, the DAP3 knockdown attenuated the radiation-induced increase of the G2/M cell population. Furthermore, western blotting analysis has revealed that the DAP3 knockdown decreased the expression of proteins related to the G2/M arrest, such as those of the phosphorylated cdc2 (Tyr15) and the phosphorylated checkpoint kinase 1 (Ser296), in irradiated A549 cells and H1299 cells. Moreover, by using a chk1 inhibitor, we were able to demonstrate that chk1 is involved in the radiation-induced G2/M arrest in both A549 and H1299 cells. Notably, the chk1 inhibitor was able to enhance the radiosensitivity of H1299 cells, while both chk1 inhibitor-abolished G2 arrest and inhibition of chk2-mediated events such as downregulation of radiation-induced p21 expression were required for enhancing radiosensitivity of A549 cells. Collectively, our findings reveal a novel role of DAP3 to regulate G2/M arrest through pchk1 in irradiated LUAD cells and suggest that chk1-mediated G2/M arrest regulates the radioresistance of H1299 cells, whereas both the chk1-mediated G2/M arrest and the chk2-mediated events contribute to the radioresistance of A549 cells.

MAPK Cascade Signaling Is Involved in α-MMC Induced Growth Inhibition of Multiple Myeloma MM.1S Cells via G2 Arrest and Mitochondrial-Pathway-Dependent Apoptosis In Vitro.

Multiple myeloma is a hematological malignancy characterized by the unrestricted proliferation of plasma cells that secrete monoclonal immunoglobulins in the bone marrow. Alpha-momorcharin (α-MMC) is a type I ribosome-inactivating protein extracted from the seeds of the edible plant Momordica charantia L., which has a variety of biological activities. This study aimed to investigate the inhibitory effect of α-MMC on the proliferation of multiple myeloma MM.1S cells and the molecular mechanism of MM.1S cell death induced through the activation of cell signal transduction pathways. The cell counting kit-8 (CCK-8) assay was used to determine the inhibitory effect of α-MMC on the proliferation of MM.1S cells and its toxic effect on normal human peripheral blood mononuclear cells (PBMCs). The effect of α-MMC on the MM.1S cells' morphology was observed via inverted microscope imaging. The effects of α-MMC on the MM.1S cell cycle, mitochondrial membrane potential (MMP), and apoptosis were explored using propidium iodide, JC-1, annexin V- fluorescein isothiocyanate/propidium iodide fluorescence staining, and flow cytometry (FCM) analysis. Western blot was used to detect the expressions levels of apoptosis-related proteins and MAPK-signaling-pathway-related proteins in MM.1S cells induced by α-MMC. The results of the CCK-8 showed that in the concentration range of no significant toxicity to PBMCs, α-MMC inhibited the proliferation of MM.1S cells in a time-dependent and concentration-dependent manner, and the IC50 value was 13.04 and 7.518 µg/mL for 24 and 48 h, respectively. Through inverted microscope imaging, it was observed that α-MMC induced a typical apoptotic morphology in MM.1S cells. The results of the FCM detection and analysis showed that α-MMC could arrest the MM.1S cells cycle at the G2 phase, decrease the MMP, and induce cell apoptosis. Western blot analysis found that α-MMC upregulated the expression levels of Bax, Bid, cleaved caspase-3, and cleaved PARP, and downregulated the expression levels of Mcl-1. At the same time, α-MMC decreased the expression levels of p-c-Raf, p-MEK1/2, p-ERK1/2, p-MSK1, and p-P90RSK, and increased the expression levels of p-p38, p-SPAK/JNK, p-c-Jun, and p-ATF2. The above results suggest that α-MMC can inhibit the proliferation of multiple myeloma MM.1S cells. MAPK cascade signaling is involved in the growth inhibition effect of α-MMC on MM.1S cells via cycle arrest and mitochondrial-pathway-dependent apoptosis.

Aurora A Kinase Plays a Key Role in Mitosis Skip during Senescence Induced by Ionizing Radiation / 生物医学与环境科学（英文）

OBJECTIVE@#To investigate the fate and underlying mechanisms of G2 phase arrest in cancer cells elicited by ionizing radiation (IR).@*METHODS@#Human melanoma A375 and 92-1 cells were treated with X-rays radiation or Aurora A inhibitor MLN8237 (MLN) and/or p21 depletion by small interfering RNA (siRNA). Cell cycle distribution was determined using flow cytometry and a fluorescent ubiquitin-based cell cycle indicator (FUCCI) system combined with histone H3 phosphorylation at Ser10 (pS10 H3) detection. Senescence was assessed using senescence-associated-β-galactosidase (SA-β-Gal), Ki67, and γH2AX staining. Protein expression levels were determined using western blotting.@*RESULTS@#Tumor cells suffered severe DNA damage and underwent G2 arrest after IR treatment. The damaged cells did not successfully enter M phase nor were they stably blocked at G2 phase but underwent mitotic skipping and entered G1 phase as tetraploid cells, ultimately leading to senescence in G1. During this process, the p53/p21 pathway is hyperactivated. Accompanying p21 accumulation, Aurora A kinase levels declined sharply. MLN treatment confirmed that Aurora A kinase activity is essential for mitosis skipping and senescence induction.@*CONCLUSION@#Persistent p21 activation during IR-induced G2 phase blockade drives Aurora A kinase degradation, leading to senescence via mitotic skipping.

JNK inhibitor IX restrains pancreatic cancer through p53 and p21.

Novel treatment options for pancreatic cancer are desperately needed. De-regulated kinases can be regularly detected in pancreatic cancer. Multiple pathway inhibitors were developed to exploit these features, among them selective inhibitors of the c-Jun N-terminal kinase isoforms 1 and 2 (JNK1 and 2). We evaluated the effectiveness of four different JNK inhibitors on pancreatic cancer cell lines. Cell mobility and migration were evaluated in scratch assay and Boyden chamber assay. Mechanism of cell death was analyzed via apoptosis assays in FACS and immunoblotting as well as cell cycle analysis via FACS, and qPCR. JNK2 knockout cells were generated using siRNA transfection. Among the inhibitors, JNK inhibitor IX (JNK-in-IX), designed as specific inhibitor against JNK2 was proven highly effective in inhibiting cell growth, mobility and migration. We were able to show that JNK-in-IX caused DNA damage resulting in G2 arrest mediated through p53 and p21. Interestingly, JNK-in-IX acted independently of its primary target JNK2. In summary, JNK-in-IX was shown highly effective in pancreatic cancer. This study underlines the need for modeling systems in testing therapeutic options as JNK2 was previously not indicated as a potential target.

Determination of Clonogenic Radiosensitivity of HeLa Cells in Early and Late G1 Phases.

BACKGROUND/AIM: Using a fluorescent ubiquitination-based cell cycle indicator (Fucci), we recently reported that post irradiation of HeLa cells, micronuclei frequency increased in the early G1 phase in comparison with that in the late G1 phase. This is inconsistent with the results of well-recognized studies that used clonogenic assays. In this study, we determined radiosensitivity of the cells using a clonogenic assay by making the best use of the Fucci property, while simultaneously characterizing cell cycle kinetics and DNA damage responses. MATERIALS AND METHODS: Early and late G1 phase cell fractions were isolated using a cell sorter by exploiting the different red fluorescence intensities of Fucci. Radiosensitivity was determined by the colony formation assay. Time-lapse imaging and immunostaining were performed to analyze cell cycle kinetics and DNA damage. RESULTS: Late G1 cells were more radioresistant than early G1 cells. Cells irradiated in the early and late G1 phases induced G2 arrest, while the latter demonstrated a significantly longer duration of G2 arrest. This difference became more evident as the radiation dose increased. Furthermore, 16 h after irradiation, a greater number of γH2AX foci remained in cells irradiated in the early G1 phase than in those irradiated in the late G1 phase. CONCLUSION: HeLa cells in the late G1 phase are more radioresistant than those in the early G1 phase, presumably because DNA damage is efficiently repaired during a longer G2 arrest in late G1 cells.

Lysosomal sequestration of weak base drugs, lysosomal biogenesis, and cell cycle alteration.

Lysosomes, now known to take part in multiple cellular functions, also respond to various stress stimuli. These include biogenesis in response to nanomolar concentrations of hydrophobic weak-base anticancer drugs. However, since lysosomal stress mediated by accumulation of weak-base drugs at such concentrations has never been proven and these drugs have diverse effects on malignant cells, we investigated whether the interpretation of the data was true. We found that lysosomal accumulation of the drugs daunorubicin, doxorubicin, mitoxantrone, symadex, chloroquine, clomipramine and sunitinib alone, was insufficient to induce lysosomal alkalization i.e., lysosomal stress-mediated biogenesis at nanomolar concentrations. Instead, we found that some of the drugs used induced G2 phase arrest and lysosomal biogenesis that is associated with activation of transcription factor EB (TFEB). Similarly, cantharidin, a control compound that does not belong to the weak base drugs, induced cell cycle arrest in the G2 phase associated with TFEB-driven lysosomal biogenesis. Overall none of the tested drugs caused stress-induced lysosomal biogenesis at nanomolar concentrations. However, daunorubicin, doxorubicin, mitoxantrone, symadex and cantharidin induced a massive block in the G2 phase of the cell cycle which is naturally associated with TFEB-driven lysosomal biogenesis.

A Novel Class of HIV-1 Inhibitors Targeting the Vpr-Induced G2-Arrest in Macrophages by New Yeast- and Cell-Based High-Throughput Screening.

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, arrests the cell cycle of the G2 phase, and this Vpr-mediated G2 arrest is implicated in an efficient HIV-1 spread in monocyte-derived macrophages. Here, we screened new candidates for Vpr-targeting HIV-1 inhibitors by using fission yeast- and mammalian cell-based high-throughput screening. First, fission yeast strains expressing the HIV-1 Vpr protein were generated and then treated for 48 h with 20 µM of a synthetic library, including 140,000 chemical compounds. We identified 268 compounds that recovered the growth of Vpr-overexpressing yeast. The selected compounds were then tested in mammalian cells, and those displaying high cytotoxicity were excluded from further cell cycle analysis and imaging-based screening. A flow cytometry analysis confirmed that seven compounds recovered from the Vpr-induced G2 arrest. The cell toxicity and inhibitory effect of HIV-1 replication in human monocyte-derived macrophages (MDM) were examined, and three independent structural compounds, VTD227, VTD232, and VTD263, were able to inhibit HIV-1 replication in MDM. Furthermore, we showed that VTD227, but not VTD232 and VTD263, can directly bind to Vpr. Our results indicate that three new compounds and their derivatives represent new drugs targeting HIV-1 replication and can be potentially used in clinics to improve the current antiretroviral therapy.

Reciprocal regulation of p21 and Chk1 controls the cyclin D1-RB pathway to mediate senescence onset after G2 arrest.

Senescence is an irreversible withdrawal from cell proliferation that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 requires p53 (also known as TP53) and retinoblastoma protein (RB, also known as RB1) family tumour suppressors, but how they are regulated to convert a temporary cell cycle arrest into a permanent one remains unknown. Here, we show that a previously unrecognised balance between the cyclin-dependent kinase (CDK) inhibitor p21 and the checkpoint kinase Chk1 controls cyclin D-CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting cyclin D1 complexed with CDK2 or CDK4. The resulting G2 exit, which precedes the appearance of senescence markers, is associated with a mitotic bypass, Chk1 downregulation and reduction in the number of DNA damage foci. In p53/RB-proficient cancer cells, a compromised G2 exit correlates with sustained Chk1 activity, delayed p21 induction, untimely cyclin E1 re-expression and genome reduplication. Conversely, Chk1 depletion promotes senescence by inducing p21 binding to cyclin D1- and cyclin E1-CDK complexes and downregulating CDK6, whereas knockdown of the checkpoint kinase Chk2 enables RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus promoting the onset of senescence induced by DNA damage in G2.

Huntingtin-Interacting Protein 1 Promotes Vpr-Induced G2 Arrest and HIV-1 Infection in Macrophages.

Human immunodeficiency virus type 1 (HIV-1) modulates the host cell cycle. The HIV-1 accessory protein Vpr arrests the cell cycle at the G2 phase in dividing cells, and the ability of Vpr to induce G2 arrest is well conserved among primate lentiviruses. Additionally, Vpr-mediated G2 arrest likely correlates with enhanced HIV-1 infection in monocyte-derived macrophages. Here, we screened small-interfering RNA to reveal candidates that suppress Vpr-induced G2 arrest and identified Huntingtin-interacting protein 1 (HIP1) required for efficient G2 arrest. Interestingly, HIP1 was not essential for Vpr-induced DNA double-strand breaks, which are required for activation of the DNA-damage checkpoint and G2 arrest. Furthermore, HIP1 knockdown suppressed HIV-1 infection in monocyte-derived macrophages. This study identifies HIP1 as a factor promoting Vpr-induced G2 arrest and HIV-1 infection in macrophages.