AR-V7 expression facilitates accelerated G2/M phase transition in castration-resistant prostate cancer.

The emergence of AR-V7, a truncated isoform of AR upon androgen deprivation therapy treatment, leads to the development of castration resistant prostate cancer (CRPC). Understanding mechanisms that regulate AR-V7 expression is critical for developing newer therapeutic strategies. In this study, we have investigated the regulation of AR-V7 during cell cycle and identified a distinct pattern of periodic fluctuation, peaking during G2/M phase. This fluctuation correlates with the expression of Cdc-2 like kinase 1 (CLK1) and phosphorylated serine/arginine-rich splicing factor 1 (p-SRSF1) during these phases, pointing towards their role in AR-V7 generation. Functional assays reveal that CLK1 knockdown prolongs the S phase, leading to altered cell cycle distribution and increased accumulation of AR-V7 and pSRSF1 in G1/S phase. Conversely, CLK1 overexpression rescues AR-V7 and p-SRSF1 levels in the G2/M phase, consistent with observed cell cycle alterations upon AR-V7 knockdown and overexpression in CRPC cells. Furthermore, overexpression of kinase-deficient CLK1 mutant leads to diminished AR-V7 levels during G2/M, underlining the essential contribution of CLK1's kinase activity in modulating AR-V7 expression. Collectively, our findings, for the first time, show periodic regulation of AR-V7 expression, its effect on cell cycle progression and the critical role of CLK1-pSRSF1 axis in modulating AR-V7 expression throughout the cell cycle.

Role of RPA Phosphorylation in the ATR-Dependent G2 Cell Cycle Checkpoint.

Cells respond to DNA double-strand breaks by initiating DSB repair and ensuring a cell cycle checkpoint. The primary responder to DSB repair is non-homologous end joining, which is an error-prone repair pathway. However, when DSBs are generated after DNA replication in the G2 phase of the cell cycle, a second DSB repair pathway, homologous recombination, can come into action. Both ATM and ATR are important for DSB-induced DSB repair and checkpoint responses. One method of ATM and ATR working together is through the DNA end resection of DSBs. As a readout and marker of DNA end resection, RPA is phosphorylated at Ser4/Ser8 of the N-terminus of RPA32 in response to DSBs. Here, the significance of RPA32 Ser4/Ser8 phosphorylation in response to DNA damage, specifically in the S phase to G2 phase of the cell cycle, is examined. RPA32 Ser4/Ser8 phosphorylation in G2 synchronized cells is necessary for increases in TopBP1 and Rad9 accumulation on chromatin and full activation of the ATR-dependent G2 checkpoint. In addition, our data suggest that RPA Ser4/Ser8 phosphorylation modulates ATM-dependent KAP-1 phosphorylation and Rad51 chromatin loading in G2 cells. Through the phosphorylation of RPA Ser4/Ser8, ATM acts as a partner with ATR in the G2 phase checkpoint response, regulating key downstream events including Rad9, TopBP1 phosphorylation and KAP-1 phosphorylation/activation via the targeting of RPA32 Ser4/Ser8.

UHRF1 promotes spindle assembly and chromosome congression by catalyzing EG5 polyubiquitination.

UHRF1 is an epigenetic coordinator bridging DNA methylation and histone modifications. Additionally, UHRF1 regulates DNA replication and cell cycle, and its deletion induces G1/S or G2/M cell cycle arrest. The roles of UHRF1 in the regulation of G2/M transition remain poorly understood. UHRF1 depletion caused chromosome misalignment, thereby inducing cell cycle arrest at mitotic metaphase, and these cells exhibited the defects of spindle geometry, prominently manifested as shorter spindles. Mechanistically, UHRF1 protein directly interacts with EG5, a kinesin motor protein, during mitosis. Furthermore, UHRF1 induced EG5 polyubiquitination at the site of K1034 and further promoted the interaction of EG5 with spindle assembly factor TPX2, thereby ensuring accurate EG5 distribution to the spindles during metaphase. Our study clarifies a novel UHRF1 function as a nuclear protein catalyzing EG5 polyubiquitination for proper spindle architecture and faithful genomic transmission, which is independent of its roles in epigenetic regulation and DNA damage repair inside the nucleus. These findings revealed a previously unknown mechanism of UHRF1 in controlling mitotic spindle architecture and chromosome behavior and provided mechanistic evidence for UHRF1 deletion-mediated G2/M arrest.

P53 status, and G2/M cell cycle arrest, are determining factors in cell-death induction mediated by ELF-EMF in glioblastoma.

The average survival of patients with glioblastoma is 12-15 months. Therefore, finding a new treatment method is important, especially in cases that show resistance to treatment. Extremely low-frequency electromagnetic fields (ELF-EMF) have characteristics and capabilities that can be proposed as a new cancer treatment method with low side effects. This research examines the antitumor effect of ELF-EMF on U87 and U251 glioblastoma cell lines. Flowcytometry determined the viability/apoptosis and distribution of cells in different phases of the cell cycle. The size of cells was assessed by TEM. Important cell cycle regulation genes mRNA expression levels were investigated by real-time PCR. ELF-EMF induced apoptosis in U87cells much more than U251 (15% against 2.43%) and increased G2/M cell population in U87 (2.56%, p value < 0.05), and S phase in U251 (2.4%) (data are normalized to their sham exposure). The size of U87 cells increased significantly after ELF-EMF exposure (overexpressing P53 in U251 cells increased the apoptosis induction by ELF-EMF). The expression level of P53, P21, and MDM2 increased and CCNB1 decreased in U87. Among the studied genes, MCM6 expression decreased in U251. Increasing expression of P53, P21 and decreasing CCNB1, induction of cell G2/M cycle arrest, and consequently increase in the cell size can be suggested as one of the main mechanisms of apoptosis induction by ELF-EMF; furthermore, our results demonstrate the possible footprint of P53 in the apoptosis induction by ELF-EMF, as U87 carry the wild type of P53 and U251 has the mutated form of this gene.

HDAC9-mediated epithelial cell cycle arrest in G2/M contributes to kidney fibrosis in male mice.

Renal tubular epithelial cells (TECs) play a key role in kidney fibrosis by mediating cycle arrest at G2/M. However, the key HDAC isoforms and the underlying mechanism that are involved in G2/M arrest of TECs remain unclear. Here, we find that Hdac9 expression is significantly induced in the mouse fibrotic kidneys, especially in proximal tubules, induced by aristolochic acid nephropathy (AAN) or unilateral ureter obstruction (UUO). Tubule-specific deletion of HDAC9 or pharmacological inhibition by TMP195 attenuates epithelial cell cycle arrest in G2/M, then reduces production of profibrotic cytokine and alleviates tubulointerstitial fibrosis in male mice. In vitro, knockdown or inhibition of HDAC9 alleviates the loss of epithelial phenotype in TECs and attenuates fibroblasts activation through inhibiting epithelial cell cycle arrest in G2/M. Mechanistically, HDAC9 deacetylates STAT1 and promotes its reactivation, followed by inducing G2/M arrest of TECs, finally leading to tubulointerstitial fibrosis. Collectively, our studies indicate that HDAC9 may be an attractive therapeutic target for kidney fibrosis.

Pan-cancer antagonistic inhibition pattern of ATM-driven G2/M checkpoint pathway vs other DNA repair pathways.

DNA repair mechanisms keep genome integrity and limit tumor-associated alterations and heterogeneity, but on the other hand they promote tumor survival after radiation and genotoxic chemotherapies. We screened pathway activation levels of 38 DNA repair pathways in nine human cancer types (gliomas, breast, colorectal, lung, thyroid, cervical, kidney, gastric, and pancreatic cancers). We took RNAseq profiles of the experimental 51 normal and 408 tumor samples, and from The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium databases - of 500/407 normal and 5752/646 tumor samples, and also 573 normal and 984 tumor proteomic profiles from Proteomic Data Commons portal. For all the samplings we observed a congruent trend that all cancer types showed inhibition of G2/M arrest checkpoint pathway compared to the normal samples, and relatively low activities of p53-mediated pathways. In contrast, other DNA repair pathways were upregulated in most of the cancer types. The G2/M checkpoint pathway was statistically significantly downregulated compared to the other DNA repair pathways, and this inhibition was strongly impacted by antagonistic regulation of (i) promitotic genes CCNB and CDK1, and (ii) GADD45 genes promoting G2/M arrest. At the DNA level, we found that ATM, TP53, and CDKN1A genes accumulated loss of function mutations, and cyclin B complex genes - transforming mutations. These findings suggest importance of activation for most of DNA repair pathways in cancer progression, with remarkable exceptions of G2/M checkpoint and p53-related pathways which are downregulated and neutrally activated, respectively.

When more is less: heritable gain-of-function chk1 mutations impair human fertility.

Heritable loss-of-function mutations in genes encoding key regulators of DNA repair and genome stability can result in degenerative progeroid and/or cancer predisposition syndromes; however, such mutations have never been found to affect the Chk1 protein kinase, despite its central role in DNA damage signalling and checkpoint activation. Remarkably, two recent reports now demonstrate that heritable, gain-of-function mutations within the Chk1 C-terminal regulatory domain can cause female infertility in humans. In vitro, oocytes from individuals heterozygous for such mutant Chk1 alleles fail to undergo the first mitotic division after fertilization owing to arrest in G2 phase of the cell cycle. This arrest results from inhibition of the master regulator of mitosis, the cyclin-dependent kinase CDK1, through the same molecular mechanisms that are engaged by activated Chk1 to impose G2 checkpoint arrest in somatic cells bearing DNA damage. Remarkably, the failure of this first zygotic division in heterozygotes in vitro can be rescued through treatment with selective Chk1 inhibitor drugs, allowing development of apparently normal blastocysts and offering hope that a pharmacological solution to this cause of infertility may be possible.

Decoupling dedifferentiation and G2/M arrest in kidney fibrosis.

Understanding the cellular mechanisms underlying chronic kidney disease (CKD) progression is required to develop effective therapeutic approaches. In this issue of the JCI, Taguchi, Elias, et al. explore the relationship between cyclin G1 (CG1), an atypical cyclin that induces G2/M proximal tubule cell cycle arrest, and epithelial dedifferentiation during fibrogenesis. While CG1-knockout mice were protected from fibrosis and had reduced G2/M arrest, protection was unexpectedly independent of induction of G2/M arrest. Rather, CG1 drove fibrosis by regulating maladaptive dedifferentiation in a CDK5-dependent mechanism. These findings highlight the importance of maladaptive epithelial dedifferentiation in kidney fibrogenesis and identify CG1/CDK5 signaling as a therapeutic target in CKD progression.

C118P exerted potent anti-tumor effects against melanoma with induction of G2/M arrest via inhibiting the expression of BUB1B.

BACKGROUND: The incidence of melanoma rapidly increased in the past decades, and the clinical treatment of melanoma met huge challenges because of tumor heterogeneity and drug resistance. C118P, a novel tubulin polymerization inhibitor, exhibited strong anticancer effects in many tumors. However, there was no data regarding the potential effects of C118P in melanoma cells. OBJECTIVE: To investigate of the efficacy and potential target of C118P in melanoma cells. METHODS: Human melanoma cells were treated with C118P, followed by assessments of proliferation, apoptosis and cell cycle distribution. Subsequently, RNA sequencing was performed to further identify the drug targets of C118P in melanoma cells. GO analysis and protein-protein interaction networks analysis were used to screen the potential targets, and verified by a series of assays. Finally, the anti-growth activity of C118P was evaluated in A375-xenografted nude mice, and the expression of BUB1B (BUB1 mitotic checkpoint serine/threonine kinase B), Ki67 and Tunel were determined. RESULTS: We found that C118P concentration-dependently inhibited proliferation of melanoma cells. Moreover, C118P simultaneously triggered dramatic G2/M arrest and apoptosis via independent mechanisms in melanoma cells in vitro. C118P exerted anti-melanoma effects by inducing potent G2/M arrest, which was mechanistically related to downregulation of the expression of BUB1B. Importantly, C118P inhibited the tumor growth in A375-xenografted nude, and increased the staining of Ki-67 and Tunel and suppressed the expression of BUB1B in melanoma tissues, which was consistent with in vitro study. CONCLUSION: C118P might provide a novel strategy for the clinical treatment of melanoma by inhibition of BUB1B.

Circular RNA circBNC2 inhibits epithelial cell G2-M arrest to prevent fibrotic maladaptive repair.

The mechanisms underlying fibrogenic responses after injury are not well understood. Epithelial cell cycle arrest in G2/M after injury is a key checkpoint for determining wound-healing leading to either normal cell proliferation or fibrosis. Here, we identify a kidney- and liver-enriched circular RNA, circBNC2, which is abundantly expressed in normal renal tubular cells and hepatocytes but significantly downregulated after acute ischemic or toxic insult. Loss of circBNC2 is at least partially mediated by upregulation of DHX9. Gain- and loss-of-function studies, both in vitro and in vivo, demonstrate that circBNC2 acts as a negative regulator of cell G2/M arrest by encoding a protein that promotes formation of CDK1/cyclin B1 complexes. Restoring circBNC2 in experimentally-induced male mouse models of fibrotic kidney and liver, decreases G2/M arrested cell numbers with secretion of fibrotic factors, thereby mitigating extracellular matrix deposition and fibrosis. Decreased expression of circBNC2 and increased G2/M arrest of epithelial cells are recapitulated in human ischemic reperfusion injury (IRI)-induced chronic kidney disease and inflammation-induced liver fibrosis, highlighting the clinical relevance. These findings suggest that restoring circBNC2 might represent a potential strategy for therapeutic intervention in epithelial organ fibrosis.

WT1 Inhibits Human Renal Carcinoma Cell Proliferation and Induces G2/M Arrest by Upregulating IL-24 Expression.

The transcription factor Wilms' tumor 1 (WT1) is involved in development, tissue homeostasis, and disease. However, the exact roles and the mechanisms of WT1 in renal carcinoma are not well understood. Therefore, in this study, we evaluated the ability of WT1 to block proliferation in renal carcinoma cells in vitro. Experimental analysis showed that WT1 overexpression inhibited the proliferation of renal carcinoma A498 cells and promoted arrest at the G2/M checkpoint. RNA-Seq identified differentially expressed genes, including IL-24, related to both the cell proliferation and the cell cycle. WT1 overexpression upregulated IL-24 expression, and IL-24 overexpression induced G2/M arrest. ChIP-Seq identified JUN as a direct target of WT1 in A498 cells, in which positive regulation was shown by RT-qPCR. It has been shown that the transcription factor JUN can regulate IL-24 expression, and therefore, we hypothesize that WT1 might regulate the IL-24 through JUN. Furthermore, analysis based on TCGA datasets showed that the expression of WT1-regulated genes, including TXNIP and GADD45A, was significantly correlated with the stage and histological grade of tumors, with high levels linked to favorable prognoses. Our results demonstrated that the overexpression of WT1 upregulates IL-24, leading to G2/M checkpoint arrest to reduce proliferation. These results indicate that regulation of IL-24 by WT1 inhibits proliferation and may represent a potential target for treating renal carcinoma.

High-dose zearalenone exposure disturbs G2/M transition during mouse oocyte maturation.

Zearalenone is a mycotoxin produced by fungi of the genus Fusarium, which has severe toxicity on animal and human health including reproduction. Previous study showed that zearalenone exposure inhibited oocyte polar body extrusion, while in present study we found that high dose zearalenone disturbed oocyte meiosis resumption. Our results showed that a high concentration of 100 µM zearalenone reduced the rate of germinal vesicle (GV) breakdown in mouse oocytes. Further analysis indicated that zearalenone caused the decrease of Cyclin B1 and CDK1 expression, indicating MPF activity was affected, which further induced G2/M arrest, and this could be rescued by the inhibition of Wee1 activity. We found that the oocytes under high concentration of zearalenone showed lower γ-H2A.X expression, suggesting that DNA damage repair was disturbed, which further activated of DNA damage checkpoints. This could be confirmed by the altered expression of CHK1 and CHK2 after zearalenone treatment. Moreover, the organelles such as mitochondria, ribosome, endoplasmic reticulum and Golgi apparatus were diffused from germinal vesicle periphery after zearalenone exposure, indicating that zearalenone affected protein synthesis, modification and transport, which further induced the arrest of G2/M transition. Taken together, our results showed that high dose of zearalenone exposure induced G2/M transition defect by affecting organelle function-related CHK1/2-Wee1-MPF pathway.

Targeted inhibition of acidic nucleoplasmic DNA-binding protein 1 enhances radiosensitivity of non-small cell lung cancer.

Acidic nucleoplasmic DNA binding protein 1 (AND-1, also known as WD repeat and HMG-box DNA-binding protein 1, WDHD1) plays an important role in DNA replication and repair, but the relationship between AND-1 and radiosensitivity is not well understood. This research explored the impact of AND-1 on the radiosensitivity of non-small cell lung cancer (NSCLC) for the first time. NSCLC cells were treated with AND-1 siRNA or a new AND-1 inhibitor, CH-3, and clonogenic survival assay was used to characterize cell radiosensitivity. Cell cycle and apoptosis were examined by flow cytometry. DNA damage was detected by comet assay, immunofluorescence, and homologous recombination (HR) repair assay. Finally, the radiosensitization effect of CH-3 was investigated in vivo in a xenograft tumor model. The results showed that AND-1 inhibition significantly increased the radiosensitivity of NSCLC cells. Mechanistically, AND-1 inhibitor (CH-3) induced G2/M phase arrest by regulating the ATM signaling pathway and enhanced irradiation-induced DNA damage by inhibiting the DNA HR repair pathway. CH-3 enhanced the radiosensitivity of NSCLC cells in vivo. The development of radiosensitizers that target AND-1 may provide an alternative strategy to inhibit NSCLC.

USP44 regulates irradiation-induced DNA double-strand break repair and suppresses tumorigenesis in nasopharyngeal carcinoma.

Radiotherapy is the primary treatment for patients with nasopharyngeal carcinoma (NPC), and approximately 20% of patients experience treatment failure due to tumour radioresistance. However, the exact regulatory mechanism remains poorly understood. Here, we show that the deubiquitinase USP44 is hypermethylated in NPC, which results in its downregulation. USP44 enhances the sensitivity of NPC cells to radiotherapy in vitro and in vivo. USP44 recruits and stabilizes the E3 ubiquitin ligase TRIM25 by removing its K48-linked polyubiquitin chains at Lys439, which further facilitates the degradation of Ku80 and inhibits its recruitment to DNA double-strand breaks (DSBs), thus enhancing DNA damage and inhibiting DNA repair via non-homologous end joining (NHEJ). Knockout of TRIM25 reverses the radiotherapy sensitization effect of USP44. Clinically, low expression of USP44 indicates a poor prognosis and facilitates tumour relapse in NPC patients. This study suggests the USP44-TRIM25-Ku80 axis provides potential therapeutic targets for NPC patients.

A Novel Role for RNF126 in the Promotion of G2 Arrest via Interaction With 14-3-3σ.

PURPOSE: Cell cycle checkpoints and DNA repair are important for cell survival after exogenous DNA damage. Both rapid blockage of G2 to M phase transition in the cell cycle and the maintenance of relatively slow G2 arrest are critical to protect cells from lethal ionizing radiation (IR). Checkpoint kinase 1 is pivotal in blocking the transition from G2 to M phases in response to IR. The 14-3-3σ protein is important for IR-induced G2 arrest maintenance in which p53-dependent 14-3-3σ transcription is involved. It has been demonstrated that Ring finger protein 126 (RNF126), an E3 ligase, is required to upregulate checkpoint kinase 1 expression. Thus, our goal was to study the role of RNF126 in the G2/M phase checkpoint. METHODS AND MATERIALS: The transition from G2 to M phases and G2 accumulation in response to IR were determined by flow cytometry through staining with phospho-histone H3 (pS10) antibody and propidium iodide, respectively. The interaction of RNF126 and 14-3-3σ was determined by GST-pulldown and coimmunoprecipitation assays. The stability of RNF126 and 14-3-3σ was determined by cycloheximide-based stability assay and ubiquitination detection by coimmunoprecipitation. The sequestering of cyclin-dependent kinase 1 and cyclin B1 from the nucleus was determined by immunofluorescence staining. RESULTS: RNF126 knockdown had no impact on the IR-induced transient blockage of G2 to M but impaired IR-induced G2 arrest maintenance in cells with or without wild-type p53. Mechanistically, RNF126 binds 14-3-3σ and prevents both proteins from ubiquitination-mediated degradation. Last, RNF126 is required for enforcing the cytoplasmic sequestration of cyclin B1 and cyclin-dependent kinase 1 proteins in response to IR. CONCLUSIONS: RNF126 promotes G2 arrest via interaction with 14-3-3σ in response to IR. Our study revealed a novel role for RNF126 in promoting G2 arrest, providing a new target for cancer treatment.

Identification of a novel catalytic inhibitor of topoisomerase II alpha that engages distinct mechanisms in p53wt or p53-/- cells to trigger G2/M arrest and senescence.

We report a novel topoisomerase IIα inhibitor, mercaptopyridine oxide (MPO), which induces G2/M arrest and senescence with distinctly different cell cycle regulators (p21 or p14ARF) in HCT116p 53WT and HCT116 p53-/- cells, respectively. MPO treatment induced defective topoisomerase IIα-mediated decatenation process and inhibition of the enzyme's catalytic activity that stalled entry into mitosis. Topoisomerase IIα inhibition was associated with ROS-mediated activation of ATM-Chk2 kinase axis in HCT116 p53WT cells, but not in HCT116 p53-/- cells displaying early Chk1 activation. Results suggest that E2F1 stabilization might link MPO-induced p53 phospho-activation in HCT116 p53WT cells or p14ARF induction in HCT116 p53-/- cells. Also, interaction between topoisomerase IIα and Chk1 was induced in both cell lines, which could be important for decatenation checkpoint activation, even upon p53 ablation. Notably, TCGA dataset analyses revealed topoisomerase IIα upregulation across a wide array of cancers, which was associated with lower overall survival. Corroborating that increased topoisomerase IIα expression might offer susceptibility to the novel inhibitor, MPO (5 µM) induced strong inhibition in colony forming ability of pancreatic and hepatocellular cancer cell lines. These data highlight a novel topoisomerase IIα inhibitor and provide proof-of-concept for its therapeutic potential against cancers even with loss-of-function of p53.

Dynamic prostate cancer transcriptome analysis delineates the trajectory to disease progression.

Comprehensive genomic studies have delineated key driver mutations linked to disease progression for most cancers. However, corresponding transcriptional changes remain largely elusive because of the bias associated with cross-study analysis. Here, we overcome these hurdles and generate a comprehensive prostate cancer transcriptome atlas that describes the roadmap to tumor progression in a qualitative and quantitative manner. Most cancers follow a uniform trajectory characterized by upregulation of polycomb-repressive-complex-2, G2-M checkpoints, and M2 macrophage polarization. Using patient-derived xenograft models, we functionally validate our observations and add single-cell resolution. Thereby, we show that tumor progression occurs through transcriptional adaption rather than a selection of pre-existing cancer cell clusters. Moreover, we determine at the single-cell level how inhibition of EZH2 - the top upregulated gene along the trajectory - reverts tumor progression and macrophage polarization. Finally, a user-friendly web-resource is provided enabling the investigation of dynamic transcriptional perturbations linked to disease progression.

Oncogenic KRAS drives radioresistance through upregulation of NRF2-53BP1-mediated non-homologous end-joining repair.

KRAS-activating mutations are oncogenic drivers and are correlated with radioresistance of multiple cancers, including colorectal cancer, but the underlying precise molecular mechanisms remain elusive. Herein we model the radiosensitivity of isogenic HCT116 and SW48 colorectal cancer cell lines bearing wild-type or various mutant KRAS isoforms. We demonstrate that KRAS mutations indeed lead to radioresistance accompanied by reduced radiotherapy-induced mitotic catastrophe and an accelerated release from G2/M arrest. Moreover, KRAS mutations result in increased DNA damage response and upregulation of 53BP1 with associated increased non-homologous end-joining (NHEJ) repair. Remarkably, KRAS mutations lead to activation of NRF2 antioxidant signaling to increase 53BP1 gene transcription. Furthermore, genetic silencing or pharmacological inhibition of KRAS, NRF2 or 53BP1 attenuates KRAS mutation-induced radioresistance, especially in G1 phase cells. These findings reveal an important role for a KRAS-induced NRF2-53BP1 axis in the DNA repair and survival of KRAS-mutant tumor cells after radiotherapy, and indicate that targeting NRF2, 53BP1 or NHEJ may represent novel strategies to selectively abrogate KRAS mutation-mediated radioresistance.

The Cyclin Cln1 Controls Polyploid Titan Cell Formation following a Stress-Induced G2 Arrest in Cryptococcus.

The pathogenic yeast Cryptococcus neoformans produces polyploid titan cells in response to the host lung environment that are critical for host adaptation and subsequent disease. We analyzed the in vivo and in vitro cell cycles to identify key aspects of the C. neoformans cell cycle that are important for the formation of titan cells. We identified unbudded 2C cells, referred to as a G2 arrest, produced both in vivo and in vitro in response to various stresses. Deletion of the nonessential cyclin Cln1 resulted in overproduction of titan cells in vivo and transient morphology defects upon release from stationary phase in vitro. Using a copper-repressible promoter PCTR4-CLN1 strain and a two-step in vitro titan cell formation assay, our in vitro studies revealed Cln1 functions after the G2 arrest. These studies highlight unique cell cycle alterations in C. neoformans that ultimately promote genomic diversity and virulence in this important fungal pathogen. IMPORTANCE Dysregulation of the cell cycle underlies many human genetic diseases and cancers, yet numerous organisms, including microbes, also manipulate the cell cycle to generate both morphologic and genetic diversity as a natural mechanism to enhance their chances for survival. The eukaryotic pathogen Cryptococcus neoformans generates morphologically distinct polyploid titan cells critical for host adaptation and subsequent disease. We analyzed the C. neoformans in vivo and in vitro cell cycles to identify changes required to generate the polyploid titan cells. C. neoformans paused cell cycle progression in response to various environmental stresses after DNA replication and before morphological changes associated with cell division, referred to as a G2 arrest. Release from this G2 arrest was coordinated by the cyclin Cln1. Reduced CLN1 expression after the G2 arrest was associated with polyploid titan cell production. These results demonstrate a mechanism to generate genomic diversity in eukaryotic cells through manipulation of the cell cycle that has broad disease implications.

Duox-generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts.

Progenitors of the thoracic tracheal system of adult Drosophila (tracheoblasts) arrest in G2 during larval life and rekindle a mitotic program subsequently. G2 arrest is dependent on ataxia telangiectasia mutated and rad3-related kinase (ATR)-dependent phosphorylation of checkpoint kinase 1 (Chk1) that is actuated in the absence of detectable DNA damage. We are interested in the mechanisms that activate ATR/Chk1 (Kizhedathu et al., 2018; Kizhedathu et al., 2020). Here we report that levels of reactive oxygen species (ROS) are high in arrested tracheoblasts and decrease upon mitotic re-entry. High ROS is dependent on expression of Duox, an H2O2 generating dual oxidase. ROS quenching by overexpression of superoxide dismutase 1, or by knockdown of Duox, abolishes Chk1 phosphorylation and results in precocious proliferation. Tracheae deficient in Duox, or deficient in both Duox and regulators of DNA damage-dependent ATR/Chk1 activation (ATRIP/TOPBP1/claspin), can induce phosphorylation of Chk1 in response to micromolar concentrations of H2O2 in minutes. The findings presented reveal that H2O2 activates ATR/Chk1 in tracheoblasts by a non-canonical, potentially direct, mechanism.