scholarly journals RACH2, a novel human gene that complements a fission yeast cell cycle checkpoint mutation.

1995 ◽  
Vol 6 (10) ◽  
pp. 1411-1421 ◽  
Author(s):  
S Davey ◽  
D Beach
Keyword(s):  
Double Mutant ◽  
Human Gene ◽  
Cell Cycle Checkpoint ◽  
Temperature Sensitive ◽  
Uv Resistance ◽  
Mitotic Entry ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Cycle Checkpoint ◽  
Dose Dependent

We have identified a novel human gene by virtue of its ability to complement the rad1-1 checkpoint mutant of Schizosaccharomyces pombe. This gene, called RACH2, rescues the temperature-sensitive lethality of a rad1-1 wee1-50 double mutant of S. pombe. Expression of RACH2 in S. pombe rad1-1 strains partially restores UV resistance to the rad1-1 mutant strain. Expression of RACH2 in a rad1-1 cdc25-22 double mutant partially restores the dose-dependent delay in mitotic entry after irradiation that is lost in rad1-1 checkpoint-deficient mutants. Overexpression of RACH2 in human tissue culture cells induces apoptosis.

scholarly journals The Yeast ULP2 (SMT4) Gene Encodes a Novel Protease Specific for the Ubiquitin-Like Smt3 Protein

2000 ◽  
Vol 20 (7) ◽  
pp. 2367-2377 ◽  
Author(s):  
Shyr-Jiann Li ◽  
Mark Hochstrasser
Keyword(s):  
Cell Function ◽  
Feedback Mechanism ◽  
Cell Cycle Checkpoint ◽  
Temperature Sensitive ◽  
Partial Recovery ◽  
Protein Ligation ◽  
Protein Conjugates ◽  
Dna Damaging Agents ◽  
Cycle Checkpoint ◽  
Specific Protease

ABSTRACT Yeast Smt3 and its vertebrate homolog SUMO-1 are ubiquitin-like proteins (Ubls) that are reversibly ligated to other proteins. LikeSMT3, SMT4 was first isolated as a high-copy-number suppressor of a defective centromere-binding protein. We show here that SMT4 encodes an Smt3-deconjugating enzyme, Ulp2. In cells lacking Ulp2, specific Smt3-protein conjugates accumulate, and the conjugate pattern is distinct from that observed in a ulp1ts strain, which is defective for a distantly related Smt3-specific protease, Ulp1. The ulp2Δ mutant exhibits a pleiotropic phenotype that includes temperature-sensitive growth, abnormal cell morphology, decreased plasmid and chromosome stability, and a severe sporulation defect. The mutant is also hypersensitive to DNA-damaging agents, hydroxyurea, and benomyl. Although cell cycle checkpoint arrest in response to DNA damage, replication inhibition, or spindle defects occurs with normal kinetics, recovery from arrest is impaired. Surprisingly, either introduction of aulp1ts mutation or overproduction of catalytically inactive Ulp1 can substantially overcome theulp2Δ defects. Inactivation of Ulp2 also suppresses several ulp1ts defects, and the double mutant accumulates far fewer Smt3-protein conjugates than either single mutant. Our data suggest the existence of a feedback mechanism that limits Smt3-protein ligation when Smt3 deconjugation by both Ulp1 and Ulp2 is compromised, allowing a partial recovery of cell function.

Activation of p53-mediated cell cycle checkpoint in response to micronuclei formation

1998 ◽  
Vol 111 (7) ◽  
pp. 977-984
Author(s):  
A.A. Sablina ◽  
G.V. Ilyinskaya ◽  
S.N. Rubtsova ◽  
L.S. Agapova ◽  
P.M. Chumakov ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Cell Activation ◽  
P53 Gene ◽  
Cell Cycle Checkpoint ◽  
Abnormal Chromosome ◽  
Brdu Incorporation ◽  
Temperature Sensitive ◽  
Cells With Micronuclei ◽  
Cycle Checkpoint

Inactivation of p53 tumor-suppressor leads to genetic instability and, in particular, to accumulation of cells with abnormal numbers of chromosomes. In order to better define the role of p53 function in maintaining genome integrity we investigated the involvement of p53 in the control of proliferation of micronucleated cells resulting from abnormal chromosome segregation. Using cell lines expressing temperature-sensitive (ts) p53 or containing p53 genetic suppressor element (p53-GSE) we showed that inhibition of p53 function increases the frequency of cells with micronuclei. Immunofluorescence study revealed that in REF52 cell cultures with both spontaneous and colcemid-induced micronuclei the proportion of p53-positive cells is considerably higher among micronucleated variants as compared with their mononuclear counterparts. Analysis of 12(1)ConA cells expressing the beta-galactosidase reporter gene under the control of a p53-responsive promoter showed activation of p53-regulated transcription in the cells with micronuclei. Importantly, the percentage of cells manifesting specific p53 activity in colcemid-treated cultures increased with an augmentation of the number of micronuclei in the cell. Activation of p53 in micronucleated cells was accompanied by a decrease in their ability to enter S-phase as was determined by comparative analysis of 5-bromodeoxyuridine (5-BrdU) incorporation by the cells with micronuclei and their mononuclear counterparts. Inhibition of p53 function in the cells with tetracycline-regulated p53 gene expression, as well as in the cells expressing ts-p53 or p53-GSE, abolished cell cycle arrest in micronucleated cells. These results along with the data showing no increase in the frequency of chromosome breaks in REF52 cells after colcemid treatment suggest the existence of p53-mediated cell cycle checkpoint(s) preventing proliferation of micronucleated cells derived as a result of abnormal chromosome segregation during mitosis.

The Wee1 Inhibitor MK1775 In Combination With Cytarabine (AraC) Has Potent Activity In AML By Completely Abrogating DNA Damage and Cell Cycle Checkpoint Repair Capacity Via The Mrn/NBS1 Complex

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3831-3831
Author(s):  
Leena Chaudhuri ◽  
James M Bogenberger ◽  
Lisa Sproat ◽  
James L Slack ◽  
Veena Fauble ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Single Agent ◽  
S Phase ◽  
Cell Cycle Checkpoint ◽  
Leukemic Cells ◽  
Repair Capacity ◽  
Strand Breaks ◽  
Mitotic Entry ◽  
Cycle Checkpoint

Abstract Cytarabine (AraC) resistance is a fundamental feature of refractory/relapsed AML. RNA interference (RNAi) screens conducted in our laboratory recently identified WEE1 kinase (WEE1) as one of the top candidate genes and target in leukemias in combination with AraC. WEE1 is a tyrosine kinase belonging to the Ser/Thr family of protein kinases and acts as a negative regulator of mitotic entry by controlling DNA damage (DDR) and cell cycle checkpoint responses. The WEE1 inhibitor MK1775 potently synergizes with AraC ex vivo and in vitro and clinical trials are in preparation. However, the mechanism of action for the anti-leukemic activity of MK1775 with AraC remains unknown. To elucidate genes mediating activity of the combination, we first performed siRNA rescue screens silencing a custom set of 44 genes involved in WEE1 regulation under combined AraC + MK1775 to identify sensitizers and markers of resistance. The MRN (MRE11, Rad51, NBS1) complex and particularly NBS1 were potent modifiers of AraC and MK1775. Focusing on NBS1 since it is proposed to centrally regulate the defense capacity of leukemic cells, we identified that NBS1 phosphorylation at Ser343 (the ATM regulation site) is significantly altered both in cell lines and primary AML samples under combined AraC+MK1775 treatment as compared to single agent MK1775. In parallel, lower phosphorylation of ATMS1981(an autophosphorylation site in response to DNA strand breaks), was observed indicating that the ATM-CHEK1 pathway is not activated under co-treatment. Further Homologous recombination (HR)-mediated repair was compromised by AraC+MK1775 shown by DR-GFP expression vector to measure intracellular HR capacity: post-transfection of the I-SceI nuclease which cleaves non-functioning GFP tandem repeats to form a functional GFP unit, the HR was reduced with the combination. Consistently other HR markers decreased as well. Delayed accumulation of Cyclin A (indicative of S-phase progression) and greater inhibition of phospho-Cdk2Y15in synchronized cells treated with AraC + MK1775 in comparison to controls was observed. In addition the cell cycle was globally dysregulated by slower S-phase kinetics (progression), a completely abrogated G2/M checkpoint/phase as well as de-regulated DNA replication origin formation and firing as evidenced by Cdt1 and Mus81. As a consequence high single and double strand breaks (ɣH2AX) were observed with an increase in phospho-histone H3 in AraC + MK1775 treated cells compared to untreated cells or MK1775 single agent, confirming faster mitotic entry. Changes were followed by massive induction of apoptosis. Since WEE1 is implicated in leukemic stem cell maintenance we examined the long term effects of the combination in colony forming assays. AraC + MK1775 treated leukemic cells obtained from patients with AML were re-plated on Methocult after drug washout and colonies counted after 14 days. While MK1775 as a single agent could reduce colony formation by 4 fold compared to controls and lower dose AraC, co-treatment with low to moderate doses of AraC and MK1775 reduced colony formation by more than 7 fold and to almost zero in some primary specimens. Taken together, these results suggest that leukemia cells co-treated with AraC + MK1775 lost their ability to activate DNA damage and repair pathways mainly by compromising the MRN complex via NBS1 with subsequently reduced HR. The combination (as opposed to single agents) almost complete dysregulated the cell cycle and its checkpoints lead to DNA damage, genomic instability and rapid exit from the cell cycle with cell death via apoptosis. Thus we have molecularly characterized the detailed mechanisms underlying the potent AraC+WEE1 inhibition in AML and describe for the first time a therapeutic combination that has the potential to abrogate the MRN and NBS1 repair capacity which is central for drug resistance in AML. A key implication of our work is to provide a clinical rationale, mechanistic understanding and suggestions for biomarkers to clinically evaluate AraC + MK1775 in patients with AML. Disclosures: No relevant conflicts of interest to declare.

Deletion of PIN4 Suppresses the Protein Transport Defects Caused by sec12-4 Mutation in Saccharomyces cerevisiae

2020 ◽  
Vol 30 (1-6) ◽  
pp. 25-35
Author(s):  
Akiko Murakami-Sekimata ◽  
Masayuki Sekimata ◽  
Natsumi Sato ◽  
Yuto Hayasaka ◽  
Akihiko Nakano
Keyword(s):  
Protein Transport ◽  
Cell Cycle Checkpoint ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Nucleotide Exchange ◽  
Casein Kinase I ◽  
Vesicle Budding ◽  
Nucleotide Exchange Factor ◽  
Cycle Checkpoint ◽  
Copii Vesicles

Newly synthesized secretory proteins are released into the lumen of the endoplasmic reticulum (ER). The secretory proteins are surrounded by coat protein complex II (COPII) vesicles, and transported from the ER and reach their destinations through the Golgi apparatus. Sec12p is a guanine nucleotide exchange factor for Sar1p, which initiates COPII vesicle budding from the ER. The activation of Sar1p by Sec12p and the subsequent COPII coat assembly have been well characterized, but the events that take place upstream of Sec12p remain unclear. In this study, we isolated the novel extragenic suppressor of <i>sec12-4</i>, <i>PIN4/MDT1</i>, a cell cycle checkpoint target. A yeast two-hybrid screening was used to identify Pin4/Mdt1p as a binding partner of the casein kinase I isoform Hrr25p, which we have previously identified as a modulator of Sec12p function. Deletion of <i>PIN4</i> suppressed both defects of temperature-sensitive growth and the partial protein transport observed in <i>sec12-4</i> mutants. The results of this study suggest that Pin4p provides novel aspects of Sec12p modulations.

Isolation and characterization of fission yeast mutants defective in the assembly and placement of the contractile actin ring

1996 ◽  
Vol 109 (1) ◽  
pp. 131-142 ◽  
Author(s):  
F. Chang ◽  
A. Woollard ◽  
P. Nurse
Keyword(s):  
Fission Yeast ◽  
Ring Formation ◽  
Cell Cycle Checkpoint ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Actin Ring ◽  
Division Site ◽  
Isolation And Characterization ◽  
Cycle Checkpoint ◽  
Actin Cables

Fission yeast cells divide by medial cleavage using an actin-based contractile ring. We have conducted a genetic screen for temperature-sensitive mutants defective in the assembly and placement of this actin ring. Six genes necessary for actin ring formation and one gene necessary for placement of the actin ring have now been identified. The genes can be further organized into different phenotypic groups, suggesting that the gene products may have different functions in actin ring formation. Mutants of cdc3 and cdc8, which encode profilin and tropomyosin respectively, display disorganized actin patches in all cells. cdc12 and cdc15 mutants display disorganized actin patches during mitosis, but normal interphase actin patterns. cdc4 and rng2 mutants display disorganized actin cables during mitosis, but normal interphase actin patterns. In mid1 mutants, the actin ring and septum are positioned at random locations and angles on the cell surface, although the nucleus is positioned normally, indicating that the mid1 gene product is required to couple the division site to the position of the nucleus. mid1 mutant cells may reveal a new cell cycle checkpoint in telophase that coordinates cell division and the proper distribution of nuclei. The actin ring forms medially in a beta-tubulin mutant, showing that actin ring formation and placement are not dependent on the mitotic spindle.

scholarly journals Dissociation of gemcitabine chemosensitization by CHK1 inhibition from cell cycle checkpoint abrogation and aberrant mitotic entry

Cell Cycle ◽  
2016 ◽  
Vol 15 (5) ◽  
pp. 730-739 ◽  
Author(s):  
Leslie A. Parsels ◽  
Daria M. Tanska ◽  
Joshua D. Parsels ◽  
Sonya D. Zabludoff ◽  
Kyle C. Cuneo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Checkpoint ◽  
Mitotic Entry ◽  
Cycle Checkpoint

scholarly journals WEE1 Inhibition Enhances Anti-Apoptotic Dependency as a Result of Premature Mitotic Entry and DNA Damage

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1743 ◽  
Author(s):  
Mathilde Rikje Willemijn de Jong ◽  
Myra Langendonk ◽  
Bart Reitsma ◽  
Pien Herbers ◽  
Marcel Nijland ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
B Cell Lymphoma ◽  
Cell Cycle Checkpoint ◽  
Cell Cycle Checkpoints ◽  
Specific Cell ◽  
M Cell ◽  
Mitotic Entry ◽  
Enhanced Sensitivity ◽  
Cycle Checkpoint

Genomically unstable cancers are dependent on specific cell cycle checkpoints to maintain viability and prevent apoptosis. The cell cycle checkpoint protein WEE1 is highly expressed in genomically unstable cancers, including diffuse large B-cell lymphoma (DLBCL). Although WEE1 inhibition effectively induces apoptosis in cancer cells, the effect of WEE1 inhibition on anti-apoptotic dependency is not well understood. We show that inhibition of WEE1 by AZD1775 induces DNA damage and pre-mitotic entry in DLBCL, thereby enhancing dependency on BCL-2 and/or MCL-1. Combining AZD1775 with anti-apoptotic inhibitors such as venetoclax (BCL-2i) or S63845 (MCL-1i) enhanced sensitivity in a cell-specific manner. In addition, we demonstrate that both G2/M cell cycle arrest and DNA damage induction put a similar stress on DLBCL cells, thereby enhancing anti-apoptotic dependency. Therefore, genotoxic or cell cycle disrupting agents combined with specific anti-apoptotic inhibitors may be very effective in genomic unstable cancers such as DLBCL and therefore warrants further clinical evaluation.

scholarly journals The Saccharomyces cerevisiae Suppressor of Choline Sensitivity (SCS2) Gene Is a Multicopy Suppressor of mec1 Telomeric Silencing Defects

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 145-154 ◽  
Author(s):  
Rolf J Craven ◽  
Thomas D Petes
Keyword(s):  
Double Mutant ◽  
Cell Cycle Checkpoint ◽  
Checkpoint Protein ◽  
Multicopy Suppressor ◽  
Telomeric Silencing ◽  
Cycle Checkpoint ◽  
A Cell ◽  
Atm Protein ◽  
Mutant Cells ◽  
Suppressor Screen

Abstract Mec1p is a cell cycle checkpoint protein related to the ATM protein kinase family. Certain mec1 mutations or overexpression of Mec1p lead to shortened telomeres and loss of telomeric silencing. We conducted a multicopy suppressor screen for genes that suppress the loss of silencing in strains overexpressing Mec1p. We identified SCS2 (suppressor of choline sensitivity), a gene previously isolated as a suppressor of defects in inositol synthesis. Deletion of SCS2 resulted in decreased telomeric silencing, and the scs2 mutation increased the rate of cellular senescence observed for mec1-21 tel1 double mutant cells. Genetic analysis revealed that Scs2p probably acts through a different telomeric silencing pathway from that affected by Mec1p.

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

2002 ◽  
Vol 22 (4) ◽  
pp. 1049-1059 ◽  
Author(s):  
Bo Xu ◽  
Seong-Tae Kim ◽  
Dae-Sik Lim ◽  
Michael B. Kastan
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
S Phase ◽  
Cell Cycle Checkpoint ◽  
Cell Cycle Checkpoints ◽  
Ionizing Irradiation ◽  
Mammalian Cell Cycle ◽  
Cycle Checkpoint ◽  
Dose Dependent ◽  
S Phase Checkpoint

ABSTRACT Cell cycle checkpoints are among the multiple mechanisms that eukaryotic cells possess to maintain genomic integrity and minimize tumorigenesis. Ionizing irradiation (IR) induces measurable arrests in the G1, S, and G2 phases of the mammalian cell cycle, and the ATM (ataxia telangiectasia mutated) protein plays a role in initiating checkpoint pathways in all three of these cell cycle phases. However, cells lacking ATM function exhibit both a defective G2 checkpoint and a prolonged G2 arrest after IR, suggesting the existence of different types of G2 arrest. Two molecularly distinct G2/M checkpoints were identified, and the critical importance of the choice of G2/M checkpoint assay was demonstrated. The first of these G2/M checkpoints occurs early after IR, is very transient, is ATM dependent and dose independent (between 1 and 10 Gy), and represents the failure of cells which had been in G2 at the time of irradiation to progress into mitosis. Cell cycle assays that can distinguish mitotic cells from G2 cells must be used to assess this arrest. In contrast, G2/M accumulation, typically assessed by propidium iodide staining, begins to be measurable only several hours after IR, is ATM independent, is dose dependent, and represents the accumulation of cells that had been in earlier phases of the cell cycle at the time of exposure to radiation. G2/M accumulation after IR is not affected by the early G2/M checkpoint and is enhanced in cells lacking the IR-induced S-phase checkpoint, such as those lacking Nbs1 or Brca1 function, because of a prolonged G2 arrest of cells that had been in S phase at the time of irradiation. Finally, neither the S-phase checkpoint nor the G2 checkpoints appear to affect survival following irradiation. Thus, two different G2 arrest mechanisms are present in mammalian cells, and the type of cell cycle checkpoint assay to be used in experimental investigation must be thoughtfully selected.

Cytoplasmic Tubules in Cells Infected with Laryngotracheitis Virus

1969 ◽  
Vol 27 ◽  
pp. 376-377
Author(s):  
A. M. Watrach
Keyword(s):  
Tissue Culture ◽  
Small Proportion ◽  
Chicken Embryo ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Morphologic Characteristics ◽  
Infectious Laryngotracheitis ◽  
Laryngotracheitis Virus ◽  
In Cells

During a study of the development of infectious laryngotracheitis (LT) virus in tissue culture cells, unusual tubular formations were found in the cytoplasm of a small proportion of the affected cells. It is the purpose of this report to describe the morphologic characteristics of the tubules and to discuss their possible association with the development of virus.The source and maintenance of the strain of LT virus have been described. Prior to this study, the virus was passed several times in chicken embryo kidney (CEK) tissue culture cells.

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