Mammalian Snm1 participates in cellular responses to genotoxic and mitotic stress

Eukaryotic cells have evolved a complex network of metabolic processes and regulatory systems to help ensure that hereditary information is protected or restored when exposed to genotoxic agents. Two members of the Snm1 protein family have been characterized; scSNM1/PSO2, a yeast gene responsible for repair of DNA interstrand crosslinks, and hARTEMIS, a human gene that is mutated in radiosensitive severe combined immunodeficiency (RS-SCID). Here we report on another member of this protein family, hSNM1, and its response to DNA damage and mitotic stress. We have found that this protein colocalizes and physically associates with 53BP1, a crucial member of the mammalian response to DNA damage. In addition, hSnm1 interacts with several proteins involved in mitosis, and mSNM1 deficiency causes a mitotic checkpoint defect in mouse embryonic fibroblasts. ^

Checkpoint mitótico como alvo terapêutico para sensibilizar células cancerígenas a agentes antimitóticos

Dissertação de Mestrado, Oncobiologia – Mecanismos Moleculares do Cancro, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

The Fate of Chrysotile-Induced Multipolar Mitosis and Aneuploid Population in Cultured Lung Cancer Cells

Chrysotile is one of the six types of asbestos, and it is the only one that can still be commercialized in many countries. Exposure to other types of asbestos has been associated with serious diseases, such as lung carcinomas and pleural mesotheliomas. The association of chrysotile exposure with disease is controversial. However, in vitro studies show the mutagenic potential of chrysotile, which can induce DNA and cell damage. The present work aimed to analyze alterations in lung small cell carcinoma cultures after 48 h of chrysotile exposure, followed by 2, 4 and 8 days of recovery in fiber-free culture medium. Some alterations, such as aneuploid cell formation, increased number of cells in G2/M phase and cells in multipolar mitosis were observed even after 8 days of recovery. The presence of chrysotile fibers in the cell cultures was detected and cell morphology was observed by laser scanning confocal microscopy. After 4 and 8 days of recovery, only a few chrysotile fragments were present in some cells, and the cellular morphology was similar to that of control cells. Cells transfected with the GFP-tagged alpha-tubulin plasmid were treated with chrysotile for 24 or 48 h and cells in multipolar mitosis were observed by time-lapse microscopy. Fates of these cells were established: retention in metaphase, cell death, progression through M phase generating more than two daughter cells or cell fusion during telophase or cytokinesis. Some of them were related to the formation of aneuploid cells and cells with abnormal number of centrosomes.

BUBR1 expression in benign oral lesions and squamous cell carcinomas: Correlation with human papillomavirus

Oral squamous Cell carcinoma (OSCC) is the most common head and neck cancer. Only in Brazil, the estimate is for 14,160 new cases in 2009. HPV is associated with increasing risk of oral cancer, but its role in carcinogenesis is still controversial. BUBR1, all important protein in the mitotic spindle assembly checkpoint (SAC), has been associated with some virus-encoded proteins and cancer. The aim of the present study was to evaluate the expression of BUBR1 in non-malignant oral lesions and OSCC with and without metastasis associated with HPV infection. We performed immunohistochemistry for BUBR1 in 70 OSCC biopsies divided into three groups (in situ tumors, invasive tumors without metastasis and invasive tumors with metastasis) with their respective lymph nodes from samples with metastasis and in 16 non-malignant oral lesions. PCR was performed in order to detect HPV DNA. Significantly higher BUBR1 expression associated with shorter survival (p=0.0479) was observed in malignant lesions. There was also it significant correlation (r=1.000) with BUBR1 expression in lesions with metastasis and their lymph nodes. Ninety percent of OSCC and 100% of benign lesions were HPV positive. HPV16 and HVP18 were present in 13 and 24% of HPV-positive OSCC samples, respectively. HPV was more prevalent (76%) in samples with a high BUBR1 expression and the absence of viral DNA had no influence oil BUBR1 expression. These findings suggest that HPV could be associated with overexpression of BUBR1 in OSCC. but not in benign oral lesions.

Metastatic lung cancer in a new mouse model inheriting p53 and latent K-ras mutations

Lung cancer is a devastating disease with very poor prognosis. The design of better treatments for patients would be greatly aided by mouse models that closely resemble the human disease. The most common type of human lung cancer is adenocarcinoma with frequent metastasis. Unfortunately, current models for this tumor are inadequate due to the absence of metastasis. Based on the molecular findings in human lung cancer and metastatic potential of osteosarcomas in mutant p53 mouse models, I hypothesized that mice with both K-ras and p53 missense mutations might develop metastatic lung adenocarcinomas. Therefore, I incorporated both K-rasLA1 and p53RI72HΔg alleles into mouse lung cells to establish a more faithful model for human lung adenocarcinoma and for translational and mechanistic studies. Mice with both mutations ( K-rasLA1/+ p53R172HΔg/+) developed advanced lung adenocarcinomas with similar histopathology to human tumors. These lung adenocarcinomas were highly aggressive and metastasized to multiple intrathoracic and extrathoracic sites in a pattern similar to that seen in lung cancer patients. This mouse model also showed gender differences in cancer related death and developed pleural mesotheliomas in 23.2% of them. In a preclinical study, the new drug Erlotinib (Tarceva) decreased the number and size of lung lesions in this model. These data demonstrate that this mouse model most closely mimics human metastatic lung adenocarcinoma and provides an invaluable system for translational studies. ^ To screen for important genes for metastasis, gene expression profiles of primary lung adenocarcinomas and metastases were analyzed. Microarray data showed that these two groups were segregated in gene expression and had 79 highly differentially expressed genes (more than 2.5 fold changes and p<0.001). Microarray data of Bub1b, Vimentin and CCAM1 were validated in tumors by quantitative real-time PCR (QPCR). Bub1b , a mitotic checkpoint gene, was overexpressed in metastases and this correlated with more chromosomal abnormalities in metastatic cells. Vimentin, a marker of epithelial-mesenchymal transition (EMT), was also highly expressed in metastases. Interestingly, Twist, a key EMT inducer, was also highly upregulated in metastases by QPCR, and this significantly correlated with the overexpression of Vimentin in the same tumors. These data suggest EMT occurs in lung adenocarcinomas and is a key mechanism for the development of metastasis in K-ras LA1/+ p53R172HΔg/+ mice. Thus, this mouse model provides a unique system to further probe the molecular basis of metastatic lung cancer.^

MAD2 associates with the cyclosome/anaphase-promoting complex and inhibits its activity

Cell cycle progression is monitored by checkpoint mechanisms that ensure faithful duplication and accurate segregation of the genome. Defects in spindle assembly or spindle-kinetochore attachment activate the mitotic checkpoint. Once activated, this checkpoint arrests cells prior to the metaphase-anaphase transition with unsegregated chromosomes, stable cyclin B, and elevated M phase promoting factor activity. However, the mechanisms underlying this process remain obscure. Here we report that upon activation of the mitotic checkpoint, MAD2, an essential component of the mitotic checkpoint, associates with the cyclin B-ubiquitin ligase, known as the cyclosome or anaphase-promoting complex. Moreover, purified MAD2 causes a metaphase arrest in cycling Xenopus laevis egg extracts and prevents cyclin B proteolysis by blocking its ubiquitination, indicating that MAD2 functions as an inhibitor of the cyclosome. Thus, MAD2 links the mitotic checkpoint pathway to the cyclin B destruction machinery which is critical in controlling the metaphase-anaphase transition.

MOB1, an Essential Yeast Gene Required for Completion of Mitosis and Maintenance of Ploidy

Mob1p is an essential Saccharomyces cerevisiae protein, identified from a two-hybrid screen, that binds Mps1p, a protein kinase essential for spindle pole body duplication and mitotic checkpoint regulation. Mob1p contains no known structural motifs; however MOB1 is a member of a conserved gene family and shares sequence similarity with a nonessential yeast gene, MOB2. Mob1p is a phosphoprotein in vivo and a substrate for the Mps1p kinase in vitro. Conditional alleles of MOB1 cause a late nuclear division arrest at restrictive temperature. MOB1 exhibits genetic interaction with three other yeast genes required for the completion of mitosis, LTE1, CDC5, and CDC15 (the latter two encode essential protein kinases). Most haploid mutant mob1 strains also display a complete increase in ploidy at permissive temperature. The mechanism for the increase in ploidy may occur through MPS1 function. One mob1 strain, which maintains stable haploidy at both permissive and restrictive temperature, diploidizes at permissive temperature when combined with the mps1–1 mutation. Strains containing mob2Δ also display a complete increase in ploidy when combined with the mps1-1 mutation. Perhaps in addition to, or as part of, its essential function in late mitosis, MOB1 is required for a cell cycle reset function necessary for the initiation of the spindle pole body duplication.

Taxol Suppresses Dynamics of Individual Microtubules in Living Human Tumor Cells

Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitotic spindle assembly, the mitotic checkpoint, and chromosome movement. We hypothesized that, in living cells, suppression of microtubule dynamics is responsible for the ability of taxol to inhibit mitotic progression and cell proliferation. Using quantitative fluorescence video microscopy, we examined the effects of taxol (30–100 nM) on the dynamics of individual microtubules in two living human tumor cell lines: Caov-3 ovarian adenocarcinoma cells and A-498 kidney carcinoma cells. Taxol accumulated more in Caov-3 cells than in A-498 cells. At equivalent intracellular taxol concentrations, dynamic instability was inhibited similarly in the two cell lines. Microtubule shortening rates were inhibited in Caov-3 cells and in A-498 cells by 32 and 26%, growing rates were inhibited by 24 and 18%, and dynamicity was inhibited by 31 and 63%, respectively. All mitotic spindles were abnormal, and many interphase cells became multinucleate (Caov-3, 30%; A-498, 58%). Taxol blocked cell cycle progress at the metaphase/anaphase transition and inhibited cell proliferation. The results indicate that suppression of microtubule dynamics by taxol deleteriously affects the ability of cancer cells to properly assemble a mitotic spindle, pass the metaphase/anaphase checkpoint, and produce progeny.

Spc98p Directs the Yeast γ-Tubulin Complex into the Nucleus and Is Subject to Cell Cycle-dependent Phosphorylation on the Nuclear Side of the Spindle Pole Body

In the yeast Saccharomyces cerevisiae, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope. Microtubule organization requires the γ-tubulin complex containing the γ-tubulin Tub4p, Spc98p, and Spc97p. The Tub4p complex is associated with cytoplasmic and nuclear substructures of the SPB, which organize the cytoplasmic and nuclear microtubules. Here we present evidence that the Tub4p complex assembles in the cytoplasm and then either binds to the cytoplasmic side of the SPB or is imported into the nucleus followed by binding to the nuclear side of the SPB. Nuclear import of the Tub4p complex is mediated by the essential nuclear localization sequence of Spc98p. Our studies also indicate that Spc98p in the Tub4p complex is phosphorylated at the nuclear, but not at the cytoplasmic, side of the SPB. This phosphorylation is cell cycle dependent and occurs after SPB duplication and nucleation of microtubules by the new SPB and therefore may have a role in mitotic spindle function. In addition, activation of the mitotic checkpoint stimulates Spc98p phosphorylation. The kinase Mps1p, which functions in SPB duplication and mitotic checkpoint control, seems to be involved in Spc98p phosphorylation. Our results also suggest that the nuclear and cytoplasmic Tub4p complexes are regulated differently.

Two genes required for the binding of an essential Saccharomyces cerevisiae kinetochore complex to DNA.

Kinetochores are DNA-protein structures that assemble on centromeric DNA and attach chromosomes to spindle microtubules. Because of their simplicity, the 125-bp centromeres of Saccharomyces cerevisiae are particularly amenable to molecular analysis. Budding yeast centromeres contain three sequence elements of which centromere DNA sequence element III (CDEIII) appears to be particularly important. cis-acting mutations in CDEIII and trans-acting mutations in genes encoding subunits of the CDEIII-binding complex (CBF3) prevent correct chromosome transmission. Using temperature-sensitive mutations in CBF3 subunits, we show a strong correlation between DNA-binding activity measured in vitro and kinetochore activity in vivo. We extend previous findings by Goh and Kilmartin [Goh, P.-Y. & Kilmartin, J.V. (1993) J. Cell Biol. 121, 503-512] to argue that DNA-bound CBF3 may be involved in the operation of a mitotic checkpoint but that functional CBF3 is not required for the assembly of a bipolar spindle.

Functional characterization of proteins required for mitotic progression and the spindle assembly checkpoint

During mitotic cell division, the genetic material packed into chromosomes is divided equally between two daughter cells. Before the separation of the two copies of a chromosome (sister chromatids), each chromosome has to be properly connected with microtubules of the mitotic spindle apparatus and aligned to the centre of the cell. The spindle assembly checkpoint (SAC) monitors connections between microtubules and chromosomes as well as tension applied across the centromere. Microtubules connect to a chromosome via kinetochores, which are proteinaceous organelles assembled onto the centromeric region of the sister chromatids. Improper kinetochore-microtubule attachments activate the SAC and block chromosome segregation until errors are corrected and all chromosomes are connected to the mitotic spindle in a bipolar manner. The purpose of this surveillance mechanism is to prevent loss or gain of chromosomes in daughter cells that according to current understanding contributes to cancer formation. Numerous proteins participate in the regulation of mitotic progression. In this thesis, the mitotic tasks of three kinetochore proteins, Shugoshin 1 (Sgo1), INCENP, and p38 MAP kinase (p38 MAPK), were investigated. Sgo1 is a protector of centromeric cohesion. It is also described in the tension-sensing mechanism of the SAC and in the regulation of kinetochore-microtubule connections. Our results revealed a central role for Sgo1 in a novel branch of kinetochore assembly. INCENP constitutes part of the chromosomal passenger complex (CPC). The other members of the core complex are the Aurora B kinase, Survivin and Borealin. CPC is an important regulatory element of cell division having several roles at various stages of mitosis. Our results indicated that INCENP and Aurora B are highly dynamic proteins at the mitotic centromeres and suggested a new role for CPC in regulation of chromosome movements and spindle structure during late mitosis. The p38 MAPK has been implicated in G1 and G2 checkpoints during the cell cycle. However, its role in mitotic progression and control of SAC signaling has been controversial. In this thesis, we discovered a novel function for p38γ MAPK in chromosome orientation and spindle structure as well as in promotion of viability of mitotic cells.

Regulation of Initiation of S Phase, Replication Checkpoint Signaling, and Maintenance of Mitotic Chromosome Structures during S Phase by Hsk1 Kinase in the Fission Yeast

Hsk1, Saccharomyces cerevisiae Cdc7-related kinase in Shizosaccharomyces pombe, is required for G1/S transition and its kinase activity is controlled by the regulatory subunit Dfp1/Him1. Analyses of a newly isolated temperature-sensitive mutant, hsk1-89, reveal that Hsk1 plays crucial roles in DNA replication checkpoint signaling and maintenance of proper chromatin structures during mitotic S phase through regulating the functions of Rad3 (ATM)-Cds1 and Rad21 (cohesin), respectively, in addition to expected essential roles for initiation of mitotic DNA replication through phosphorylating Cdc19 (Mcm2). Checkpoint defect in hsk1-89 is indicated by accumulation of cut cells at 30°C. hsk1-89 displays synthetic lethality in combination with rad3 deletion, indicating that survival of hsk1-89 depends on Rad3-dependent checkpoint pathway. Cds1 kinase activation, which normally occurs in response to early S phase arrest by nucleotide deprivation, is largely impaired in hsk1-89. Furthermore, Cds1-dependent hyperphosphorylation of Dfp1 in response to hydroxyurea arrest is eliminated in hsk1-89, suggesting that sufficient activation of Hsk1-Dfp1 kinase is required for S phase entry and replication checkpoint signaling. hsk1-89 displays apparent defect in mitosis at 37°C leading to accumulation of cells with near 2C DNA content and with aberrant nuclear structures. These phenotypes are similar to those of rad21-K1 and are significantly enhanced in a hsk1-89 rad21-K1 double mutant. Consistent with essential roles of Rad21 as a component for the cohesin complex, sister chromatid cohesion is partially impaired in hsk1-89, suggesting a possibility that infrequent origin firing of the mutant may affect the cohesin functions during S phase.

TRAIP is a regulator of the spindle assembly checkpoint.

Accurate chromosome segregation during mitosis is temporally and spatially coordinated by fidelity-monitoring checkpoint systems. Deficiencies in these checkpoint systems can lead to chromosome segregation errors and aneuploidy, and promote tumorigenesis. Here, we report that the TRAF-interacting protein (TRAIP), a ubiquitously expressed nucleolar E3 ubiquitin ligase important for cellular proliferation, is localized close to mitotic chromosomes. Its knockdown in HeLa cells by RNA interference (RNAi) decreased the time of early mitosis progression from nuclear envelope breakdown (NEB) to anaphase onset and increased the percentages of chromosome alignment defects in metaphase and lagging chromosomes in anaphase compared with those of control cells. The decrease in progression time was corrected by the expression of wild-type but not a ubiquitin-ligase-deficient form of TRAIP. TRAIP-depleted cells bypassed taxol-induced mitotic arrest and displayed significantly reduced kinetochore levels of MAD2 (also known as MAD2L1) but not of other spindle checkpoint proteins in the presence of nocodazole. These results imply that TRAIP regulates the spindle assembly checkpoint, MAD2 abundance at kinetochores and the accurate cellular distribution of chromosomes. The TRAIP ubiquitin ligase activity is functionally required for the spindle assembly checkpoint control.

The DNA damage response to adeno-associated virus : centrosome overduplication and induction of cell death independently of the spindle checkpoint

Summary: Adeno-associated virus type 2 (AAV2) is a small virus containing single-stranded DNA of approximately 4.7kb in size. Both ends of the viral genome are flanked with inverted terminal repeat sequences (ITRs), which serve as primers for viral replication. Previous work in our laboratory has shown that AAV2 DNA with ultraviolet radiation-generated crosslinks (UV-AAV2) provokes a DNA damage response in the host cell by mimicking a stalled replication fork. Infection of cells with UV-AAV2 leads to a p53-and Chk1-mediated cell cycle arrest at the G2/M border of the cell cycle. However, tumour cells lacking the tumour suppressor protein p53 cannot sustain this arrest and enter a prolonged impaired mitosis, the outcome of which is cell death. The aim of my thesis was to investigate how UV-inactivated AAV2 kilts p53-deficient cancer cells. I found that the UV-AAV2-induced DNA damage signalling induces centriole overduplication in infected cells. The virus is able to uncouple the centriole duplication cycle from the cell cycle, leading to amplified centrosome numbers. Chk1 colocalises with centrosomes in the infected cells and the centrosome overduplication is dependent on the presence of Chk1, as well as on the activities of ATR and Cdk kinases and on the G2 arrest. The UV-AAV2-induced DNA damage signalling inhibits the degradation of cyclin B 1 and securin by the anaphase promoting complex, suggesting that the spindle checkpoint is activated in these mitotic cells. Interference with the spindle checkpoint components Mad2 and BubR1 revealed that the UV-AAV2-provoked mitotic catastrophe occurs independently of spindle checkpoint function, This work shows that, in the p53 deficient cells, UV-AAV2 triggers mitotic catastrophe associated with a dramatic Chk1-dependent overduplication of centrioles and the consequent formation of multiple spindle poles in mitosis. Résumé Le virus associé à l'adénovirus type 2 (AAV2) est un petit virus contenant un simple brin d'ADN d'environ 4.7kb. Des expériences antérieures dans notre laboratoire ont montré que les liens intramoléculaires sur l'ADN de AAV2 provoqués paz l'irradiation aux ultraviolets (UV) ressemblent à une fourche de réplication bloquée, ce qui provoque une réponse aux dommages à l'ADN dans la cellule hôte. L'infection des cellules avec UV-AAV2 résulte en un arrêt du cycle cellulaire à la transition G2/M entraîné par les protéines ATR et Chk1. Cependant, les cellules tumorales auxquelles il manque le suppresseur de tumeur p53 ne peuvent pas tenir cet arrêt et entrent dans une mitose anormale et prolongée qui se terminera par la mort cellulaire. Le but de ma thèse était d'étudier comment l'AAV2 inactivé par l'irradiation UV tue les cellules cancéreuses n'ayant pas p53. Je montre ici que le signal de dommages à l'ADN induit par UV-AAV2 génère une surduplication des centrioles dans les cellules infectées. Le virus est capable de dissocier le cycle de duplication du centriole du cycle cellulaire ce qui crée un nombre amplifié de centrosomes. Chk1 est co-localisé avec le centrosome dans les cellules infectées et la swduplication du centrosome est dépendante de la présence de Chk1, de l'activité des kinases ATR et Cdk et de l'arrêt en G2 de la cellule. Le signal d'ADN endommagé induit par UV-AAV2 réprime la dégradation des protéines cycline B1 et securine par le complexe promoteur de l'anaphase (APC), ce qui suggère que le point de contrôle du fuseau mitotique est activé dans ces cellules en mitose. L'étude d'interférence avec des éléments du point de contrôle du fuseau mitotique, Mad2 et BubR1, a révélé que la catastrophe mitotique provoquée paz UV-AAV2 survient indépendamment du point de contrôle du fuseau mitotique. Ce travail montre que dans les cellules déficientes en p53, UV-AAV2 induit une catastrophe mitotique associée à une surduplication des centrioles dépendant de Chk1 et ayant pour conséquence dramatique la formation de multiples fuseaux mitotiques dans la cellule en mitose.

Mitotic regulation by Polo-like kinase 1 and the Chromosomal Passenger Complex

During mitosis, the duplicated genome must be accurately divided between two daughter cells. Polo-like kinase 1 (Plk1) and Aurora B kinase, together with its binding partners Incenp, Survivin and Borealin (chromosomal passenger complex, CPC), have key roles in coordinating mitotic events. The accuracy of cell division is safeguarded by a signaling cascade termed the mitotic spindle checkpoint (SC), which ensures that chromosomes are not physically separated before correct bipolar attachments have been formed between kinetochores and spindle microtubules (MT). An inhibitory “wait anaphase” signal, which delays chromosome separation (anaphase onset), is created at individual kinetochores and broadcasted throughout the cell in response to lack of kinetochore-microtubule (kMT) attachment or proper interkinetochore tension. It is believed that the fast turnover of SC molecules at kinetochores contributes to the cell’s ability to produce this signal and enables rapid responses to changing cellular conditions. Kinetochores that lack MT attachment and tension express a certain phosphoepitope called the 3F3/2 phosphoepitope, which has been linked to SC signaling. In the experimental part, we investigated the regulation of the 3F3/2 phosphoepitope, analyzed whether CPC molecules turn over at centromeres, and dissected the mitotic roles of the CPC using a microinjection technique that allowed precise temporal control over its function. We found that the kinetochore 3F3/2 phosphoepitope is created by Plk1, and that CPC proteins exhibit constant exchange at centromeres. Moreover, we found that CPC function is necessary in the regulation of chromatid movements and spindle morphology in anaphase. In summary, we identified new functions of key mitotic regulators Plk1 and CPC, and provided insighs into the coordination of mitotic events.